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A LIBRARIES
MICHIGAN STATE UNIVERSITY
EAST LANSING, MICH 48824-1048

times?

This is to certify that the
thesis entitled

EFFICACY OF CHEMICAL SANITIZERS TO INACTIVATE
ESCHERICHIA COLI 0157IH7, SALMONELLA
TYPHIMURIUM DT104, AND LISTER/A MONOCYTOGENES
ON ALFALFA SEEDS AND SPROUTS

presented by
Pascale Marie-Michele Pierre

has been accepted towards fulfillment
of the requirements for the

Masters degree in Food Science

 

 

W 3. fight

Major Professor’s Sfinature

3/1.; /c 5’

Date

MSU is an Affirmative Action/Equal Opportunity Institution

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE

DATE DUE

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6/01 cJCIRC/DaloDuepGS—p. 15

 

EFFICACY OF CHEMICAL SANITIZERS To INACTIVATE ESCHERICHIA
COLI 0157:H7, SALMONELLA TYPHIMURIUM DT 104 AND LISTERIA
MONOCYTOGENES ON ALFALFA SEEDS
AND SPROUTS
By

Pascale Marie-Michele Pierre

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Food Science and Human Nutrition

2004

ABSTRACT
EFFICACY OF CHEMICAL SANITIZERS TO INACTIVATE ESCHERICHIA COLI
0157:H7, SALMONELLA TYPHIMURIUM DT 104 AND LISTERIA
MONOCYTOGENES ON ALFALFA SEEDS AND SPROUTS
By

Pascale Marie-Michele Pierre

Alfalfa seeds and sprouts were inoculated with a 3-strain cocktail of Escherichia
coli 01572H7, Salmonella Typhimurium DT104 or Listeria monocytogenes by immersion
so as to contain ~ 6 to 8 log CFU/g and subjected to various sanitizer treatments to
reduce the pathogen load 2 5 logs, while maintaining acceptable seed germination and
sprout quality. Exposing seeds and sprouts for S 10 minutes to CloroxTM (sodium
hypochlorite, 200 to 20,000 ppm), TsunamiTM (peroxyacetic acid / hydrogen peroxide, 80
and 800 ppm) or Vegi-CleanTM (anionic surfactant, 1%, 2%, 5%) was generally unable to
decrease pathogen populations 5 logs. No appreciable differences in pathogen reduction
were observed using either sonication (20 kHz) or copper ions (1 ppm) alone or in
combination with the previous sanitizers. An FDA-approved fatty acid based-sanitizer
containing 3,750 ppm peroxyacid, 15,000 ppm caprylic and capric acid (E 658), 15,000
ppm lactic acid and 7,500 ppm glycerol monolaurate reduced E. coli, Salmonella and
Listeria > 5.45, > 5.62 and > 6.92 logs, respectively, on seeds, after 3 minutes, with no
injury and no Significant loss in seed germination rate or Sprout yield. The combination
of lactic acid (chelator of metal ions from bacterial cell membrane) and E 658, which was
responsible for the observed reduction, may provide a viable alternative to the

recommended 20,000 ppm chlorine.

To the two dearest microbes of my life: my sons, Laurent-David and Alain-Nicolas César

iii

ACKNOWLEDGMENTS

I would like to thank God, with all my soul, for my faith that provides me with
comfort, strength, wisdom, confidence and the deep certainty that no situation is too
complicated that He and I cannot get through together. I am thanka for all the
opportunities that were offered to me and all the miracles that happen so often in my life.

I would like to extend my heartfelt gratitude to Dr Jerry N. Cash and his wife,
Stella and to Dr. Eleanor Bossi for their determinant impact at critical times of my life at
Michigan State University. They were the compass that kept the boat of my life afloat and
on track.

I am genuinely thankful to Dr. Elliot T. Ryser for his careful and patient guidance
throughout my research, his support and his dedication to create a comfortable and
enjoyable work atmosphere for his lab group. The deep relationship that we built
together during these years is so meaningful to me.

I would like to thank Dr. Randolph Beaudry for being part of my graduate
Committee and for all his suggestions and recommendations. His ability to listen and
understand are greatly appreciated.

I also like to thank my previous Committee members, Dr. Perry K.W. Ng,
Dr.Wanda Chenoweth and Dr. Robert Ofoli.

I had one of the most interesting learning experience with Dr. Maija Zile. Her
guidance style contributes to reinforce my self-confidence in my ability to explore new

frontiers.

iv

I am thankful to all the professors who have contributed to my academic
experience at Michigan State University. A particular thought goes for Dr. Gale
Strasburg, Dr. Maurice Bennink, Dr. Janice Harte, Dr. Zeynep Ustunol, Dr. John Linz and
Dr. James Pestka.

I would like to extend my Sincere appreciation to the Haitian Ministry of
Agriculture, Natural Resources and Rural Development, the College of Agriculture and
Veterinary Medecine (FAMV) of the State University of Haiti, the Fulbright-Laspau
Scholarship program and the Inter-American Foundation Fellowship program of the
United States Government, the National Association of Mango Exporters of Haiti
(ANEM), the Office of International Students and Scholar (0188) at Michigan State
University for sponsoring and fimding my graduate studies, contributing to my research
projects or providing the required papers and authorizations

I am deeply thankful to my family members, my mother, Marie-Lisette David, my
sons, Laurent-David and Alain-Nicolas, my sisters, Danielle, Brigitte and Murielle for
their love, encouragement, caring and support. Thank you Marnan for always being there
for me and believing in my abilities, my goals and my dreams. Thank you Laurent and
Alain for your maturity and your love. You are the blessing of my life.

I am grateful to all the friends that gave me so precious token of friendship and
encouragement during this step of my life. It is impossible to mention all the names here
but my heart will always remember. Among them, my childhood fiiends, Régine and
Jean-Richard Beauboeuf, Marcelle Chevallier, Fabienne and Alix Fleury, Claudine
Gourgues Jeannot my family’s friend, Claude and Adrien Nicolas, Norma-Jean Ek, Lully

Thélémaque, Monique Paul Garrity who, despite her responsibilities as Resident

 

 

 

Representative of the World Bank at the United Nations took the time to call me each and
every day during more than two months at a critical moment of my Master program, my
friends and colleagues of the Center for Agricultural Research and
Documentation(CRDA), particularly Colette Blanchet, Myrlene Chrysostome and
Emmanuel Prophete. A special thought for my mentors, my fiiend and CRDA boss,
Danielle Avin and my BS advisor, Dr. Frantz F lambert.

I will dearly remember my labmates, the “Ryser’s Research Monkeys”, Arzu,
RajeSh, F inny, Lindsey, Keith, Alyssa, Yan, Iuliano and Geetha for all the memories that

we shared during this step of our academic life.

vi

 

 

 

TABLE OF CONTENTS

LIST OF TABLES .................................................................................... x
1.- INTRODUCTION .............................................................................. 1
2.- LITERATURE REVIEW ....................................................................... 5
2.1 .- ALFALFA SEEDS AND SPROUTS ................................................................ 5
2.1.1.-Characterization of Alfalfa ............................................................... 5
2.1.2.- Sprouting Process ......................................................................... 5
2.1.3.- Significance of Alfalfa Sprouts ......................................................... 6
A.- Nutritional value ...................................................................... 6
B.- Economic significance ............................................................... 7
2.1 .4.- Health Concerns .......................................................................... 8
A.- Nature of the problem with sprouts ................................................ 8
B.- Source of alfalfa sprout contamination ............................................. 9
C.- State of present research ........................................................... 10
a.- Chemical sanitizers .............................................................. 11
b.- Heat treatment ................................................................... 20
c.- Gamma irradiation .............................................................. 21
d.- Pre-soaking ...................................................................... 22
e.- Surfactants ....................................................................... 23
f.- Addition of antimicrobials to inigation water ............................... 24
g.- Effect of duration of seed storage ............................................. 25
h.- Examination of sprouts using scanning electron microscopy ............. 25
i.- Sensory evaluation ............................................................... 26
D.- Sprout industry regulations ........................................................ 27

2.2.- BACTERIAL PATHOGENS ASSOCIATED WITH ALFALFA SPROUTS .......................... 28
2.2.1.- Escherichia coli 0157:H7 ............................................................ .28
A.- Health significance of Escherichia coli 0157:H7 .............................. 28
B.- Evidence of Escherichia coli 0157:H7 in alfalfa seeds and Sprouts ......... 28

C.- Generalities about Escherichia coli 0157:H7 ................................... 33
D.- Basis for laboratory identification of Escherichia coli 0157:H7 ............ 34

vii

2.2.2.- Salmonella Typhimurium DT104 .................................................... 35

A.- Health significance of Salmonella Typhimurium DT104 ..................... 35

B.- Evidence of Salmonella T yphimurium DT104 in alfalfa seeds and
sprouts ................................................................................ 38
C.- Generalities about Salmonella Typhimurium DT104 .......................... 45
D.- Basis for laboratory identification of Salmonella Typhimurium DT104. . .50
2.2.3.- Listeria monocytogenes ................................................................ 50
A.- Health significance of Listeria monocytogenes ................................ 50
B.- Evidence of Listeria monocytogenes in fi'esh produce ......................... 53
C.- Generalities about Listeria monocytogenes ..................................... 54
D.- Basis for laboratory identification of Listeria monocytogenes ............. 56
2.3.- BACTERICIDAL AGENTS ....................................................................... 57
2.3.1.- Sodium hypochlorite (NaClO) ......................................................... 57
A.- General characteristics ............................................................. 57
B.- Mechanism of action ............................................................... 58
C.- Antimicrobial performance ......................................................... 59
2.3.2.- TsunamiTM ................................................................................ 61
A.- General characteristics ............................................................. 61
B.- Mechanism of action ................................................................ 61
C.- Antimicrobial performance ...................................................... .61
2.3.3.- Vegi-CleanTM ............................................................................. 62
A.- General characteristics ............................................................. 62
B.- Mechanism of action ................................................................ 63
C.- Antimicrobial performance ....................................................... .63
2.3.4.- Copper ion solution .................................................................... 63
A.- General characteristics ............................................................. 63
B.- Mechanism of action ................................................................ 64
C.- Antimicrobial performance ........................................................ 65
2.3.5.- Sonication ................................................................................ 67
A.- Characterization of the technique ................................................. 67
B.- Mechanism of action ................................................................ 67
C.- Antimicrobial performance ........................................................ 67
2.3.6.- Fatty acid-based sanitizer ............................................................... 68
A.- General characteristics .............................................................. 68
B.- Mechanism of action ................................................................ 73
C.- Antimicrobial performance ........................................................ 76

viii

 

 

 

 

3.- SYNERGIS'I'IC EFFECTS BETWEEN COMMERCIAL CHEMICAL SANrrIZERS (CLOROXTM,
TSUNAMITM, VEGI-CLEANTM) AND SONICATION OR COPPER IONS FOR REDUCTION OF
ESCHERICHIA COLI 0157:H7, SALMONELLA TYPHIMURIUM DT104 AND LISTERIA

MONOCYTOGENES ON INOCULATED ALFALFA SEEDS AND SPROUTS ............................ 81

Abstract ............................................................................................. 81
3. l .- Introduction ................................................................................... 82
3.2.- Materials and Methods ...................................................................... 84
3.3.- Results and Discussion ...................................................................... 89
3.4.- Conclusion ................................................................................... 106

4.- EFFICACY OF A FATTY ACID-BASED SANITIZER To INACTIVATE ESCHERICHIA COLI
0157:H7, SALMONELLA TYPHIMURIUM DT 104 AND LISTERIA MONOCYTOGENES ON

ARTIFICIALLY CONTAMINATED ALFALFA SEEDS ................................................ 108

Abstract ............................................................................................. 108
4.1.- Introduction ................................................................................. 109
4.2.- Materials and Methods ................................................................... .112
4.3.- Results ....................................................................................... 119
4.4.- Discussion ................................................................................. .134

5.- CONCLUSION .................................................................................... 146

APPENDIX A.- REDUCTION OF MESOPHILIC AEROBIC BACTERIA ON ALFALFA SEEDS AND
SPROUTS PREVIOUSLY INOCULATED WITH ESCHERICHIA COLI 0157:H7, SALMONELLA
TYPHIMURIUM DT104 AND LISTERIA MONOCYTOGENES USING COMMERCIAL CHEMICAL
SANITIZERS (CLOROXTM, TSUNAMI”, VEGI-CLEANTM) AND SONICATION OR COPPER
IONS .................................................................................................. 149

APPENDD< B.- EFFICACY OF A FATTY ACID-BASED SANmZER TO INACTIVATE MESOPHILIC
AEROBIC BACTERIA ON ALFALFA SEEDS ARTIFICIALLY CONTAMINATED ESCHERICHIA COLI
0157:H7, SALMONELLA TYPHIMURIUM DT104 AND LISTERIA MONOCYTOGENES .......... 155

REFERENCES .................................................................................... 163

ix

LIST OF TABLES
CHAPTER 2

TABLE 2.1.- INACTIVATION OF E. COLI 0157 :H7 ON ARTIFICIALLY CONTAMINATED ALFALFA
SEEDS USING CHEMICAL SANITIZERS .............................................................. 12

TABLE 2.2.- INACTIVATION OF SALMONELLA ON ARTIFICIALLY CONTAMINATED ALFALFA
SEEDS USING CHEMICAL SANITIZERS .............................................................. 12

TABLE 2.3.- SUMMARY OF E. COLI 0157:H7 AND E. COLI 0157: NM OUTBREAKS ASSOCIATED
WITH RAW SPROUT CONSUMPTION ................................................................. 30

TABLE 2.4.- RECALLS OF SPROUT PRODUCTS LINKED To CONTAMINATION BY E. COLI
0157:H7 AND E. COLI 0157:NM ................................................................... 32

TABLE 2.5.- SUMMARY OF SALMONELLA OUTBREAKS ASSOCIATED WITH RAW ALFALFA
SPROUT CONSUMPTION ............................................................................. 39

TABLE 2.6.- RECALLS OF SPROUT PRODUCTS AND SPROUTING SEEDS LINKED TO
CONTAMINATION BY SALMONELLA ................................................................. 46

TABLE 2.7.- RECALLS OF SPROUT PRODUCTS LINKED TO CONTAMINATION BY L.
MONOCYTOGENES .................................................................................... 58

CHAPTER 3

TABLE 3.1.- REDUCTION OF ESCHERICHIA COLI 0157 :H7 (logro CFU/g) ON INOCULATED
ALFALFA SEEDS USING VARIOUS ANTIMICROBIAL TREATMENTS ............................... 90

TABLE 3.2.- REDUCTION OF SALMONELLA TYPHIMURIUM DT104 (logic CFU/g) ON
INOCULATED ALFALFA SEEDS USING VARIOUS ANTIMICROBIAL TREATMENTS ............... 91

TABLE 3.3.- REDUCTION OF ESCHERICHIA COLI 0157:H7 (logro CFU/g) ON INOCULATED
ALFALFA SPROUTS USING VARIOUS ANTIMICROBIAL TREATMENTS ............................ 93

TABLE 3.4.- REDUCTION OF SALMONELLA TYPHIMURIUM DT104 (logio CFu/g) ON
INOCULATED ALFALFA SPROUTS USING VARIOUS ANTIMICROBIAL TREATMENTS ........... 94

TABLE 3.5.- REDUCTION OF LISTERIA MONOCYTOGENES (logio CFU/g) ON INOCULATED
ALFALFA SPROUTS USING VARIOUS ANTIMICROBIAL TREATMENTS ............................ 9S

CHAPTER 4
TABLE 4.1 .- COMPOSITION (ML) OF THE VARIOUS SANITIZER WORKING SOLUTIONS ...... 114

TABLE 4.2.- ACTIVE ANTIMICROBIAL COMPONENTS (PPM) OF VARIOUS CONCENTRATION OF
THE DILUTED FATTY ACID-BASED SANITIZER WORKING SOLUTIONS ......................... 114

TABLE 4.3.- ANTIMICROBIAL SOLUTIONS FORMULATIONS (PPM) INCLUDING PEROXY ACID
(PA), EMERY 658 (E65 8), LACTIC ACID (LA), GLYCEROL MONOLAURATE (GM) ALONE OR
IN VARIOUS COMBINATIONS ....................................................................... 115

TABLE 4.4.- INACTIVATION OF E. COLI 0157:H7 ON ARTIFICIALLY CONTAMINATED ALFALFA
SEEDS INOCULATED To CONTAIN 6.20 i 0.113 LOGS USING 5x, 10x AND 15x
CONCENTRATIONS OF A FATTY ACID-BASED SANITIZER ....................................... 120

TABLE 45- INACTIVATION OF E. COLI 0157 :H7 ON ARTIFICIALLY CONTAMINATED ALFALFA
SEEDS INOCULATED TO CONTAIN 6.15 i 0.08a USING 5x, 10x AND 15x CONCENTRATIONS
OF A FATTY ACID-BASED SANITIZER ....................................... 121

TABLE 46- INACTIVATION OF S. TYPHIMURIUM DT104 ON ARTIFICIALLY CONTAMINATED
ALFALFA SEEDS INOCULATED TO CONTAIN 6.32 i 0 .14a LOGS USING 5x, 10x AND 15x
CONCENTRATIONS OF A FATTY ACID-BASED SANITIZER ....................................... 122

TABLE 4.7 .- INACTIVATION OF LISTERIA MONOCYTOGENES ON ARTIFICIALLY CONTAMINATED
ALFALFA SEEDS INOCULATED TO CONTAIN 7.62 :l: 0 .11a LOGS USING 5x, 10x AND 15x
CONCENTRATIONS OF A FATTY ACID-BASED SANITIZER ............. , .......... 124

TABLE 48- INACTIVATION OF S. TYPHIMURIUM DT 104 ON ALFALFA SEEDS INOCULATED TO
CONTAIN 7.60 i 0.50 ‘ LOGS USING PEROXYACID (PA), CAPRIC/CAPRYLIC ACID (EMERY
65 8), LACTIC ACID (LA), GLYCEROL MONOLAURATE (GM) ALONE OR IN COMBINATION. 125

TABLE 49- INACTIVATION OF E. COLI 015 7:117 ON ALFALFA SEEDS INOCULATED To
CONTAIN 7.66 i 0.42 “ LOGS USING PEROXYACID (PA), CAPRlC/CAPRYLIC ACID (EMERY
65 8), LACTIC ACID (LA), GLYCEROL MONOLAURATE (GM) ALONE OR IN COMBINATION. 126

TABLE 4.10.- INACTIVATION OF L. MONOCYTOGENES ON ALFALFA SEEDS INOCULATED To
CONTAIN 7.31 i 0.09a LOGS USING PEROXYACID (PA), CAPRIC/CAPRYLIC ACID (EMERY 65 8),
LACTIC ACID (LA), GLYCEROL MONOLAURATE (GM) ALONE OR IN
COMBINATION ...................................................................................... 128

TABLE 4.11.- EFFECT OF 3 CONCENTRATIONS OF A FATTY ACID- BASED SANITIZER ON THE
GERMINATION RATE OF ALFALFA SEEDS ........................................................ 131

TABLE 412- GERMINATION RATE (%) FOR ALFALFA SEEDS AFTER A 3-MINUTE EXPOSURE TO
TREATMENTS A AND C ........................................................................ 132

xi

 

TABLE 413- EFFECT OF 3 CONCENTRATIONS OF A FATTY ACID-BASED SANITIZER ON
ALFALFA SPROUT YIELD ........................................................................... 133

TABLE 414- INACTIVATION OF 2 TO 3 LOGS E. COLI 0157:H7 ON ARTIFICIALLY
CONTAMINATED ALFALFA SEEDS USING CHEMICAL SANITIZERS ............................. 135

TABLE 415- INACTIVATION OF 5 1.70 LOGS E. COLI 0157:H7 ON ARTIFICIALLY
CONTMINATED ALFALFA SEEDS USING CHEMICAL SANITIZERS ............................... 135

TABLE 416- INACTIVATION OF ~1 TO 2 LOGS SALMONELLA ON ARTIFICIALLY
CONTAMINATED ALFALFA SEEDS USING CHEMICAL SANITIZERS ............................. 135

TABLE 4.17.- INACTIVATION OF ~ 2 .8 To 3.2 LOGS SALMONELLA ON ARTIFICIALLY
CONTAMINATED ALFALFA SEEDS USING CHEMICAL SANITIZERS ............................. 135

APPENDD( A

TABLE A.1.- REDUCTION OF MESOPHILIC AEROBIC BACTERIA (10810 CFU/g) ON ALFALFA
SEEDS PREVIOUSLY INOCULATED WITH ESCHERICHIA COLI 0157 :H7 USING VARIOUS
ANTIMICROBIAL TREATMENTS. .................................................................. 150

TABLE A.2.- REDUCTION OF MESOPHILIC AEROBIC BACTERIA (logro CFU/g) ON ALFALFA
SEEDS PREVIOUSLY INOCULATED WITH SALMONELLA TYPHIMURIUM DT104 USING VARIOUS
ANTIMICROBIAL TREATMENTS .................................................................. 151

TABLE A.3.- REDUCTION OF MESOPHILIC AEROBIC BACTERIA (logro CFU/g) ON ALFALFA
SPROUTS PREVIOUSLY INOCULATED ESCHERICHIA COLI 0157:H7 USING VARIOUS
ANTIMICROBIAL TREATMENTS .................................................................... 152

TABLE A.4.- REDUCTION OF MESOPHILIC AEROBIC BACTERIA (logio CFU/g) ON ALFALFA
SPROUTS PREVIOUSLY INOCULATED WITH SALMONELLA TYPHIMURIUM DT104 USING
VARIOUS ANTIMICROBIAL TREATMENTS ........................................................ 153

TABLE A.5.- REDUCTION OF MESOPHILIC AEROBIC BACTERIA (logio CFU/g) ON ALFALFA

SPROUTS PREVIOUSLY INOCULATED WITH LISTERIA MONOCYTOGENES USING VARIOUS
ANTIMICROBIAL TREATMENTS ................................................................... 154

xii

APPENDIX B

TABLE B.1.- INACTIVATION OF MESOPHILIC AEROBIC BACTERIA ON ALFALFA SEEDS
ARTIFICIALLY CONTAMINATED WITH E. COLI 0157:H7 USING 5x, 10x AND 15x
CONCENTRATIONS OF A FATTY ACID-BASED SANITIZER ....................................... 156

TABLE B.2.- INACTIVATION OF MESOPHILIC AEROBIC BACTERIA ON ALFALFA SEEDS
ARTIFICIALLY CONTAMINATED WITH E. COLI 0157:H7 USING 10x AND 15x
CONCENTRATIONS OF A FATTY ACID-BASED SANITIZER ....................................... 157

TABLE B.3.- INACTIVATION OF MESOPHILIC AEROBIC BACTERIA ON ALFALFA SEEDS
ARTIFICIALLY CONTAMINATED WITH SALMONELLA TYPHIMURIUM DT 104 USING 10x AND
15x CONCENTRATIONS OF A FATTY ACID-BASED SANITIZER .................................. 158

TABLE B.4.- INACTIVATION OF MESOPHILIC AEROBIC BACTERIA ON ALFALFA SEEDS
ARTIFICIALLY CONTAMINATED WITH LISTERIA MONOCYTOGENES USING 10x AND 15x
CONCENTRATIONS OF A FATTY ACID-BASED SANITIZER ....................................... 159

TABLE B.5.- INACTIVATION OF MESOPHILIC AEROBIC BACTERIA ON ALFALFA SEEDS
PREVIOUSLY INOCULATED WITH S. TYPHIMURIUM DT 104 USING PEROXYACID (PA),
CAPRIC/CAPRYLIC ACID (EMERY 658), LACTIC ACID (LA), GLYCEROL MONOLAURATE (GM)
ALONE OR IN COMBINATION ...................................................................... 160

TABLE B.6.- INACTIVATION OF MESOPHILIC AEROBIC BACTERIA ON ALFALFA SEEDS
PREVIOUSLY INOCULATED WITH E. COLI 0157:H7 USING PEROXYACID (PA),
CAPRIC/CAPRYLIC ACID (EMERY 658), LACTIC ACID (LA), GLYCEROL MONOLAURATE (GM)
ALONE OR IN COMBINATION ....................................................................... 161

TABLE 87- INACTIVATION OF MESOPHILIC AEROBIC BACTERIA ON ALFALFA SEEDS
PREVIOUSLY INOCULATED WITH LISTERIA MONOCYTOGENES USING PEROXYACID (PA),
CAPRIC/CAPRYLIC ACID (EMERY 658), LACTIC ACID (LA), GLYCEROL MONOLAURATE (GM)
ALONE OR IN COMBINATION ....................................................................... 162

xiii

1.- INTRODUCTION

With the growing trend toward a healthier diet, market demand and consumption
of fresh or minimally processed fruits and vegetables continue to increase. Moreover,
new production and packaging technologies allow for year-long availability of numerous
of fruits and vegetables. Presence of salad-bars in restaurants, displays of fresh produce
in receptions and availability of fresh cut ready-to-eat salads in supermarkets have
become increasingly common.

A wide range of technologies have been developed to increase the shelf-life of
fresh fruits and vegetables. However, none of these technologies are without some
negative side effects. Changes in consumption patterns, delays in storage and
consumption of fruits and vegetables and numerous manipulations required by new
technologies have led to increased risks of cross-contamination with added shelf-life
raising additional concerns.

The emergence of new pathogens is an undeniable fact. Pathogens that were
previously less virulent have increased in virulence and are now considered public health
concerns to certain high risk segments of the population. Some of these organisms have
acquired antibiotic resistance while others have been able to grow in environments and
conditions (low temperature, low pH) where they were presumed to be unable to survive.
Thus, the number of produce-associated food-bome disease outbreaks Where fruits and
vegetables have been clearly identified as vectors of bacterial infections and the number
of cases of illness due to food pathogens have significantly increased in recent years.
(Tauxe 1997; Xu, 1999). The bacterial pathogens of greatest concern include

Escherichia coli 0157:H7, Salmonella Typhimurium DT104 and Listeria

 

 

monocytogenes, all three of which are targeted in this study. These microorganisms have
been linked to, at least, 20 outbreaks and 22 Class I recalls involving Sprouts (CDC 1997;
FDA, 2004) and have thus gathered Significant public and government attention. In one
such outbreak involving Salmonella Muenchen in Wisconsin, over 12,500 pounds of
alfalfa seeds and 2,700 pounds of sprouts were recalled. In a Similar outbreak involving
E. coli 0157:H7, 30,000 pounds of sprouts were recalled by a Michigan grower.
Improvements in health care have significantly increased the number of immuno-
compromised, elderly and chronically ill patients. Infants and pregnant women are very
susceptible to acute bacterial infections. Fruits and vegetables are known to be a good
source of vitamins and minerals and because of their high digestibility and good
nutritional value, they are recommended as part of the daily diet. Therefore, more
efficient strategies are clearly needed to enhance the safety of fresh links and vegetables.
Raw alfalfa sprouts, formerly considered to be a “safe”,“healthy” and nutritious
product, have been repeatedly incriminated in food-borne outbreaks involving E. coli
0157:H7 and Salmonella sp. The first consumer warning about Sprouts was issued by
the CDC in 1997 (Powell et al., 2002). CDC and FDA recommended that individuals at
high risk for systemic infections (i.e., the elderly, young children and
immunocompromised persons) not eat raw sprouts. III July of 1999, the FDA advised all
consumers to be aware of the risks associated with raw sprouts and informed the public
that, at that time, the best way to control this safety risk was to avoid eating raw sprouts
(Powell et al., 2002). For people who continued to eat sprouts, FDA recommended

cooking Sprouts before consumption, to reduce the risk of illness (Mohle-Boetani et al.,

2002; Powell et al., 2002).

Since then, considerable research efforts have focused on increasing the
microbial safety of raw alfalfa Sprouts by identifying consumer-acceptable strategies that
can reduce the microbial load on alfalfa seeds or alfalfa sprouts by 5 logs or greater. Any
proposed seed treatment must also maintain a commercially acceptable seed germination
rate. Although, some progress has been made in regard to bean sprouts, no effective
treatment has been developed to guarantee safe consumption of organoleptically
acceptable alfalfa Sprouts. Therefore, the need for further investigations still remains.

Sonication is a physical technique involving the use of ultrasound. Stresses and
strains produced during cavitation result in mechanical disruption of bacterial cells
(Shukla, 1992; Lillard, 1993). One hypothesis for the present study is that sonication
may play a role in damaging the bacterial cell wall, thereby making such organism more
susceptible to chemical sanitizers. Nevertheless, sonication is, at least, expected to
contribute in declumping and dislodging of bacteria from the surface of alfalfa seeds and
sprouts and more effectively expose the pathogens to the bactericidal action of various
chemical S anitizers including C loroxTM (sodium hypochlorite), TsunamiTM (peroxyacetic
acid), Vegi-CleanTM (anionic surfactant) and copper ions. This synergistic effect between
sonication and the chemical sanitizers should lead to enhanced microbial reduction.

Copper has long been known for its antimicrobial properties (Yeager 1991). The
second hypothesis in the present study is that copper ions alone will exert a certain level
of toxicity toward bacterial pathogens on alfalfa seeds and sprouts. Moreover, a
synergistic antimicrobial effect is expected from the combined use 0 f c opper ion w ith
Cloroxm, TsunamiTM or Vegi-Cleanm. Copper ions are generated through an electrolytic

process and dispersed into a circulating water stream. The formation of electrostatic

bonds between positively charged ions and negatively charged sites on the bacterial cell
surface (Superior Water Solutions, Inc.) Should enhance sanitizer contact and
performance, thereby leading to greater bacterial reductions.

As part of this work, a novel FDA-approved fatty acid-based sanitizer is also
being assessed for inactivation of pathogens on alfalfa seeds. This novel sanitizer
concentrate is diluted in water to a reference concentration of 1x, so as to contain 250
ppm peroxyacid, 1000 ppm fatty acid [caprylic (octanoic,C3) and capric (decanoic,Clo)
acids], 1000 ppm lactic acid and 5 00 ppm glycerol m onolaurate. T he c ombination of
these ingredients produces a synergistic effect, providing a much more potent biocide
than what could be obtained using these components separately and offers the unique
advantage of having antimicrobial activity at substantially lower concentrations. In
addition, lactic acid and glycerol monolaurate also react with peroxyacids and free fatty
acids to enhance antimicrobial activity (Guthery, 2002). All components in this fatty
acid-based sanitizer have attained Generally Recognized AS Safe (GRAS) status. These
fatty acids and their esters, which are non-toxic, naturally occurring substances in foods
(Kabara, 1984; Oh and Marshall, 1993), carry a considerable advantage over other types
of chemical sanitizers developed to control microorganisms. The goal of this research is
to determine the optimal concentration and length 0 f exposure to this fatty acid-based
sanitizer for decreasing E. coli 0157 :H7, Salmonella Typhimurium DT 104 and Listeria
monocytogenes populations 5 logs on alfalfa seeds while maintaining an optimal

germination rate and sprout yield.

2.- LITERATURE REVIEW
2.1.- ALFALFA SEEDS AND SPROUTS
2.1.1.- CHARACTERIZATION OF ALFALFA.

Alfalfa (Medicago sativa, Linn) is a perennial legume (botanical family
Leguminosae) mostly grown for forage production. Originally from southwestern Asia, it
has spread throughout the world and can be found in very diverse ecological and
agricultural areas. It has been grown for forage since the Roman era. (Ivanov, 1988;
Scheaffer et al., 1993). Alfalfa is considered the most nutritious and palatable forage
Species because of its high protein content and balance of amino acids as well as vitamins
and minerals (Orloff, 1995).

The alfalfa plant herbage originates from a large crown and usually reaches 1 to 4
feet or more in height, depending on the soil, climate or cultivation technique. The root
system may grow as deep as 20 feet into the soil. Being a legume, this plant enriches the
soil in nitrogen. The leaves are comprised of three leaflets. The flowers, most oflen
purple, resemble pea blossoms and are dispersed through the alfalfa branches and stems.
The seeds, which are small, usually kidney shaped, brown-olive-green in color measure

approximately 2 mm x 1 mm. (Coburn, 1907; Sheaffer, 1993) and weigh 2-3 mg each.

2.1.2.- SPROUTING PROCESS

In commercial settings, alfalfa seeds are sanitized before being processed
according to recommendations from the International Food Growers Association (ISGA
website, 2004) and FDA requirements (FDA/CFSAN, 1999). The seeds are sprouted

hydroponically, at ambient temperature, under dim light, in flat open trays or in relatively

 

 

closed rotating drums. Treated potable water is applied through sprinklers at regular
intervals. After harvest, fresh sprouts are thoroughly washed, centrifuged and packed
(Hooper, 2000; Fett, 2000). T ypical s eed to sprout yield ratios range from 1 :5 to 1 :9
(DeVitto, 1982). Fresh yield and nutritive value of alfalfa sprouts are affected by light,
temperature, moisture conditions, cultivar and length of sprouting time (Bass et al.,1988).

A variety of home-sprouting devices are also available.

2.1.3.- SIGNIFICANCE OF ALFALFA SPROUTS
A.- Nutritional value

Sprouts benefit from a strong public perception as being a very healthy product
since alfalfa sprouts are most often viewed as an “unprocessed” or “ natural” food
(Hooper, 2000). Sprouts are considered a health food because they are low in fat and
calories and high in fiber (Weissinger and Beuchat 2000), with the nutritional content of
dry seeds increasing as a result of the sprouting process (Rajkowski and Thayer, 2000).

Alfalfa sprouts contain several nutrients of interest: minerals, proteins and
Vitamins such as ascorbic acid, thiamine, riboflavin and niacin (Bass et al., 1988).
DeVitto (1982) reported the following nutritional composition for 100 g of alfalfa
Sprouts: 16 mg of ascorbic acid, 5 g of protein, 2 g of fiber, 28 mg of calcium and 1.6 mg
of niacin. Nutritional content of alfalfa sprouts was investigated by Pennington (1989)
who published the following data for 33 g (1 cup) of sprouts : 10 Kcal, 30.1 g of water,
0.2 g of fat, 0.1 g of polyunsaturated fatty acids, 1.3 g of protein, 1.3 g of carbohydrate,
0.7 g of dietary fiber, 5 Retinol Equivalent / 51 International Unit of vitamin A, 3 mg of

ascorbic acid, 2 mg of sodium, 10 mg of calcium, 9 mg of magnesium, 0.30 mg of zinc,

26 mg of potassium, 23 mg of phosphorus, 0.32 mg of iron, 44 mg of threonine, 47 mg of
isoleucine, 88 mg of leucine, 71 mg of lysine and 48 mg of valine.

Vitamin C (ascorbic acid) is considered a key nutrient in alfalfa sprouts. DeVitto
(1982) found a mean initial ascorbic acid level of 15 mg/ 100 g of alfalfa sprouts followed
by a statistically significant decreased to 10.7 mg/ 100 g of product after 9 days of storage.
On a fresh-weight basis, alfalfa sprouts provide higher amounts of ascorbic acid and iron
than 0 ther v egetables s uch a s c abbage, lettuce and c aITots ( DeVitto, 1982; Bass et al.,
1988). Nevertheless, their ascorbic acid content is far less than high vitamin C-fruits like

citrus (DeVitto, 1982; Bass et al., 1988).

B.- Economic significance

The sprouting industry encompasses approximately 475 sprout growers in the
United States and Canada, 850 in Japan, 200 in Europe, 35 in Australia and New-
Zealand, 3000 in Korea and more than a million in China (Snider, 2000).

On a world-wide basis, sales of Sprouts are generating revenues in the range of 1
billiofi dollars, with sprouts representing total sales of $250 millions / year in the United
States and Canada from an annual production of 600 millions pounds of sprouts (Snider,
2000). In 1984, an estimated 32,000 Kg of alfalfa seeds were processed for commercial

sprouting with an estimated farm value of $63 millions (Bass et a1, 1988).

2.1.4.- HEALTH CONCERNS
A.- Nature of the problem with the sprouts

Alfalfa sprouts found in retail salad bars are minimally processed and most often
eaten raw or briefly cooked (Weissinger and Beuchat, 2000). As is true for many other
types of flesh produce, alfalfa sprouts typically contain high levels of bacteria, some of
which may be pathogenic. The Sprouting process itself involves extensive use of water.
The sprouting environment, characterized by high moisture and warm temperatures,
creates a favorable environment for growth and Spread of bacteria (Taorrnina and
Beuchat, 1999a; Fett, 2000). Therefore, contamination of seeds with low levels of
pathogens can result in a final product that supports rapid grth of these organisms
(Fett, 2000). A pathogen population of 2.5 loglo CFU/g on alfalfa seeds would be
considered large in a commercial setting and, in reality, would be unlikely to occur. On
alfalfa seeds, pathogen populations would more likely be at least 100-fold lower (J aquette
et al., 1996).

Seeds and sprouts have been recognized as an important cause of foodbome
illness (FDA/CFSAN, 1999). They have been linked to, at least, 20 outbreaks of E. coli,
Salmonella or L. monocytogenes infection and targeted by 22 Class I recalls (CDC 1997;
FDA, 2004). The potential for sprouts to transmit pathogenic microbes has been linked,
especially, to the fact that they are usually eaten uncooked (Piemas and Guiraud, 1997).
The first consumer warning about sprouts was issued by the CDC in 1997 (Powell et al.,
2002). CDC and FDA recommended that people at high risk for systemic infections (i.e.,
the elderly, young c hildren and immunocompromised individuals) 11 ot e at raw Sprouts,

with the California Department of Health Services (1998) issuing an interim advisory on

 

raw alfalfa sprouts. In July of 1999, the US Department of Health and Human Services
(1999) advised consumers about the risks associated with raw sprouts and informed them
that, at that time, the best way to control this safety risk was to avoid raw Sprouts. For
persons who continue to eat sprouts, the FDA recommended cooking as a means to
reduce the risk of illness (FDA, 2002; Mohle-Boetani et al., 2002; Powell et al., 2002).
The Califomia Department of Health Services and the California Department of
Education also recommended that schools stop serving uncooked sprouts to young

children (Mohle-Boetani et al., 2002).

B.-Source of contamination of Alfalfa sprouts

Alfalfa seeds may become contaminated flom animal waste while growing in the
field. Contaminated irrigation water, run—off water, sewage and improperly composted
manure may also serve as sources of contamination (Como-Sabetti et al., 1997; Mahon et
al. 1997; Taonnina and Beuchat, 1999a ; Beuchat, 1999; Mohle-Boetani et al., 2 002).
Harvest, transportation, storage and distribution operations must be considered potential
points of contact with pathogens (Beuchat, 1996; Park et al., 2000; Mohle-Boetani et al.,
2002)

The procedures inherent to the Sprouting process make contamination very easy.
Sprouting involves the use of large quantities of water and creates an environment
characterized by high moisture and warm temperatures which encourage the growth of
bacteria (Mahon et al., 1997; Taonnina and Beuchat, 1999a; Mohle-Boetani et al., 2002).
While contaminated water, contaminated equipment on the farm or at the processing

facility, poor handling and poor hygiene can serve as vectors for contamination

(FDA/NACMCF, 1999; Rajkowski and Thayer, 2000; Park et al. 2000; Mohle-Boetani et
al., 2002), most Sprout-related outbreaks have been traced to contaminated seeds (F ett,
2000; Mohle-Boetani et al., 2002). Considering this issue, Mohle-Boetani et al. (2002)
suggested the designation of Sprout seed production lots for human consumption at seed
planting, in order to encourage producers to focus on reducing potential seed
contamination during production and harvest.

Pathogens can grow on sprouts w ithout In odifying their appearance (Taormina,
1999). Jaquette (1996) reported that Salmonella Stanley populations of102 to 103 CFU/g,
on seeds, began increasing after 6 hours of soaking, increasing 3 and 4 logs during the
first 24 and 48 hours of germination, respectively. The bacterial population eventually
stabilized at 6.75 to 7.08 logs, 54 hours after Sprouting with populations decreasing only
slightly during 10 days of refligerated storage at 5 °C.

Therefore, it can be easily understood why seeds contaminated with very low
levels of pathogens can be implicated in outbreaks. This observation may explain why, in
a 1994 Salmonella outbreak, in Sweden and Finland, the organism was found in Sprouts
but not in seeds (Taormina and Beuchat, 1999a). In cases where spot contamination
occurs in one lot of seeds, sprouting water can act as a vector for contaminating the entire

batch (Taormina and Beuchat, 1999a).

C.- State of present research
No bactericidal treatment has been developed to effectively guarantee safe
consumption of organoleptically acceptable bean sprouts (Rajkowski and Thayer, 2000,

Mohle-Boetani et al., 2002). After a multistate Salmonella Muenchen outbreak in

10

 

 

 

September 1999, even the U S F ood and D rug A dministration (FDA) reported that the
recommended 20,000 ppm Ca (00;) soak for 15 minutes did not completely eliminate
the safety risk as this outbreak occured despite the incriminated sprouts having been
grown flom seeds that were previously sanitized with the recommended treatment
(Proctor, 2001). While several other options have led to a better understanding of the

problem, alternative decontamination methods are still needed.

a.- Chemical sanitizers

Various disinfectants have been used on seeds (Tables 2.1 and 2.2) and on
Sprouts, most notably, sodium hypochlorite (section 2.3.1), peroxyacetic acid (section
2.3.2) and acid-anionic surfactants (section 2.3.3). However, no chemical or water rinse
treatments have thus far been able to completely decontaminate flesh fruits and
vegetables 1 caving a satisfying edible raw product (FDA/NACMCF, 1999; Rajkowski
and Thayer, 2000). Moreover, cracks and crevices in alfalfa seeds may trap pathogenic
bacteria, making them less accessible to lethal concentrations of disinfectants (Taormina
and Beuchat, 1999a; Mohle-Boetani et al., 2002). Therefore potential hazards will likely

remain after treatment, particularly for the high risk populations.

Hydrogen peroxide (H303)

The antimicrobial activity of hydrogen peroxide is due to its oxidizing capacity
(Piemas and Guiraud, 1997). These authors Showed that post-harvest treatment of
biologically cultivated brown rice with a 1% solution of hydrogen peroxide for 10

minutes decreased the aerobic plate count by 2 logs. No significant improvement

11

TABLE 2.1.- INACTIVATION OF E. COLI 0157:H7 ON ARTIFICIALLY CONTAMINATED ALFALFA
SEEDS USING CHEMICAL SANITIZERS.

 

 

SANITIZERS REDUCTION (bogs) EXPOSURE TIME (Min)
Ca (0C1); (20,000 ppm chlorine) ~ 2 to 3 3 and 10
Hydrogen peroxide (1%) ~ 3 3 and 10
Trisodium phosphate (4%) ~ 2 0.5 and 2
Vortexxm (40 and 80 ppm) ~ 2 3 and 10
TsunamiTM (80 ppm) > 1.70 3 and 10
Vegi-CleanTM (1 and 2%) ~ 1.50 to 2.1 3 and 10

 

From Taormina and Beuchat (1999a)

TABLE 22- INACTIVATION OF SALMONELLA ON ARTIFICIALLY CONTAMINATED ALFALFA
SEEDS USING CHEMICAL SANITIZERS.

 

 

SANITIZERS REDUCTION (Logs) EXPOSURE TIME (Min)
Ca (0C1); (20,000 ppm) 1.95 10
TsunamiTM (530 and 1,060 ppm) 1.12 and 1.50, respectively 10
Trisodium phosphate (2 and 5%) 0.90 and 1.99, respectively 10
Acid. NaClO (500 & 1,200 ppm) 1.26 and 1.43, respectively 10
VortexxTM (530 and 1,060 ppm) 1.29 and 1.62, respectively 10
Lactic acid (2%) 1.19 10
Lactic (5%) 2.98 10
Citric acid (5%) 2.98 10
Acetic acid (5%) 1.74 10
Hydrogen peroxide (8%) 3.22 10
Calcium hydroxide (1%) 2.84 10
Calcinated calcium (1%) 2.88 10

 

From Weissinger and Beuchat (2000)

12

resulted either flom increasing the soaking time or flom raising the concentration of the
bactericidal agent. Hydrogen peroxide at 1% did not affect germination of treated rice
seeds or growth of seedlings.

Taormina and B euchat ( 1999a) 11 sed 1 % hydrogen p eroxide to decrease E. coli
0157:H7 flom 3.21 to < 0.3 loglo CFU/g in 10 minutes on alfalfa seeds. These seeds
were artificially contaminated by dipping 1 kg dry seeds in an 5-Strain E. coli inoculum
cocktail for 1 minute and then drying the seeds for 48 hours. However, even 8% H202
did not eliminate the pathogen, which was detected by enrichment of alfalfa seeds after
treatment

When alfalfa seeds inoculated with Salmonella were exposed to 10% hydrogen
peroxide for 30 seconds, Beuchat (1997) reported that populations decreased flom 3.57
loglo CFU/g to <1 CFU/g; however, the pathogen was not eliminated. Weissinger and
Beuchat (2000) found that 0.2%, 2% and 8% hydrogen peroxide respectively reduced
Salmonella populations 0.22, 0.67 and 3.22 logs on experimentally contaminated alfalfa
seeds, following a 10 minute exposure. These concentrations did not affect the
germination rate which was 93.3% for the water control compared to 96.1 to 96.5% for

the treated seeds (Weissinger and Beuchat, 2000).

Trisodium phosphate (NagPOAL

Taormina and Beuchat (1999a) did not recover E. coli 0157:H7 by direct plating
when alfalfa seeds artificially contaminated to contain 2.20 logto CFU/ g were treated with
2 4% trisodium phosphate. However, E. coli 0157:H7 was detected in all samples, after

enrichment.

13

Weissinger and Beuchat (2000) reduced the number of Salmonella by 0.90 and
1.99 logm CFU/g (compared to water control) by respectively exposing Salmonella-
inoculated alfalfa seeds to 2% and 5% Na3PO4 for 10 minutes. The seed germination rate

was not significantly affected by these treatments.

Calcium hydroxide CQLQLI);

Weissinger and Beuchat (2000) artificially contaminated alfalfa seeds with
Salmonella by mixing 1 kg of dry seeds in a 6-Strain cocktail inoculum, followed by
drying 24 hours. Salmonella population on the previously inoculated seeds decreased by
0.31, 2.06 and 2.84 logs following a 10 minute exposure to 0.1%, 0.5% and 1% calcium

hydroxide, respectively. The seed germination rate remained unaffected.

Calcium hypochlorite CalOCllz

Taormina and Beuchat (1999a) reported that E. coli 0157:H7 populations
decreased flom 2.68 and 2.80 logs to < 0.3 loglo CPU/g, respectively, after 3 and 10
minute applications of Ca(OC1)2 (20,000 ppm of active chlorine) in 0.05 M potassium
phosphate buffer (pH 6.8) to previously inoculated alfalfa seeds, with no Significant loss
of germination rate (70.3 and 70.7%, respectively compared to their corresponding water
control 78.3 and 77.0%). FDA currently recommends a 20,000 ppm calcium hypochlorite
soak before sprouting to reduce the risk for sprout-related illnesses (FDA/NACMCF,
1999). However, this high dose w as n ot c ompletely e ffective in p reventing o utbreaks
(Proctor, 2001) and doses > 20,000 ppm Ca (00;) can impair seed germination (Mohle-

Boetani et al. 2002).

14

 

Weissinger and B euchat (2000) reported a 1.95 log reduction in Salmonella on
alfalfa seeds after a 10 minute exposure to 20,000 ppm of flee chlorine as Ca(OC12) in
0.05 M potassium phosphate buffer (pH 6.8), with a significantly lower germination rate
of 91.6% compared to 94.8% for the control. Beuchat et al. (2001) observed a
germination loss of approximately 10% when 20,000 ppm chlorine were applied to

inoculated alfalfa seed for 30 minutes, which led to a 2.3 log reduction in Salmonella.

Cplcium oxide (Cafl)

Bari et al. (1999) found that the addition of 0.4% calcium oxide to a radish seed
Sprouting medium containing 3.0 to 3.2 loglo CFU of E. coli 0157:H7/ml completely
inhibited the growth or inactivated the pathogen.

Weissinger and Beuchat (2000) reported a 2.88 log reduction in Salmonella after
alfalfa seeds were exposed to 1% calcinated calcium for 10 minutes with the seed

germination rate unaffected by this treatment.

Acidified sodium hypochlorite

Piemas and Guiraud (1997) applied acidified sodium hypochlorite to biologically
cultivated brown rice seeds destined for sprouting. Exposure to solution of acidified
sodium hypochlorite (pH 4 to 7) had little effect on the mesophilic aerobic bacterial load
until 1 ,000 ppm when aerobic p late c ounts d ecreased by 2 to 3 logs after 20 minutes.
Decontamination efficacy was not improved by extending contact time, nor by increasing
the solution concentration 10,000 ppm. Decreasing the pH of sodium hypochlorite to 4 to

7 did not increase the microbial reduction. Weissinger and Beuchat (2000) tried acidified

15

sodium hypochlorite on seeds contaminated with low level of Salmonella spp. Reductions
of 1.26 and 1.43 logs were observed after 10 nrinute exposure to concentrations of 500

and 1,200 ppm, respectively.

m

Ethanol at high concentrations will denature bacterial proteins and seed enzymes
involved in germination (Piemas and Guiraud, 1997). These authors assessed the efficacy
of 70% ethanol for disinfecting biologically cultivated brown rice seeds. A 10 minute
exposure to 70% ethanol decreased mesophilic aerobic populations 2 to 4 logs in 10
minutes, after which populations stabilized. Germination was, however, greatly affected,
as only 11.5% of disinfected seeds germinated after 24 h, producing abnormal seedlings.
Reducing the ethanol concentration to 10% suppressed the undesirable effects on
germination. However, decontamination at that concentration was no more efficacious

than washing with water.

Active oxygen solution (V ortexx. Ecolab. Mendota Heights, Minn.)

Vortexx (40 and 80 ppm) was applied by Taormina and Beuchat (1999a) to
inactivate E. coli 0157:H7 on alfalfa seeds. After either 3 or 10 minutes of exposure, E.
coli 0157:H7 populations of 0.30 log remained on the seeds, compared to ~ 2 logs for the
corresponding water controls.

Vortexx (270, 530 and 1,060 ppm) was used by Weissinger and Beuchat (2000) to

reduce populations of Salmonella on alfalfa seeds. After a 10 minute exposure, microbial

16

reductions of 0.78, 1.29 and 1.62 logs, respectively, compared to the water control, were

observed without any significant reduction in germination rate.

Organic acids

The antimicrobial effect of organic acids is partly attributed to low pH.
Weissinger and Beuchat (2000) used 2% and 5% acetic, lactic or citric acid to
decontaminate alfalfa seeds experimentally inoculated with Salmonella. The lethal
effects of the 5% organic acid treatments were substantial: 1.74, 2.98 and 2.98 log
reduction, respectively, after 10 minutes of exposure. Nevertheless, seed germination
rates were also significantly reduced: 46.7%, 56.8%, 81.4%, respectively, compared to
92.3% for the water control. Sprouts produced flom these seeds were slightly etiolated

and were inferior to the control sprouts.

Allyl isothiocym

Allyl isothiocyanate (AIT) results flom hydrolysis of glucosinolates by
myrosinase in cruciferous plants, including mustard and horseradish (Park et al., 2000).
Although the antimicrobial activity of AIT varies widely (Delaquis and Mazza, 1995;
Park et al., 2000), the volatile compound has been shown to inhibit the growth of E. coli
(Kyung and Fleming, 1997; Park et al., 2000), including serotype 0157 :H7 (Delaquis and
Scholberg, 1997; Park et al., 2000).

Park et al. (2000) hypothesized that a volatile compound such as allyl
isothiocyanate, which is potentially lethal to microorganisms, could more easily reach E.

coli 0157:H7 cells in areas otherwise protected flom contact with aqueous solutions.

17

Incubation of Trypic Soy Agar (TSA) disks inoculated with E. coli 0157:H7 in a 950 ml
jar containing 8 p1 of AIT, at 37 0C, for 48 hours, resulted in reductions > 7 logs, with
these reductions lower when Similar experiments were repeated at 20 °C. Nevertheless, 8
pl AIT was not completely lethal to E. coli 0157:H7 cells inoculated on the agar disk, as
subsequent incubation at 37 °C for 48 h in an atmosphere flee of AIT resulted in growth
of the pathogen. Colonies developed slowly, however, indicating that exposure of cells to
AIT may have caused sublethal injury (Park et al., 2000).

These authors applied 50 p1 of AIT on 2 g of dry (6.8% moisture) alfalfa seeds
with an initial E. coli 0157:H7 population of 2.9 logs CFU/g and on 2 g of wet (22.5%
moisture) alfalfa seeds with an initial pathogen population of 2.7 logs CFU/g at 25, 37
and 47 °C for 24 h: The pathogen was not recovered by direct plating or by enrichment
flom wet seeds held at 37 and 47 °C for 24 h. However, E. coli 0157:H7 was recovered
flom wet seeds held at 25 °C, after enrichment, indicating that the effect of AIT is
temperature dependent. AIT is clearly more effective in killing E. coli 0157:H7 on wet
seeds than on dry seeds as enrichment of the treated dry seeds revealed the presence of the
pathogen, and as exposure to 100 pl of AIT at 47 0C for 24 h did not eliminate the
pathogen flom dry seeds.(Park et al., 2000).

The drawback of this method is that AIT drastically reduced the germination rate
which fell flom 90% to 3% after wet seeds were exposed to 50 p1 AIT at 25 °C for 24 h.
(Park et al., 2000). As the mechanism of action is believed to involve respiratory
inhibition in bacterial cells, AIT may also adversely affect the respiratory mechanism of

alfalfa seeds (Park et al., 2000).

18

Fit TM. (Procterfi and Gamble, Cincinnati, OH)

FitTM is a GRAS alkaline produce-sanitizing solution composed of water, 0 leic
acid, glycerol, ethanol, potassium hydroxide, sodium bicarbonate, citric acid and distilled
grapefluit oil. Beuchat et al. (2001) compared the antimicrobial performance of F itTM to
20,000 ppm chlorine solution as Ca(OCl)2 in 0.05 M potassium phosphate buffer (pH 7.0)
These authors found that treatment with 2% chlorine or FitTM applied for 15 or 30
minutes reduced Salmonella populations by 2.3 to 2.9 logs. Treating seeds with 2%
chlorine for 15 or 30 minutes resulted in equal or a significantly greater reduction in
viable E. coli 0157:H7 (1.6 and 2.0 logs, respectively) compared to FitTM (1.5 to 1.7 logs,
respectively). The actual difference in numbers of E. coli recovered flom seeds treated
with 2% chlorine or FitTM for 30 minutes, although significant, was only 0.3 loglo CFU/g.
However, exposing seeds to 2% chlorine for 15 or 30 nrinutes or to F itTM for 30 minutes

Significantly reduced the seed germination rate flom 95.7% to 85.7 — 87.7%.

Chlorine dioxide

Applying 500 ppm of acidified C102 under the form of USS-1400 (Universal
Sanitizers and Supplies, Knoxville, TN), for 3 and 10 minutes reduced E. coli 0157:H7
on alfalfa seeds flom approximately 2.65 logs to <0.30 logs, with the pathogen detected
after enrichment. After the 3 minute exposure, the germination rate declined flom 73.7%
to 55% (Taormina and Beuchat, 1999a,b). Although chlorine dioxide Will effectively
destroy microorganisms p resent in s olutions and those attached to e quipment s urfaces,
this sanitizer is less efficacious for fluits and vegetables (Reina et al., 1995). Mari et al.

(1999) mentioned that some limitations affect the efficacy of chlorine. They are mostly

19

related to a rapid drop in chlorine activity in the presence of organic substances that

modify the pH of the solution.

b.- Heat treatment

Alfalfa seeds have been exposed to high temperatures, with heat also having been
combined with various chemical treatments to assess possible synergistic effects.

Jaquette et a1. ( 1996) studied the effect of a mild heat treatment on the microbial
load of alfalfa seeds inoculated with Salmonella Stanley. No reduction in populations of
S. Stanley was seen on seeds soaked in water at 21 °C for 5 or 10 minutes. Treating seeds
in w ater at 5 4 ° C for 5 and 1 0 minutes reduced S. Stanley p opulations flom 263 to 9
CFU/g and flom 261 to 6 CFU/g, respectively. All other treatment temperatures (57, 60,
63, 66 and 71 °C) led to populations of <1CFU/g after 5 minutes.

Jaquette et al. (1996) also assessed the effect of temperature on alfalfa seed
viability. Compared with dipping seeds in water at 21 °C (control), immersing seeds in
water at 54, 57, or 60 °C for 5 minutes did not substantially reduce the germination rate
after 48 h of storage at 30 °C. However, treatments at 54, 57 and 60 °C for 10 minutes
reduced seed viability flom 96% (control) to 88, 84 and 42%, respectively. Treating
seeds at 63 and 66 °C for 5 minutes reduced the germination rate to 83 and 82%,
respectively, while treatment at the same temperature for 10 minutes reduced viability to
21 and 6 %, respectively. While heating appears to effectively inactivate S. Stanley on
alfalfa seeds, the range of temperatures that can be used is narrow, i.e., between 57 and 60
°C for no longer than 5 minutes because lower temperatures may not kill S. Stanley and

perhaps other Salmonellae with higher temperatures or a longer exposure time (10

20

minutes) decreasing germination. Therefore, this hot water treatment is not practical in
commercial settings.

Piemas and Guiraud (1997) experimented with heat as an antimicrobial treatment
for rice seeds. These authors reported that temperatures of 70 or 90 0C inactivated the
seeds after 30 seconds. Irnmersing the seeds at 60 °C for 5 minutes led to a 3 log decrease
in total aerobes without affecting germination.

Piemas and Guiraud (1997) observed substantially lower aerobic plate counts on
biologically cultivated brown rice seeds destined for sprouting afier soaking in a 1, 000

ppm sodium hypochlorite solution at 60 °C for 5 minutes.

c.- Gamma irradiation

Rajkowski and Thayer (2000) reported D-values of 0.54 and 0.46 kGy when
radish sprouts were inoculated with a Salmonella cocktail and irradiated. D-values were
0.34 and 0.30 kGy when alfalfa Sprouts were inoculated with’E. coli 0157:H7. These
sprouts were previously irradiated at 6 kGy/ 19 °C to eliminate background flora before
inoculation. After enrichment, Salmonella was not detected on sprouts irradiated at 2 0.5
kGy. Nevertheless, only a 4 log reduction in total aerobes was observed using 3.0 kGy
(Rajkowski and Thayer, 2000). Although radish sprouts used in these experiments kept
their structure following sterilization by irradiation, inoculation and irradiation, the
authors acknowledged that further research is necessary to determine the effect of
ionizing radiation on the structure and keeping quality of other Sprout varieties. They
suggested the combination of low level radiation, an effective chemical wash and

modified atmosphere packaging to control pathogens on flesh fluits and vegetables

21

The organoleptic quality of sprouts should be unaffected by gamma irradiation.
This process may be economically feasible for Sprout growers. However, a major
drawback is that sprout growers have small operations scattered over large areas, and
therefore, 2 to 3 days may be required for irradiation. Considering the relatively Short
shelf-life of Sprouts, this solution may not be commercially Viable for all growers.
Moreover, the germination rate and production yield are essential concerns for Sprout
growers. Irradiation does not affect germination rate while Sprout production yield is

affected (Rajkowski and Thayer, 2001).

d.- Pre-soaking treatment

Experiments conducted either on seeds or Sprouts actually show that the microbial
reduction is sometimes less after longer rather than Shorter exposure times. This
observation is drawn flom work by Taormina and Beuchat (1999a) and Weissinger and
Beuchat (2000). The explanation offered is that, in the case of longer solution-seed
contact times, the seeds imbibe more water and therefore release bacterial cells that have
been more strongly attached to the seed surface or were previously trapped and hidden
within crevices or between the testae and the cotyledons. This explanation can also be
extended to sprouts.

In order to verify the previous hypothesis, Weissinger and Beuchat (2000)
assessed the effect of pre-soaking alfalfa seeds in water for 30 minutes on the release of
bacteria hidden in their crevices. The effect of pre-soaking on the efficacy of subsequent
exposure to 2,000 ppm chlorine was negligible; however, it might slightly enhance the

efficacy of lactic acid.

22

e.- Surfactants

Surfactants were u sed b y Z hang and F arber ( 1996) to increase 5 urface w etting.
Surfactants have a capacity to penetrate and adhere to porous surfaces (Piemas and
Guiraud, 1997).

Use of Orenco Peel 40 (Rio Linda Chemical Co., Inc., Sacramento, CA) and
Tergitol (Sigma Chemical Co., St Louis, MO) as surfactants added to a chlorine
treatment did not lead to a lower microbial load in comparison with the use of chlorine
alone (Zhang and Farber, 1996).

Addition of 1 g/L Tween 80, as a surfactant, to a sodium hypochlorite solution by
Piemas and Guiraud (1997) improved the disinfection of rice seeds, although this
microbial reduction was not due to any bactericidal effect of Tween 80 at that
concentration. No Significant decrease in quantity of flee available chlorine was observed
after adding Tween 80 at concentrations up to 1 g/L.

Although benzalkonium chloride, as a quaternary ammonium compound, is not
permitted as a food sanitizer due to the possibility of generating toxic residues, Piemas
and Guiraud (1997) assessed its use as a surfactant. Dipping biologically cultivated
brown rice seeds in a solution containing 0.1 mg/L of benzalkonium chloride for 30
minutes decreased the natural microflora about 2 logs. Decontamination was more
effective at 1 mg/L when aerobic plate counts decreased 2.5 to 3 logs after 10 minutes.

Prolonged soaking had no effect with neither germination nor seedling growth affected.

23

f.- Addition of antimicrobials to irrigation water

Addition of antimicrobials to Sprout irrigation water was investigated as a means
to inhibit the grth of human pathogens introduced after seed decontamination due to
inadequate water quality or insufficient worker hygiene (F ett, 2000).

Fett (2000) reported that it is very difficult to achieve a significant reduction in
the natural microflora of growing Sprouts by addition of antimicrobials to irrigation water.
Such a treatment would likely be unable to kill human pathogens that are located in the
internal tissues of sprouts (Fett 2000).

Work by Taormina and Beuchat (1999b) led to the same conclusions. These
authors treated alfalfa seeds previously inoculated with E. coli 0157:H7. The seeds were
soaked in one of the following chemicals for 20 minutes : NaOCl (200 and 2,000 ppm
active chlorine), Ca(OCl)2 (200 and 2,000 ppm active chlorine), acidified NaClO2 (100,
500 and 1,200 pg/ml), Na3PO4 (1%), Vegi-CleanTM (1%), TsunamiTM (40 and 80 ppm),
VortexxTM (40 and 80 ppm) or H202 (1%). After soaking, the seeds were drained for 2
minutes, immersed in sterile tap water for 1 hour, and then placed in plastic boxes for
germination. During sprouting, 40 to 45 ml of each test chemical was sprayed evenly
onto sprouts after 24 hours of germination and 24, 48 and 72 hours later. Chemical
solutions sprayed onto sprouts were rinsed 5 minutes after application by spraying 20 to
25 m1 of sterile tap water evenly. Samples of sprouts were removed flom the sprouting
boxes and analyzed for E. coli populations prior to and immediately after spray
applications. No chemical treatment reduced numbers of E. coli 0157:H7 on Sprouts
when compared with numbers recovered flom water-treated sprouts. With the exception

of NaClO2 at 1200 pg/ml, Spray applications of these chemicals did not Significantly

24

reduce populations or control the growth of E. coli 0157:H7 on alfalfa sprouts during the
sprouting process. Populations of E. coli 0157 :H7 peaked at ca. 6 to 7 logs, 48 hours
after initiation of the Sprouting process and remained stable despite further Spraying with

chemicals. No difference were seen between various treatments after 6 days of cold

storage at 9 i 2 °C.

g.- Effect of duration of seed storage

Storing dry alfalfa seeds at 8 °C for 9 weeks reduced populations of Salmonella
Stanley flom 2.53 to 1.81 logs. Inactivation was enhanced at 21 °C. Storing these dry
seeds at 8 °C for 1 week and then at 21 °C for 8 weeks decreased the Salmonella

population flom 2.53 to 0.92 log (J aquette et al., 1996).

h.- Examination of Sprouts using scanning electron microscopy

Scanning electron microscopy allows for observing biofilrn formation on all parts
of alfalfa sprouts (Fett, 2000). Mature biofilms are structured communities of microbes
adherent to a surface and embedded in a self-produced glycocalyx material composed
primarily of exopolysaccharides. The process of biofilrn formation is believed to start
with adhesion of individual microbes to a surface, aggregation into microcolonies,
intercellular communication and finally maturation into structured biofilms (Costerton et
al., 1999; Fett, 2000). Biofilrn bacteria are 2 500 times more resistant to antimicrobial
compounds and interact physiologically with other microbes as a complex community
(Costerton et al., 1995; Fett, 2000). Rod-Shaped bacteria of various sizes were the

predominant microbes observed on all Sprout surfaces (cotyledons, hypocotyls and roots).

25

Cocci-shaped bacteria, as well as yeast, were rarely seen, while structures resembling
filamentous fungi were not observed (Fett, 2000). After 2 days of growth, biofilms were
already seen on laboratory grown sprouts. Rinsing under nmning tap water did not
remove these biofilms flom the surface of Sprouts. By the 4th day of growth, 29 to 59%

of the total mesophyllic bacteria were present in biofihns (Fett, 2000).

i.- Sensory evaluation

Taormina and Beuchat (1999b) evaluated the overall visual appearance of mature
alfalfa sprouts that had been dipped in chemical solutions and rinsed with tap water or not
rinsed. A hedonic scale ranging flom 1 (inedible) to 5 (excellent quality) was used to rate
the appearance of the sprouts during 10 days of storage at 9 i 2 °C. Chemical treatments
used u sed in this study included N aClO at 2 00, 1 ,000 and 2 ,000 ppm active chlorine;
Ca(OCl)2 at 200, 1,000 and 2,000 ppm active chlorine; acidified NaOCl2 at 850 and
1,200 ppm; Na3P04 at 40,000 and 120,000 ppm; TsunamiTM at 40 and 80 ppm; VortexxTM
at 40 and 80 ppm, and H202 at l and 5%. Rinsing with tap water after applying the
chemical sanitizer clearly helped to maintain overall appearance. After 1 day of storage,
the rinsed sprouts usually (except Na3PO4 120,000 ppm) compared well to the water
control. By the 6th day, the rinsed sprouts previously treated with NaClO, Tsunami and
H202 were still similar to the water control while some alterations were detected in the

appearance of those treated with VortexxTM and Na3POA.

26

D.- Sprouts industry regulations

In order to counteract the “sprout problem”, several guidelines and
regulations has been published. On October 27, 1999, the FDA and the Center for Food
Safety and Applied Nutrition (CFSAN) published a series of papers as “guidance for the
industry” (FDA/CFSAN, 1999). A paper entitled “Reducing microbial food safety
hazards for sprouted seeds” identifies the preventive controls that the FDA believes
Should be taken immediately to reduce the public health risks associated with raw sprouts
and to ensure that Sprouts are not adulterated under the food safety provisions of the
Food, Drug and Cosmetic (FDC) Act. Failure to adopt effective preventive methods of
control assumes that the product w as prepared under unsanitary conditions which may
render it injurious to health. Food produced under such conditions is adulterated under
the act [21 U.S.C. 342 (a) (4)]. The FDA would consider enforcement actions against any
party who does not have effective preventive controls in place, particularly, microbial
testings. This paper provides guidelines and recommends good agricultural practices
(GAPS) for seed production, conditioning, storage and transportation and good
manufacturing practices (GMPS) for sprout production. It also advocates seed treatment
with an approved antimicrobial (i.e., use of the recommended 20,000 ppm calcium
hypochlorite soak), testing for pathogens in the spent irrigation water, and

implementation of traceback systems (FDA/CFSAN, 1999).

27

2.2.- BACTERIAL PATHOGENS ASSOCIATED WITH ALFALFA SPROUTS
2.2.1.- ESCHERICHIA COL10157:H7
A.- Health significance of E. coli 0157 :H7

Escherichia coli 0157:H7 was first isolated in 1975 flom the stool of a woman
with bloody diarrhea. In 1982, E. coli 0157:H7 was identified as a human pathogen
when it was involved in two outbreaks of hemorrhagic colitis in Oregon and Michigan.
Since these years, E. coli 0157:H7 has gone flom a little known enteric pathogen to a
foodbome pathogen of international importance. Indeed, it iS the predominant cause of
enterohemorrhagic-associated diseases in the United States and in many other countries
(Doyle et al., 1997; Ryser, 1998).

Issues of concern with E. coli 0157:H7 include a very low infective dose (< 100
cells), unusual tolerance to acid and the development of antibiotic resistance. Recent
evidence suggests that isolates of E. coli 0157 :H7 have developed a trend toward
resistance to streptomycin, sulfisoxazole and tetracycline (Doyle et al., 1997; Ryser,
1998)

Infections with E. coli 0157:H7 lead to symptoms ranging flom a mild
nonbloody diarrhea to hemorrhagic colitis (characterized by grossly bloody diarrhea
accompanied by severe appendicitis-like abdominal pain), and hemolytic uremic
syndrome (HUS) which can produce renal failure, thrombocytopenic purpura and
eventually death. These symptoms are related to the adherence of the pathogen to the
intestinal tract lining followed by production of one or more verotoxins (V TS) also called

Shiga-like toxins (SLTs) (Doyle et al., 1997; Ryser, 1998).

28

B.- Evidence of E. coli 0157:H7 and E. coli 0157: NM in fresh produce

Contamination of flesh produce by E. coli has been well documented (Beuchat,
1996). Alfalfa sprouts are reported among foods often linked to E. coli outbreaks (Table
2.3) and, rather than ground beef, have been found by the CDC to be incriminated in the
greatest number of E. coli 0157:H7-related illnesses. Of 285 cases of E. coli 0157 :H7
infection reported by CDC as of 1998, 108 were caused by alfalfa sprouts whereas 20
were attributed to tainted ground beef and 52 to other sources (FDA, 1998). Cattle and
chickens are well known reservoirs of E. coli 0157: H7. Manure, in general, and manure
flom cattle and chickens, in particular, Should be avoided in fertilization of flesh produce
intended for raw consumption.

The world’s largest E. coli 0157:H7 oubreak occured in Japan, flom May to
August 1996, and involved about 10,000 people, including 6,000 primary school children,
in Sakai City, Osaka Prefecture and factory workers in Kyoto. E. coli 0157:H7 isolates
flom S akai City and Kyoto had identical PFGE and RAPD patterns. Based on strong
evidence, white radish (daikon) sprouts were incriminated as the source of infection
although E. coli could not be isolated flom either the seeds or the Sprouts. As a
consequence of this outbreak, 12 deaths were reported and at least one person died of
HUS-associated encephalopathy (Gutierrez, 1997; Como-Sabetti et al., 1997; Itoh et al.,
1998; Watanabe et al., 1999; Taormina and Beuchat, 1999a; Taormina 1999; Taormina et
al., 1999; FDA/NACMCF, 1999).

The following year, E. coli 0157:H7 was again implicated through radish sprouts
in two different outbreaks in Yokohama and Gamagori City, Japan (Gutierrez, 1997; Itoh

et al., 1998, Watanabe et al., 1999; Taormina and Beuchat, 1999a; Taormina, 1999).

29

 

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Hara-Kudo et a1. (1997) reported an E. coli 0157:H7 population of 7.8 loglo CFU/g on
Sprouts obtained flom the implicated seeds. This outbreak consisted of 126 cases,
including one fatality (Gutierrez, 1997).

The first reported outbreak of E. coli 0157:H7 infection associated with alfalfa
sprouts occurred in Michigan and Virginia during June and July of 1997. This outbreak
was traced to alfalfa Sprouts p roduced from a s eed lot c ommon to both Michigan and
Virginia producers and supplied by the same seed distributor. Forty to 60 persons were
affected in Michigan, with an additional 48 cases in Virginia. The strains implicated in
both states were identical by molecular subtyping (Como-Sabetti et al., 1997; Taorrrrina
and Beuchat 1999a; FDA/NACMCF, 1999; Taormina et al., 1999). Following these E.
coli 0157:H7 outbreaks, a class I recall (Table 2.4) was issued for an undetermined
volume of alfalfa sprouts grown in Norfolk, VA that were distributed in North Carolina,
Maryland, Virginia and the District of Columbia (FDA, 2004).

In June 1998, a cluster of infections by a non-motile E. coli 0157 strain (lacking
the flagellar antigen) producing Shiga toxins I and H occurred in Northern California and
Arizona. These cases were associated with eating an alfalfa and clover sprout mixture
produced by the same Sprouter implicated in a simultaneous Salmonella Sefienberg
outbreak in California and Nevada. E. coli 0157:NM isolates flom 8 patients had the
same PFGE pattern but laboratory analysis of seeds, sprouted seeds and environmental
samples did not yield E. coli 0157:NM. This producer inconsistently used chlorine
disinfection before sprouting (Taormina et al., 1999, FDA/NACMCF, 1999; Mohle-
Boetani et al., 2001). A class I recall (Table 2.4) was issued to recover the Sprouts jointly

associated with the two E. coli 0157:NM and Salmonella Seftenberg outbreaks.

31

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32

An outbreak of E. coli 0157:H7 associated with alfalfa sprouts occurred during
the summer of 2002 in San Luis Obispo County, California (FDA, 2002, Huff, 2002c).
The victims, four women and one teenage girl, experienced mild symptoms with one of
them hospitalized and all quickly recovering. Four of the victims were students at
California Polytechnical State University and three had their meals at the university
cafeteria. These infections were reported during the first days of August 2002 (Prado and
Huff, 2002; Huff, 2002a,b,c; Stone, 2002; Wootson, 2002). The source of the outbreak
was traced back to the sprouts by interviewing the victims with no lab report

incriminating the sprouts (Huff, 2002c).

C.- Generalities about Escherichia coli 0157:H7

Escherichia coli 0157:H7 is a gram-negative, non-sporeforming, motile rod—
shaped organism, possessing the ability to ferment lactose with production of gas within
48 hours at 35 °C and showing optimal grth at 30 to 42 °C (Hitchins et al., 1995;
Doyle, 1997; Ryser, 1998). E. coli 0157:H7, also called enterohemmorhagic E. coli
(EHEC), presents some specific characteristics which allow for differentiation from most
other E. coli. These features include the inability to grow well, if at all at > 44.5 °C, poor
growth at < 10 °C, inability to ferment sorbitol within 24 h unlike 80 to 93% of other E.
coli strains, inability to produce B-glucuronidase (i.e., inability to hydrolyze 4-methy1-
umbelliferyl-D-glucuronide) contrarily to 92 to 96% of all other E. coli strains, possession
of an attaching and effacing (eae) gene, carriage of a 60-Mda plasmid and expression of
an uncommon 5000 to 8000-molecular-weight outer membrane protein (Hitchins et al.,

1995; Doyle et al., 1997; Ryser, 1998).

33

E. coli 0157:H7 shows an exceptional tolerance for acidic conditions. It has been
found to survive in apple cider at pH 3.6 to 4.0 for 10 to 31 days and 2 to 3 days at 8 or
25 °C, respectively (Conner and Kotrola 1995; Doyle et al. 1997). Concentrations of up
to 1.5% acetic, citric, or lactic acid used as antibacterial sprays on beef were not effective
in reducing E. coli 0157:H7 populations (Brackett et al., 1994; Doyle, 1997). Although
not fully understood, the mechanism of acid tolerance seems to be related to a protein (s)
that can be induced by preexposing the organism to acid conditions (Doyle, 1997). E.
coli 0157:H7 is also known for its ability to form homogeneous biofihns on sprouts and

other plants (F ett 2000).

D.- Basis for laboratory identification of E. coli 0157:H7

Differentiation of E. coli 0157:H7 using Cefixime - Tellurite Sorbitol McConkey
Agar (CT-SMAC) is based on the inability of this serovar to ferment d-sorbitol, unlike
most other strains of E. coli. On Sorbitol McConkey agar (SMAC) non-sorbitol
fermenting bacteria produce pale gray or colorless colonies compared to the bright pink-
red colonies produced by sorbitol fermenters such as non-0157:H7 E. coli strains and
other enterics (Hitchins et al. / FDA-BAM, 1995; Difco, 1998). Two antibiotics,
cefixime and potassium tellurite (CT Supplement), (Dynal, Lake Success, NY) added to
Sorbitol McConkey agar increase the selectivity of the medium for E. coli 0157:H7 by
suppressing the growth of normal flora (Hitchins et al. / FDA-BAM, 1995). Bile salts
and crystal violet are selective agents that inhibit growth of gram-positive organisms

(Difco, 1998).

34

2.2.2.- SALMONELLA TYPHIMUva DT104
A.- Health significance of Salmonella Typhimurium DT104

Strains of Salmonella that are resistant to antimicrobial agents have become a
world-wide problem (Glynn et al., 1998). Salmonella subsp. enterica serovar
Typhimurium Definitive Type104 is a non-enteric fever (non-typhoid) Salmonella strain
featuring a rare multiantibiotic-resistant plasmid. In the US, it has shown resistance to 5
antibiotics: arnpicillin, chlorarnphenicol, streptomycin, sulfonamides and tetracyclines.
This penta-antibiotic resistance pattern is used as a screening method for S. Typhimurium
DT104 with chloramphenicol-resistance being the most specific marker for this pattern of
resistance. Moreover, in England such strains have developed additional resistances to
trimethoprim (24%), fluoroquinolones and ciprofloxacin (14%) (Glynn et al. 1998). This
resistance is believed to be induced by sub-therapeutic use of these drugs by veterinarians
in animal rations (D’Aoust, 1989; Glynn et al 1998), with the antibiotic resistant strains
being transmitted to humans by a very healthy population of animal carriers (D’Aoust,
1997)

In the US, prevalence of penta-resistant S. Typhimurium DT104 isolates
increased from 0.6% in 1979-1980 to 34 % in 1996, with the highest prevalence seen in
western states. In 1998, S. Typhimurium DT104 accounted for ca. 75% of the
multiresistant Salmonella infections. The highest attack rate occurred among children,
the elderly, immuno-compromised individuals and antibiotic users (Glynn et al.,1998).
These authors suggested that infections caused by antibiotic resistant S. Typhimurium
DT104 might be associated with greater morbidity and mortality than other Salmonella

infections.

35

The contamination route is mostly fecal to oral (D’Aoust, 1989). Symptoms of
Salmonella infection usually appear after 8-72 hours of incubation (D’Aoust, 1997;
Ryser, 1998). The clinical manifestations of non-typhoid Salmonella infections in
humans can range from mild and self-limited gastroenteritis to septicemia and death.

In mild cases, nausea and vomiting are the first signs of gastroenteritis or
enterocolitis, also named “ Salmonella food poisoning “. These symptoms generally
subside within a few hours. Development of mild fever, chills, prostration, myalgia and
abdominal pain sometimes resembling acute appendicitis, is soon followed by diarrhea,
the most prominent symptom, which can range from a few loose stools to overtly bloody
and rice-water cholera-like stools in more severe cases (D’Aoust, 1989 and 1997; Ryser,
1998). The clinical condition is generally self-limiting with remission of diarrhea and
abdominal pain typically occurring without intervention within 5 days of the onset of the
symptoms (D’Aoust, 1997; Ryser, 1998). Another public health concern at this stage is
the shedding of Salmonella in infected patients’ stools at concentrations of 106 to 109
CFU/g (Ryser, 1998). In the case of uncomplicated enterocolitis, supportive therapy such
as fluid and electrolyte replacement is usually sufficient (D’Aoust, 1997; Ryser, 1998).
Antibiotics are not recommended for gastroenteritis as they will also disturb or destroy
the normal gut microflora which normally competes with Salmonella for nutrients and
intestinal binding sites and produces bacteriocins that limit the growth or survival of
Salmonella. Administration of antibiotics at this stage tends to prolong the asymptomatic
carrier state and the intermittent excretion of salmonellae possessing greater antibiotic

resistance (D’Aoust, 1989 and 1997; Ryser,1997).

36

S. Typhimurium DT 104 enterocolitis may also proceed to septicemia among pre-
disposed individuals and precipitate chronic conditions. The population at risk
encompasses infants, young children, the elderly, people with a disturbed intestinal
microflora and patients with pre-existing physiological, anatomical or immunological
disorders, such as cancer, liver disease, sickle-cell anemia, gastric disorders, gallblader
diseases and AIDS (D’Aoust, 1989 and 1997; Ryser, 1998). The defense mechanism
possessed by these patients is unable to counteract the effect of invasive Salmonella
(D’Aoust, 1997). Fever is the primary symptom of Salmonella septicemia. At this stage
the action of an efficient antibiotic is strongly recommended.

Unusually virulent cases of S. Typhimurium DT104 infection, and prolonged and
untreated septicemia can lead to serious complications such as osteomyelitis, brain
abscesses, meningitis and other neural infections, pneumonia, pyelonephritis,
endocarditis, suppurative arthritis and splenomegaly (D’Aoust 1989, Ryser, 1998).

In England. S. Typhimurium DT104 leads to a high percentage of hospitalizations
(41%) and fatalities (3%) compared to less than 3% hospitalizations and 0.1% fatalities
for other strains of Salmonella (D’Aoust 1989). Evidence from outbreaks suggests that as
few as 1 to 10 cells can constitute a sufficient infectious dose, particularly for populations
at risk (D’Aoust, 1997).

Despite an obvious need for immunogenic preparations against non-typhoid
salmonellae, the development of prophylactics is hampered by the multiplicity of
serovars, the rapid succession of serovars in the human population and the unpredictable

pathogenicity of infective strains (D’Aoust, 1997).

37

B.- Evidence of S. Typhimurium DT104 in fresh produce

Salmonella is widely known as a contaminant of fresh produce (Beuchat,1996).
Fruits and vegetables are one of the reservoirs for non-typhoid salrnonellae. Global
export, questionable hygienic conditions during production, harvesting and distribution,
irrigation water and untreated fertilizers, all increase the risk of contamination. Cross-
contamination with other foods or from infected workers is also an important means of
spreading Salmonella. Alfalfa sprouts have been incriminated in at least 14 Salmonella
outbreaks since 1994 (Table 2.5).

In a bacteriological survey of sixty “health” foods in the Baltimore-Washington,
DC. area, Andrews et al. (1979) found Salmonella poona at a level of 6.3 x 104 CFU/g
after sampling a brand of organically labeled alfalfa seeds.

An outbreak of Salmonella Bovismorbificans occurred in Sweden (103 cases) and
Finland (210 cases) in March, 1994. From May through November 1994, 282 additional
cases were recorded in Sweden. The bacterium was isolated from alfalfa sprouts but not
fi'om the implicated seeds which came from the same importer and, probably, the same
lot originating from Australia (Ponkéi et al., 1995; Taormina and Beuchat, 1999a).

Salmonella Stanley was responsible for an international sprout-related o utbreak
which peak occurred during May - June 1995, and involved alfalfa sprouts in Finland and
17 US states, including Michigan. Isolates from Finland and the United States yielded
the same DNA profile and one particular antibiotic resistance pattern (resistance to
trimethoprim-sulfarnethoxazole, tetracycline, sulfisoxasole, streptomycin and kanarnycin)
(Jaquette, 1996; Mahon et al., 1997; Tauxe et al., 1997; FDA/NACMCF, 1999). T be

implicated sprouts, eaten by 50 patients in 6 states were traced through 9

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growers and one distributor, to one Dutch shipper. The Finnish sprouts were also traced
to seeds from the same Dutch shipper (Tauxe et al., 1997; Mahon et al., 1997; Taormina
and Beuchat, 1999a; FDA/NACMCF, 1999 ). Approximately 242 culture-confirmed
cases were reported in this outbreak (Tauxe, 1997;Taormina et al., 1999). Based on the
rate of underreporting from other Salmonella outbreaks, Mahon et al., (1997) estimated
the actual number of cases at 5,000 to 24,000.

A S. Newport outbreak of approximately 133 cases, linked to alfalfa sprouts, was
reported in Oregon, U SA and B ritish C olumbia, C anada, in late 1 995 and early 1 996.
The pathogen was isolated from both alfalfa seeds and sprouts. Using PFGE, isolates
fiom the aforementioned outbreaks were indistinguishable from each other (Tauxe et al.,
1997; Van Beneden et al., 1999; Taormina and Beuchat, 1999a; FDA/NACMCF, 1999)
and from isolates from previous S. Newport outbreaks in late 1995 in Georgia and
Vermont, and in June 1995 in Denmark (Taormina et al., 1999). This observation is
coherent with the fact that these seeds came from the same Dutch shipper also involved in
the S. Stanley outbreak (Tauxe et al., 1997; Van Beneden et al., 1999; Taormina and
Beuchat, 1999a; FDA/NACMCF, 1999). Cultures of the implicated seeds yielded S.
Newport (Taormina et al., 1999).

Nevada and California were sites of a series of infections involving Salmonella
Montevideo and Meleagridis. In May through July 1996, ~ 500 culture-confirmed cases
were recorded. A case-control study showed that consumption of alfalfa sprouts was
associated with the outbreak. Moreover, the same strain of S. Meleagridis was isolated
from p atients and from s prouts obtained from retail stores and the sprouting facilities,

although the seed samples did not yield either serotype (FDA/NACMCF, 1999). In

42

October, 1997, S. Meleagridis was again the cause of 78 cases of illness linked to
consmnption of alfalfa sprouts in Canada (Taormina et al., 1999).

In 1997, alfalfa sprouts were incriminated in an outbreak of Salmonella Infantis
and Salmonella Anatum in Kansas and Missouri that resulted in 109 culture-confirmed
cases. S. Anatum was recovered fi'om the seeds, whereas the sprouts yielded both
Salmonella serovars (FDA/NACMCF, 1999).

From late 1997 through July 1998, 52 to 60 culture-confirmed patients showing
infections with the same strain of Salmonella Senftenberg were reported in the states of
Nevada and California and linked to consumption of an alfalfa / clover sprout mixture.
Cultures of clover and alfalfa seeds used to grow the implicated sprouts did not yield the
pathogen (FDA/NACMCF, 1999; Taormina et al., 1999).

Alfalfa sprouts were again incriminated in an outbreak of 18 cases of Salmonella
Havana in Arizona and California in May 1998 and, also, in an outbreak of 22 cases of
Salmonella Cubana in Arizona, California, Maryland, New-Mexico and Utah during May
to August 1998. The sprouts were grown from the same seed lot yielding S. Havana, S.
Cubana and S. Tennessee which were indistinguishable from the patient isolates. These
sprouts came from one large California producer who claimed to soak the seeds in 2,000
ppm chlorine for 30 minutes, followed by a 300 ppm chlorine soak for several hours
before sprouting (FDA/NACMCF, 1999; Taormina et al., 1999).

Alfalfa sprouts were associated with about 75 cases of Salmonella Mbandaka
infection in Oregon, Washington, Idaho and California during January through March
1999. The pathogen was isolated from the traced seed lot which was grown in California

and distributed to the implicated sprout growing facilities (F DA/NACMCF , 1999).

43

Health Canada (2001) reported laboratory-confirmed infections of Salmonella
paratyphi B var. java which were potentially linked to c onsumption o f alfalfa sprouts
during August and September of 1999, in Alberta (43 cases), British Columbia (6 cases)
and Saskatchewan (2 cases).

During September of 1999, a multistate outbreak of Salmonella Muenchen
associated with eating raw alfalfa sprouts was identified in Wisconsin (Kuenn, 2000;
Proctor, 2001). In this state, 62 case patients were observed with six hospitalized and no
deaths reported. Ultimately, 95 additional outbreak-related cases were identified in 6
states other than Wisconsin (California, 23; Idaho,11; Michigan, 35; Missouri, 19;
Nevada, 5; Washington, 2). Due to underreporting, the total number of cases was
estimated at 3,500 to 16,200. Dates of illness for the Wisconsin patients were between
August 20 and October 20 and onsets of non-Wisconsin cases occurred between July 8
and November 29. This is the first specific documentation (signed FDA affidavit) of an
outbreak occurring despite the incriminated sprouts having been grown from seeds that
were previously sanitized with the recommended FDA 2% calcium hypochlorite
treatment for 15 minutes. Trace back procedures indicated that all contaminated sprouts
were grown from the same seed lot. Salmonella Muenchen isolates from the 157 patients
and from intact packages of alfalfa sprouts grown from the traced lot of seed were
indistinguishable by PFGE (Proctor et al., 2001). Following trace back after the
Wisconsin outbreak, an FDA Class I recall (Table 2.6) was issued for 32,900 lb of alfalfa
seeds.

During February to April 2001, Salmonella Kottbus, a relatively rare strain of

Salmonella, was implicated in a multi-state oubreak (California, 24 cases; Arizona, 6

cases, Colorado, 1 case and New—Mexico, 1 case) linked to consumption of alfalfa sprouts
produced at a single facility. The S. Kottbus isolates from patients, seeds and the
production environment were indistinguishable by pulsed-field gel electrophoresis.
Twenty-one (21) patients developed an acute diarrheal illness, three patients had urinary
tract infections and three patients were hospitalized (Mohle-Boetani et al., 2002).

In early June 2004, 12 cases of Salmonella bovismorbificans infection reported in
the states of Oregon and Washington have been possibly linked to consumption of raw
alfalfa sprouts. Following this outbreak, an undetermined amount of sprouts have been
recalled in Washington, Oregon and North California (FDA, 2004).

These numerous sprout contaminations by various Salmonella strains have led to
at least 2 Class II and 18 Class I recalls fiom 1990 to date, involving more than 83,000
pounds of sprouts and 58,000 pounds of seeds (Table 2.6). These numbers give a realistic
account of the acute financial and public health concerns that underlies the critical need to
find a more reliable alternative to the currently recommended 20,000 ppm calcium

hypochlorite treatment.

C.- Generalities about salmonella Typhimurium DT104

Salmonella are facultatively anaerobic gram-negative rods belonging to the family
of Enterobacteriaceae. These bacteria grow optimally at 37 oC and catabolize D-glucose
and other carbohydrates with the production of acid and gas, but do not utilize lactose and
sucrose. Salmonellae are oxidase negative and catalase positive, grow on citrate as the
sole carbon source, generally produce hydrogen sulfide, decarboxylate lysine and

omithine, and do not hydrolize urea (D’Aoust, 1997).

45

 

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49

Salmonella adapts well to extreme environmental conditions and actively grows
at temperatures as high as 54 °C. Some strains also exhibit psychotrophic properties , as
reflected by their ability to grow in foods stored at 2 to 4 °C (D’Aoust, 1997). Salmonella

can form homogeneous biofihns on sprouts (Korber et al., 1997; Fett, 2000).

D.- Basis for laboratory identification of S. Typhimurium DT104

The use of Xylose Lysine Desoxycholate agar (XLD) as a specific media to select
for Salmonella is based on the following principles. Xylose is fermented by most enteric
organisms except Shigella and Providencia. Addition of lysine in the medium
formulation allows for differentiation of Salmonella. As xylose is exhausted, Salmonella
decarboxylates lysine to cadaverine causing a reversion to alkaline conditions. Alkaline
reversion by other lysine-positive organisms is prevented by excess acid production fi'om
fermentation of lactose and sucrose. Sodium thiosulfate and ferric ammonium citrate
allow for visualization of hydrogen sulfide production under alkaline conditions. After
growth on XLD agar, S. Typhimurium cells appear red with black centers due to
hydrogen sulfide production. Moreover, sodium desoxycholate inhibits the growth of

gram-positive organisms (D’Aoust, 1997; Difco, 1998).

2.2.3.- LISTEIUA MONOCYTOGENES
A.- Health significance of Listeria monocytogenes

Listeria monocytogenes emerged as one of the major foodbome pathogen during
the 1980’s with the first confirmed foodbome outbreak of listeriosis occurring in 1981, in

Nova-Scotia, Canada (Schlech et al., 1983; Rocourt and Cossart, 1997). Listeriosis is a

50

major public health concern and may be the leading fatal foodbome disease in the United
States (Farber and Peterkin, 1991; Fisher et al., 2000). Despite the availability of suitable
antibiotic therapy, the treatment of listeriosis is complicated by growth of L.
monocytogenes as an intracellular pathogen within macrophage cells of the spleen and
liver with many antibiotics unable to effectively penetrate the blood-brain barrier (Ryser,
1998). The highly significant case-fatality rate (~ 20%), the severity of the illness and
severe complications make listeriosis the second most costly foodbome disease in the
United States (Rocourt and Cossart, 1997; Ryser, 1998).

Development of listeriosis in humans is affected by host susceptibility, gastric
acidity, inoculum size, the strain of L. monocytogenes and various virulence factors of the
organism (Ryser, 1998). Host susceptibility is a determinant factor in Listeria infections.
Although Listeria is a ubiquitous pathogen, healthy individuals seldom acquire listeriosis
(Ryser, 1998). Ingestion of food containing 2 102 Cng poses a significant health risk
for susceptible individuals including pregnant women and newborn infants, the elderly,
immunocompromised adults including cancer patients, transplant patients under cortico—
steroids, human immunodeficiency virus (HIV)-positive individuals and acquired
immunodeficiency syndrome (AIDS) patients and also patients with underlying illnesses,
such as heart disease, diabetes, cirrhosis of the liver and alcoholism (Rocourt and Cossart,
1997; Ryser, 1998)

L. monocytogenes produces very serious systemic infections in this high risk
population. In non-pregnant adults, the bacterium has a particular tropism for the central
nervous system but some cases of mild gastrointestinal illness have been observed

(Rocourt and Cossart, 1997). In immunocompromised adults, Listeria can cause

51

meningitis, encephalitis and septicemia (Ryser, 1998). After an initial incubation period
of 2 days to 3 months, the symptoms develop suddenly with severe headache, dizziness,
stiff neck or back, incoordination and other disturbances of the central nervous system
most often observed (Ryser, 1998). The prognosis of listeriosis depends on both the type
of infection and the underlying conditions (Rocourt and Cossart, 1997). A favorable
prognosis is influenced b y a rapid diagnosis and appropriate antibiotic therapy (Ryser,
1998). Without proper treatment about 20 % of those infected will die (38 to 40% among
immunocompromised adults, the elderly and patients with other predisposing conditions),
with some survivors developing permanent neurologic complications (Rocourt and
Cossart, 1997; Ryser, 1998). Oral administration of large doses of ampicillin or penicillin
together with an aminoglycoside for 2 to 4 weeks is the recommended treatment.

Pregnant women are most frequently affected in the third trimester of pregnancy
(Rocourt and Cossart, 1997; Ryser, 1998). In this group, Listeria infection may be either
asymptomatic (Rocourt and Cossart, 1997) or flu-like leading to infection of the fetus.
These patients experience symptoms such as sudden chills, fever, sore throat, headache,
dizziness, myalgia, lower back pain, discolored urine and occasionally diarrhea. Pregnant
women almost invariably recover without complications (Rocourt and Cossart, 1997;
Ryser, 1998).

Infection of the fetus or the infant carries more serious consequences.
Contamination from the mother can result in abortion, fetal death, stillbirth or the
premature delivery of an infant with neonatal septicemia, a severe infection of the
respiratory, circulatory and central nervous systems that c an either terminate fatally o r

lead to permanent retardation (Rocourt and Cossart, 1997; Ryser, 1998).

52

The possibility that cold and prolonged storage may enhance virulence of some L.
monocytogenes strains (Lou and Yousef, 1999) increases the acuteness of this public
health problem. Finding L. monocytogenes in a ready-to-eat food is grounds for
immediate rejection, as the US has adopted a policy of “zero tolerance” for this pathogen

in all RTE or cooked foods (Ryser, 1998).

B.- Evidence of L. monocytogenes in fresh produce

Listeria monocytogenes is widely distributed on plant vegetation (Beuchat, 1996).
Fruits and vegetables can be contaminated by soil, water, manure, decaying vegetation
and fertilization with effluents from sewage treatment plants containing the pathogen (Al-
Ghazali and Al-Azawi 1990; Reina et al., 1995; Rocourt and Cossart, 1997). The
aforementioned 1981 listeriosis outbreak in Canada, with 41 cases reported, was linked
to consumption of coleslaw prepared from cabbage fertilized with manure from a flock of
Listeria-infected sheep (Schlech et al., 1983; Rocourt and Cossart, 1997). Fresh produce
can also become contaminated during harvest and further processing (Zhang and Farber
1996)

This pathogen has been isolated from fresh cut vegetables and, being a “hardy”
organism, survives well on produce (Zhang and Farber 1996). As a psychotrophic
organism that can grow at refiigeration temperature, this pathogen can develop on
refi'igerated foods (Lou and Yousef, 1999). Carlin and Nguyen-the (1994) and Beuchat
(1996) reported that L. monocytogenes grew on lettuce and endive during storage at 10
°C. Indeed, Listeria spp. has been found on a wide range of retail fi'uits and vegetables,

including potatoes, radishes, cabbage, cucumbers, mushrooms and lettuce (Heisick et al.,

53

1989; Reina et al., 1995; Rocourt and Cossart, 1997). Raw celery, tomatoes and lettuce
were incriminated in a listeriosis outbreak involving 23 patients from eight Boston
hospitals in 1979 (Ho et al. 1986; Beuchat, 1996).

No L. monocytogenes outbreaks has thus far been linked to consumption of alfalfa
sprouts. However, this pathogen have been detected on various types of sprouts, leading
to two Class I recalls, in 1998 and 2002 (Table 2.7), as recorded in FDA enforcement

reports (FDA, 2004).

C.- Generalities about Listeria monocytogenes
Listeria monocytogenes is a ubiquitous, gram-positive, non-spore-forming,
aerobic to facultatively anaerobic, short diphteroid-like, rod-shaped bacterium (Ryser,
1998; Fisher, 2000). Biochemically, Listeria species produce catalase, ferment glucose to
acid without gas and hydrolize esculin. Typical L. monocytogenes isolates ferment
rhamnose but not xylose and are weakly B-hemolytic (Ryser, 1998). Hemolysis is used to
differentiate between L. monocytogenes and L. inocua, the most frequently encountered
non-pathogenic Listeria specie (Rocourt and Cossart, 1997). When grown at room
temperature, L. monocytogenes exhibits a characteristic tumbling motility when viewed
under the microscope (Ryser, 1998).
In general, temperatures > 5 O 0C are lethal to L. monocytogenes. In a suitable
laboratory media, this pathogen will grow at O to 45 °C with optimal grth occurring at

30 to 38 °C. Frozen storage at temperatures as low as -18 °C, and even repeated freezing

54

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and thawing have little negative impact, and these conditions are more likely to injure
than inactivate L. monocytogenes, with cold storage possibly enhancing virulence of some
strains isolated from refrigerated foods (Rocourt and Cossart, 1997; Lou and Yousef,
1999)

L. monocytogenes is also acid tolerant and able to grow at pH 4.3 -10. This
bacterium exhibits a high salt tolerance, being able to grow in the presence of 10% salt
and survive in a 23% brine solution (Rocourt and Cossart, 1997, Ryser 1998) and persist

at nitrite concentrations allowed in foods (Rocourt and Cossart, 1997).

D.- Basis for laboratory identification of L. monocytogenes

Modified Oxford Agar (MOX), one of the most popular media for selective
isolation of Listeria spp, is prepared by adding Bacto Modified Oxford Antimicrobic
Supplement to Bacto Oxford Medium Base. The principle underlying the selectivity
action of MOX is the hydrolysis of esculin by all Listeria species. The reaction between
ferric ions present in MOX as ferric ammonium citrate, and the 6,7-dihydroxycoumarin
produced by hydrolysis of esculin results in black colonies surrounded by a black halo
(Difco, 1998). Lithium chloride is added to this medium to inhibit the growth of
enterocci. Modified Oxford Antimicrobic Supplement contains moxalactam and colistin
methane sulfonate or colistin sulfate. These antibiotics increase the selectivity of the
medium by completely inhibiting gram-negative organisms and most gram-positive

organisms after 24 hours of incubation (Difco, 1998).

56

2.3.- BACTERICIDAL AGENTS

Disinfection of seeds prior to germination is a possible method of eliminating
pathogens, even though, it may not necessarily affect the final count on sprouts (Piemas
and Guiraud, 1997). A major problem with disinfectants, in the case of ready to eat
foods, is the retention of toxic residues. Since germination requires that seeds be
frequently rinsed with water, such rinses could reduce toxic residues, thus allowing
higher concentrations of sanitizer to be used (Piemas and Guiraud, 1997). However, as
low levels of pathogens on seeds can grow to high numbers during sprouting, disinfection

of the mature sprouts must also be considered.

2.3.1.- CLOROX 1ml SODIUM HYPOCHLORITE (NACLO)
A.- General characteristics

Chlorine is a well-known and potent microbicide (Reina et al., 1995) that is
extensively used for treating water. Sodium hypochlorite (N aClO) is the active ingredient
of CloroxTM which is a disinfectant as well as a bleaching and oxidizing agent. Sodium
hypochlorite is available in either powder or liquid formulations.

When chlorine is added as calcium or sodium hypochlorite to water, a mixture of
hypochlorous (HOCI) and hydrochloric acid is formed. HOCl has the highest oxidation
potential of all chlorine species formed (Wei et al.,1985; Dychdala, 1991; Ong et al.,
1996). These authors have shown that the percentage of HOCl, in solution, is greatest at
low pH and decreases as the pH and temperature of the solution increase.

Environmental health communities have expressed concerns about residual

chlorine by-products and the fact that they are returned to the environment through

57

wastewater (Xu, 1999). Important by-products found in chlorinated drinking water
include chloroform, bromodichloromethane and MX [3-chloro-4-(dichloromethyl)-5-
hydroxy-2(5H)-furanone] ( Richardson et al. 1998). Trihalomethane formed in foods as a
result of chlorination is carcinogenic (Wei et al., 1985; Kim et al., 1999). The produce
industry is concerned about the possibility of future regulatory constraints on the use of
chlorine-based sanitizing agents. Chlorine, even when used at a low concentration, may

cause taste and odor defects in treated products (Kim et al., 1999).

B.- Mechanism of action

The mechanism of action of chlorine is not completely understood (Dychdala,
1991). It is suggested that hypochlorous acid is responsible for the antimicrobial potency
of sodium hypochlorite:

NaClO + H20 —> Na+ + OCl' + H20
NaClO + H20 —) NaOH + HOCL
HOCl <:> H+ + OCl'

The dissociation of hypochlorous acid, which depends on the pH and equilibrium
between HOCL and OCL', is maintained even though HOCL is constantly consumed
through its germicidal function (Dychdala, 1991). Several theories have been proposed to
explain the mechanistic action of chlorine. It is generally believed that chlorine inhibits
essential c ell e nzyme s ystems through 0 xidation (Dychdala, 1 991; A lasri et a 1., l 992).
HOCl liberates nascent oxygen, which in turn, combines with components of cytoplasm

to destroy the cell. According to another theory, chlorine destroys bacteria by combining

58

with cell membrane proteins, forming N-chloro compounds, which change the cell

membranes to allow the cell contents to diffuse outward (Dychdala, 1991).

C.- Antimicrobial performance

Low concentrations of chlorine are used to sanitize fresh produce. However,
chlorine has a limited effectiveness in killing bacteria on fruit and vegetables surfaces due
to its inactivation by other organic materials (Xu, 1999; Mari et al., 1999), with maximum
reductions of 2 - 3 logs being reported (Park et al.,1991; Sapers, 1998; Xu, 1999).

Chlorine solutions prepared by adding appropriate amounts of 5% sodium
hypochlorite stock solution to 0.05 M potassium phosphate buffer (pH 6.8) were used at
21 °C, by Jaquette et al. (1996), to reduce populations of Salmonella Stanley on alfalfa
seeds. Dipping alfalfa seeds containing an initial population of 339 CFU/g for 10
minutes in a 100 ppm chlorine solution reduced the population to 197 CFU/g. Dipping in
290 ppm chlorine further reduced the population to 99 CFU/g. Dipping in 480 ppm
caused a reduction to 64 CFU/ g and a 1,010 ppm treatment reduced the microbial load to
37 CFU/g which means that no further appreciable reductions were found between 290
and 1,010 ppm chlorine. Jaquette et al. (1996) needed to use concentrations containing
2,040 and 3,990 ppm flee chlorine to d ecrease a Salmonella Stanley p opulation o f 6 5
CFU/ g to an undetectable level when analyzed by direct plating. However, these samples
were not enriched. Jaquette et al. (1996) also reported a 1995 personal communication by
D. Caudill stating that treatment of alfalfa seeds with up to 5,000 ppm chlorine did not
substantially reduce seed viability. J aquette et al. (1996) have suggested a 2,000 to 4,000

ppm chlorine treatment to reduce Salmonella populations on alfalfa seeds while not

59

adversely affecting germination. However, these authors also warned that because of the
dramatic increase which occurs during sprouting, even 2,000 to 4,000 ppm chlorine can
not guarantee that sprouts will be free of Salmonella.

Beuchat (1997) reported that Salmonella populations decreased from 3.9 logs to
<1 CFU/g after soaking seeds for 30 seconds in 1,800 ppm Ca(ClO); or 2,000 ppm
NaClO. Piemas and Guiraud (1997) found that adding active chlorine as sodium
hypochlorite to the wash water had little effect on the bacterial load of rice seeds until
1000 ppm, when aerobic plate counts were reduced by 2 to 3 logs over 20 minutes.
Weissinger and Beuchat (2000) reported a 0.33 log reduction in Salmonella, alter a 10
minute exposure to 200 ppm NaClO and a 0.72 log reduction using 2,000 ppm of NaClO.
These authors indicated that chemical sanitizers, other than 20,000 ppm of chlorine, can
be used to achieve similar reductions in populations of Salmonella without reducing the
germination rate.

In the multi-state, February-April, 2001 Salmonella kottbus outbreak, the grower
indicated that heated seeds were subjected to a 2,000 ppm sodium hypochlorite soak for
15 minutes The outbreak suggests that the aforementioned technique was inadequate to

eliminate Salmonella from the seeds (Mohle-Boetani et al., 2002).

60

2.3.2.- TSUNAMI / PEROXYACETIC ACID
A.- General characteristics

TsunamiTM is the brand name of a commercial sanitizing solution that contains a
combination 0 f p eracetic acid and hydrogen peroxide. This product is mainly used as
sanitizer and oxidizing agent.

Peracetic acid (PAA) shows better stability than chlorine dioxide and acts faster as
a biocide with its action not pH dependent. Low temperatures decrease the sanitizing
effectiveness of PAA (Mari et a1 1999). The toxicity level of PAA is lower than C102
with PAA exhibiting an LDso in rats of 1,540 mg/kg. The corrosiveness of PAA on
stainless steel can be alleviated by using a commercial formulation containing a lower

concentration of PAA without sulfuric acid (Mari et al., 1999).

B.- Mechanism of action

The mechanism of action of peracetic acid is covered in more detail in section
2.3.6.3. After contact with organic substrates, peracetic acid decomposes to yield oxygen
and acetic acid (Doores, 1983). The antibacterial action of acetic acid is partially due to
the lowering of pH below the optimum level for growth. Indeed, proteins, nucleic acids,

phospholipids can be structurally altered by pH changes (Doores, 1983).

C.- Antimicrobial performance

Various concentrations of PAA were tested by Mari et a1 (1999) for their

fungistatic effect on fruits and vegetables and may be used as references to evaluate the

61

bacteriostatic effect of PAA. The nature and pH of the fruit surface influence the
decomposition rate 0 f P AA and m ay e xplain differences observed in the antimicrobial
action of PAA (Mari et a1. 1999).

Two concentrations of TsunamiTM (40 and 80 ppm) were applied to alfalfa seeds
as 20 minute soaking treatments and also sprayed at regular intervals during sprouting
and subsequent storage of the sprouts. Using 80 ppm Tsunami”, a 1.61 log reduction in
numbers of E. coli 0157:H7 was observed after a 20 minute soak in 80 ppm Tsunami”.
The antimicrobial action of T sunamiTM 40 or 80 ppm did not lead to reductions >1 log.
Thus, treatment with TsunamiTM can not guarantee the safety of sprouts (Taormina and

Beuchat, 1999a,b).

2.3.3.- VEGI CLEANTM/ ACID-ANIONIC SURFACTANT
A.- General characteristics

Vegi-CleanTM is the brand name for a chemical sanitizer manufactured by
Microcide, Inc. (Detroit, MI) under US Patent # 5,143,720 and 5,280,042. This highly
water soluble white powder is intended as a sanitizer for fruits and vegetables. Active
ingredients in Vegi-CleanTM include citric acid, sodium acid phosphate and sodium
dodecyl benzene sulfonate with the latter acting as an anionic surfactant. Acid-anionic
surfactants combine the advantages of strong bactericidal action and low toxicity. The
antimicrobial properties of acid-anionic surfactants are better expressed at low pH (pH 2

to 3) (Dychdala and Lopes, 1991).

62

B.- Mechanism of action

The mechanism of action of acid-anionic surfactants has not yet been fully
elucidated (Dychdala and Lopes, 1991). However, these sanitizers may function by
inhibiting key enzymes, disorganizing the cell membrane, interrupting cellular transport

and/or denaturating cellular proteins (Dychdala and Lopes, 1991 ).

C.- Antimicrobial performance

The efficacy of 1 and 2% Vegi-CleanTM during sprouting of alfalfa seeds and
storage of the sprouts was studied by Taormina and Beuchat (1999b). Seeds were soaked
for 20 minutes in Vegi-CleanTM which was, then, sprayed at regular intervals on the
germinating and growing sprouts. Although populations of E. coli 0157:H7 decreased
0.67 logs afier spraying, this treatment was unable to eliminate the pathogen. Other than
the aforementioned germination step, spraying of Vegi-CleanTM during sprout production

and storage failed to significantly reduce the pathogen load.

2.3.4.- COPPER ION SOLUTION
A.- General characteristics

Copper is well known, for centuries, for its antimicrobial properties (Yeager,
1991). Copper ions generated through an electrolytic process can be dispersed into a
circulating water stream with this copper ion solution intended as a disinfectant. As
stated by copper ion system manufacturers, copper ion water is environmentally safe, user

fi'iendly and cost effective. These previously mentioned systems are also registered, as

63

meeting all requirements, with the Enviromnental Protection Agency (Superior Water

Solutions, Inc., advertising booklet).

B.- Mechanism of action

Copper ion water is believed to form electrostatic bonds between positively
charged copper ions and negatively charged sites on the bacterial cell surface. Reactions
at the cell surface allow the metal ion to penetrate the cell membrane and induce copper
toxicity (Superior Water Solutions Inc., advertising booklet).

In 1989, Yayha et al. acknowledged that the bactericidal mechanism of copper ion
was not fully understood. At that time, copper ion was assumed to bind to the sulfhydril
groups of respiratory enzymes and therefore, impair respiratory activity. It is also
believed that copper, like other metals, can take up key functional thiol groups from
enzymes (Lukens, 1991).

Years later, these assumptions have been supported by Riggle and Kunamoto
(2000) who reported that copper, an essential cofactor for enzymes, is implicated in
respiration, destruction of free radicals, iron homeostasis and neurological development.
Nevertheless, copper, at excess levels can induce a high toxicity by generating reactive
oxygen species via the Fenton reaction, disrupting metal ion binding and homeostasis and
binding macromolecules, such as proteins. Like other prokaryotes, bacteria resist copper
toxicity by reducing influx and/or utilizing efflux mechanisms by way of ATPase

transporters to control intracellular copper levels.

64

C.- Antimicrobial performance

As a bactericide, copper ion acts slower than chlorine. However, copper appears
to injure coliforrns more effectively than chlorine, requiring longer recovery times (Yayha
et al., 1989). Concentrations of 250 ug/L can induce 90% injury in E. coli, in less than 24
hours at 4 °C with more rapid injury morning at room temperature. (Yayha et al., 1989).
Landeen et al. (1989) reported a 3.3 log reduction in E. coli after a 1 hour exposure to 302
jig/L of copper ions. The same authors also reported that a contact time of at least 24
hours was necessary for a solution containing 400 rig/L and 40 rig/L of copper and silver
ion, respectively, to achieve a 3 log reduction in numbers of Legionella pneumophila. In
contrast, a solution of 0.4 mg/L of free chlorine was able to achieve a 2.6 log reduction in
1.5 minutes.

No studies assessing the efficacy of copper ion water solution for seed or sprout
disinfection have appeared in the literature. Nevertheless, Rodrick and Hultstrand in a
November 1998 correspondence to Superior Aqua Enterprises Inc., acknowledged a 2 log
reduction in total surface bacteria and at least a 1 log reduction in yeasts and molds on
tomatoes, after a 5 minute exposure to a copper ion concentration of 0.5 - 1.0 mg /L.
Superior Water Solutions Inc. claims reductions of ~ 3.5 logs for microorganisms on raw
fruits and vegetables when their Superior Aqua Agriculture System was used in fruit and
vegetable packing stations. A letter by Debra A. Sanders to James Mulha of Superior
Aqua Enterprises Inc. reports an experiment involving the washing of scallions
containing 1 x 105 CFU/g of E. coli. The scallions were dipped in an aqueous solution 1

ppm copper ion produced by a Superior Aqua Agricultural System installed on a

65

recirculated tank. The microbial load fell to less than 100 CFU/g which means
approximately a 3.5 log reduction.

Yayha et al.(1989) reported a reduction of only 0.12 log for E.coli in an aqueous
model system after 6 minutes of exposure to a copper-silver ion solution having a
concentration of 433ug/L : 43ug/L. In a similar model system, these authors observed a
2.8 log reduction in E.coli, after 0.5 minutes, using 0.20 mg/ L of flee chlorine and a 3.5
log decrease in 0.5 minutes when the previously mentioned concentration of copper-silver
ion and flee chlorine were combined. Thus, the combination treatment decreased E. coli
populations an additional 0.7 log. Inactivation by combined copper-silver ion is relatively
slow compared with to flee chlorine (Y ahya et al., 1989). However, when these metal
ions are added to low levels of flee chlorine, inactivation rates for indicator organisms,
usually used to judge water quality were greater than those for flee chlorine alone, at
comparable levels. In the letter previously mentioned, Debra Sanders also suggested that
the use of chlorine in conjunction with Superior Aqua Agricultural System achieved
additional reductions in microorganisms, with the work of Landeen let a1. (1989)
supporting this observation. With the aqueous model system previously mentioned, these
authors reported a 3.7 log reduction in Legionella pneumophila after 1.5 minutes when
0.4 mg/L of flee chlorine was combined with a 400 ug and 40 ug copper and silver ion
solution, compared to a reduction of 2.6 log using flee chlorine alone and 3 logs, using

copper and silver ion alone.

66

2.3.5.- SONICATION
A.- Characterization of the technique

Sonication is a physical technique involving the use of ultrasound. Shukla (1992)
and Lillard (1994) indicated that the active force in microwave ultrasonics (sound waves
pitched above the level of human hearing at 16 kHz or 16,000 cycles/sec) is mechanical
as opposed to heat in microwave heating. Large stresses, strains and high shear forces are
induced during cavitation when high frequency vibrations result in alternate compressions
and expansions of microscopic bubbles which implode violently, releasing large amounts

of energy and generating high temperatures and pressures.

B.- Mechanism of action
Cavitation is the mechanical effect responsible for the destruction of bacterial
cells. The magnitude of ultrasonic waves is sufficiently high to produce protein
breakdown and hydrolysis, simple cell lysis and protein particulation (Shukla, 1992).
Sonication can be used to break up bacterial cell clumps and to dislodge bacteria
flom solid surfaces and possibly flom the surface of fluits and vegetables. Sonication at
higher m agnitudes p lays a m ajor role in disrupting the b acterial c ell wall. Hence, the

aforementioned effects may help to improve the usefulness of sanitizers.

C.- Antimicrobial performances of sonication
Lillard (1993) showed that bactericides lethal to salmonellae in processing water,
do not access bacteria that are firmly attached or entrapped. Bacteria are not easily

removed flom poultry skin. Sonication of poultry skin for 15 or 30 minutes in a chlorine

67

solution containing 0.5 ppm free residual c hlorine increased the n umbers o f d islodged
bacteria and reduced the microbial load by 2.44 to 3.93 logs. This author also reported
that ultrasound in combination with chlorine is more effective in reducing bacterial
counts than either ultrasound or chlorine alone.

Sonication was used by Walker et al. (1998) in combination with other
techniques to recover bacteria flom peas and beans. Sonication flom a 40 kHz ultrasonic
generator also was used by Burleson et al. (1975) alone or in combination with ozone or
oxygen against Salmonella Typhimurium and enteropathogenic E. coli 0126:Bl6
suspended in phosphate buffer solution or in a secondary effluent flom a wastewater
treatment plant.

Lillard (1993) reported experiments involving the use of sonication to reduce
microbial populations in milk. Cell destruction occured at high flequencies, whereas
increases in total counts were observed at low flequencies and were probably due to
declumping of bacteria normally found in milk. Ultrasonic waves (80 kHz/sec) were

reported to remove most bacteria flom milk fihns on metal surfaces.

2.3.6.- NOVEL FDA-APPROVED FATTY ACID-BASED SANITIZER
A.- Characterization of the product

The major antimicrobial product examined in this study is a novel F DA-approved
sanitizer concentrate which is diluted 1:200 in water so as to contain 250 ppm
peroxyacid, 1000 ppm fatty acid (caprylic and capric acids), 500 ppm glycerol

monolaurate and 1000 ppm lactic acid as active ingredients.

68

This fatty acid-based sanitizer contains peroxyacetic acid conjugated to
peroxycaprylic and peroxycapric acids (Guthery, 2001). Peracetic acid is bactericidal at
fairly high concentrations, generally greater than 100 ppm. Similarly, peroxyfatty acids
are also biocidal at concentrations greater than 200 ppm (0akes et al., 1993). The
combination of these acids produces a synergistic effect, providing a much more potent
biocide than that obtained when these components are used separately and offers the
unique advantage of having antimicrobial or biocidal activity at substantially lower
concentrations (0akes et al., 1993). These lower concentrations minimize cost, odor and
potential toxic effects to the user. An effective antimicrobial solution is formed when the
concentrate is diluted in water at a pH of 2 to 8. At pH < 5, peroxyacids, including
peroxyfatty acids, are very potent biocides at low levels. This solution may be used at
temperatures ranging flom 4 to 60 °C (0akes et al., 1993).

Peroxyacetic acid is obtained after reacting glacial acetic acid with hydrogen
peroxide (50%) overnight, in the d ark (Guthery, 2 001). I t is formed w hen an o xygen
molecule is bound to the carboxyl atom of acetic acid (Doores, 1983). Peroxyacetic acid
is reportedly superior to other forms used in this type of sanitizer (0akes et al., 1993).
Acetic acid is a monocarboxylic acid with a pungent odor and taste which limits its use.
It is the principal component of vinegar and is highly soluble in water (Doores, 1983).
Acetic acid is generally regarded as safe (GRAS) for miscellaneous and general purpose
use (21 CFR 182.1005). The acceptable daily intake for human consumption is not
limited (Doores, 1983). Peracetic acid is effective as a biocide in aqueous solutions. It is

not time dependent, making it a fast disinfectant (Alasri et al., 1992).

69

Peroxyfatty acids are formed by reacting hydrogen peroxide (50% v/v) with a
mixture of 60% caprylic acid and 40% capric acid, commercially available under the
brand name Emery 658 (Guthery, 2001). C aprylic acid (octanoic, C3) and capric acid
(decanoic, Cm) are structurally represented by the formula Rl-C03H. These linear,
monoperoxy aliphatic fatty acids are particularly appropriate and preferred among other
eligible fatty acids for this type of sanitizer (0akes et al., 1993). Caprylic acid is a
colorless liquid which has a slightly unpleasant o dor and a b urning rancid taste. I t is
slightly soluble in water and has a pka of 4.89. The mouse LDso mouse is estimated at 600
mg/kg of body weight after intravenous administration. It is approved as a GRAS
substance for miscellaneous and general purpose use. Caprylic acid may be used to flavor
many foods at levels ranging flom 0.001% to 0.16% (21 CFR 184.1025). It is also
approved as an antimicrobial agent for indirect use in cheese wraps (21 CFR 186.1025)
(Doores, 1983).

The diluted sanitizer contains an equilibrium mixture of hydrogen peroxide,
peroxyacetic acid, peroxycaprylic acid, peroxycapric acid, flee acetic acid, flee caprylic
and flee capric acid and water. Moreover, the sanitizer also contains glycerol
monolaurate, lactic acid and some additional function enhancing components with
sulfuric acid added as a catalyst (Guthery, 2002).

Hydrogen peroxide, generally used as an antiseptic in dilute solutions (Baldry,
1983; Alasri et al.,1992) is GRAS-approved for use at concentrations of 0.05% as an
antimicrobial agent in cheese-making and whey processing (Davidson et al. 1983;

Venkitanarayanan et al., 1999). However, the actual concentration of hydrogen peroxide

7O

present in this sanitizer, as a result of the equilibrium reaction, falls below the level where
microbicidal action could be expected (Guthery 2002).

Glycerol monolaurate, also called monolaurin and sold under the brand name
Lauricidin® (Lauricidin, Inc., 0kemos, MI), is the monoacyl derivative resulting from
esterification of lauric acid to glycerol. This food-grade monoester is approved in the
United States as a food emulsifier by the FDA (21 CFR GRAS 182.4505). In addition to
its emulsification properties, glycerol monolaurate also possess antimicrobial activity
which has been extensively investigated (Oh and Marshall 1992, 1993; Kabara, 1972,
1975, 1979; Kabara et al., 1977). The addition of an acidulant can increase the
antimicrobial spectrum and activity of glycerol monolaurate (Oh and Marshall, 1992).

Lactic acid (pka, 3.8) is a hygroscopic, syrupy liquid having a moderately strong
acid taste. It is one of the most widely distributed acids in nature as it is the primary acid
produced during natural fermentation (Doores, 1983 and 1993). L actic acid is GRAS
approved (21 CFR 182.1061) for miscellaneous and general purpose (Doores, 1983,
1993; Anderson and Marshall,l990; Greer and Dilts, 1992). The oral LD50 for rats and
guinea pigs is, respectively, 3,730 and 1,810 mg/kg of body weight. The acceptable
human daily intake is unlimited (Doores, 1983). Lactic acid is inhibitory to various
pathogenic and spoilage organisms (Anderson and Marshall,1990; Greer and Dilts, 1992).
The antimicrobial activity of organic acids can be increased or potentiated when
combined with other food preservatives (Bogh-Serensen 1994; Venkitanayaranan et al.,
1999; Marshall and Kim, 1996).

Other components can be added to this sanitizer, such as hydrotrope coupling

agents, stabilizers and chelating agents (0akes et al., 1993). The hydrotrope solubilizer

71

acts to solubilize the fatty acids in both the concentrate and the ready-to-use solution
(0akes et al., 1993). Propylene glycol is present as a solvent in this sanitizer. Although
propylene glycol can exhibit antimicrobial properties at concentrations >70%, the
concentration used in this sanitizer (40%) is too low for this purpose (Guthery, 2002,
personal communication).

The components of this F DA-approved fatty acid-based sanitizer are characterized
as GRAS. Fatty acids and their esters are naturally occurring substances in foods and are
non-toxic, unless used at “heroic doses” (sic. Kabara, 1975), that are not applicable in
food processing (Freese et al., 1973; Kabara, 1975, 1984; Shibasaki and Kato, 1978; Oh
and Marshall, 1993). Organic acids are metabolized by the body primarily through lipid
oxidation and via the tricarboxylic acid cycle (Doores, 1983). Therefore, they offer a
considerable advantage over other types of chemical sanitizers to control microorganisms.
The adverse impact of chemical sanitizers on humans and the environment has been often
overlooked. Nowadays, safety and environmental concerns surrounding chemical
products are being raised by governments, industry and consumers. The concern is about
being able to control microbial pathogens while minimizing the side effects created by
these powerful agents which have become more a part of the problem than the
solution(e.g., resistance of microorganisms to germicides) (Kabara, 1984). Some
chemical agents are being seriously considered for removal flom the market because of
their potential or perceived toxic or carcinogenic effects (Oh and Marshall, 1993). The
solution to this problem is to consider natural, non-toxic, agents, such as fatty acids, that

inactivate bacteria and fungi without harming humans and the environment.

72

B.- Mechanism of action

Peroxyfatty acids have the ability to form peroxides and other flee radicals that
inhibit bacterial growth (Kabara, 1979; Hinton and Ingram, 2000). P eroxyacetic acid,
like other peroxides and oxidizing agents, is assumed to oxidize sensitive sulfliydryl and
sulfur bonds in proteins, enzymes and other metabolites. It is suggested that peroxyacetic
acid disrupts the chemiosmotic function of the cytoplasmic membrane and transport
through dissociation or rupture of cell walls (Baldry and Fraser, 1988; Block, 1991).

Other than sulfirric acid, most of the remaining components of this sanitizer are
weak organic acids. In aqueous solution, weak acids are only slightly ionized and do not
readily give up their proton(s) to water (Doores, 1983). At a pH lower than the pka, the
equilibrium reaction shifts toward a higher concentration of undissociated acid. Based on
experiments reported by Doores (1983) comparing the antimicrobial performance of weak
and strong acids at higher pH, toxicity was due, not to hydrogen ion alone, but was a
function of the undissociated molecule. Bacterial membranes are less permeable to
charged molecules than uncharged molecules (Doores, 1983). Therefore, the inhibition
by organic acids used as antimicrobial agents increases with decreasing pH, in agreement
with the pka values (Freese et al., 1973; Doores, 1983). According to Galbraith and
Miller (1973a,b,c) and Oh and Marshall (1992, 1993), lowering the pH of the suspending
medium increased fatty acid uptake by Bacillus megaterium and reduced the interfacial
tension at the bacterial lipid membrane-aqueous medium interface. Hunter and Segel
(1973) and Doores (1983) suggest that weak acids at or below their pk, could discharge
the proton gradient and ionize within the cell to acidify the interior. It was postulated that

the rate of proton leakage into a cell versus proton ejection would determine inhibition

73

(Freese et al., 1973; Doores, 1983). Peracetic acid, upon contact with organic substrates,
decomposes to yield oxygen and acetic acid (Doores, 1983). Antibacterial action of
acetic acid is partially due to the lowering of pH below the optimum level for growth.
The same is also true for lactic acid. Indeed, proteins, nucleic acids and phospholipids
can be structurally altered by pH changes (Doores, 1983).

In general, fatty acids function as surface-active anionic detergents (Kabara et al.,
1972). Fatty acids uncouple both substrate transport and oxidative phosphorylation flom
the electron transport system (Freese et al., 1973). They act by disrupting the bacterial
cell membrane and lysing protoplasts as evidenced by the leakage of 260nm-absorbing
material, protein and other internal metabolites flom both bacteria and protoplasts. Thus,
fatty acids prevent bacterial growth by modifying cell membrane permeability which
leads to changes and/or inhibition in oxygen, aminoacid, nucleic acid, organic acid,
phosphate and other substrate molecule uptake (Freese et al., 1973; Kabara, 1979; Oh and
Marshall, 1992, 1993; Galbraith and Miller, 1973 a,b,c; Doores,l983; Hinton and Ingram,
2000). Although saturated fatty acids inhibit cellular oxygen consumption, they do not
inhibit NADH oxidation by isolated membranes, that is by the cytochrome-linked
electron transport system. Therefore, inhibition of oxygen consumption in whole cells
must result flom the deficiency of compounds that donate electrons to the electron
transport chain. This deficiency results flom the inability to transport necessary
substrates into cells (Freese et al., 1973). Inhibition of cellular uptake, however, does not
necessarily prove that transport itself is inhibited; it may merely reflect inhibition of
metabolism with unmetabolized compounds preventing their own uptake (Freese et al.,

1973)

74

long chain fatty acids show greater antibacterial activity against Gram positive
organisms (Galbraith and Miller, 1973a; Sheu and Freese,1973; Kabara et al., 1977).
Except for c ertain s hort c hains, gram-negative organisms are g enerally 11 ot affected by
fatty acids (Sheu and Freese,1973; Kabara et al., 1977). When compared to the
concentrations of short chain fatty acids up to C5 needed for inhibition of E. coli, twice
the concentration of C3 and about 50 times the concentration of C10 is required with no
inhibition observed for longer chain fatty acids (Freese et al., 1973). As a possible
explaination, the lipopolysaccharide layer that typically surrounds the wall of gram-
negative organisms could screen out the larger chain fatty acids (Freese et al.,1973,
Doores, 1983). Another theory proposes that gram-negative organisms can rapidly
metabolize these fatty acids, thereby preventing their accumulation within the cell
(Kabara et a1. 1972; Freese et al., 1973; Doores, 1983 and 1993). The concentration of
fatty acids, up to C3 that completely inhibits the growth of E. coli does not reduce the
amount of ATP/A600 measured after 20 or 40 minutes of incubation. Apparently, in E.
coli, some other factor acts to limit microbial viability (Freese et al., 1973).

Vadehra et a1. (1985) and Oh and Marshall (1992) reported that glycerol
monolaurate causes extensive leakage of 260 nm-absorbing intracellular proteins flom
Staphylococcus aureus. Lower pH may also increase the uptake of glycerol monolaurate

(Oh and Marshall, 1993).

75

C.- Antimicrobial performance

Peer-reviewed publications regarding the performance of this novel FDA-
approved fatty acid-based sanitizer are not yet available. A patent process is currently
ongoing for this disinfectant.

Peracetic acid exhibits excellent antimicrobial properties, especially under acidic
conditions. It is less effective at pH 8 because of its pk, of 8.2 (Baldry, 1983). When
peracetic acid was tested against populations of 106 CFU/ml of Pseudomonas aeruginosa
ATCC 15442, Klebsiella pneumoniae ATCC 4352, Streptococcus faecalis 10541 or
Staphylococcus aureus ATCC 538 in aqueous solutions, 66 umol/L failed to produce a
complete kill within 30 minutes; whereas 330 umol/L and 1.3 mmol/L were effective
aflerlO and < 1 minutes, respectively (Baldry, 1983).

Acetic acid is more effective in limiting bacterial and yeast growth than mold
grth (Doores, 1983). As gram-negative bacteria were more inhibited than gram-
positive bacteria at pH 6 and 5, acetic acid may effectively select for gram-positive
organisms (Doores, 1 983).

Peroxycaprylic and peroxycapric acids reportedly provide antibacterial activity
against a wide variety of gram-positive (e. g., Staphylococcus aureus) and gram-negative
(e.g., Escherichia coli) bacteria as well as yeasts, molds and bacterial spores (0akes et al.,
1993). The present blend of peroxyacetic, peroxycaprylic and peroxycapric acids reduced
microbial populations 5 logs after 30 seconds of exposure to concentrations ranging
between 100 ppm and 20 ppm of the peracid blend (0akes et al., 1993).

Inhibition by fatty acids generally increases about 10-fold when the concentration

is increased by a factor of 10, indicating an essentially linear concentration dependence

76

(Freese et al.1973). As an anionic surfactant, fatty acids are less potent at physiological
pH values (Kabara et al., 1972). The inhibitory action of fatty acids can be largely
diminished by the presence of reversing agents including Ca2+ and Mg”. The reversing
action of other cations such as Fe3+ is also a possibility (Galbraith and Miller, 1973a).
These authors studied the effect of cations on bactericidal activity of lauric and linoleic
acid against fatty acid sensitive strains of Bacillus megaterium and Micrococcus
lysodeictikus at p H 7 .0. W ith lauric acid, c onsiderable reversal o f i nhibition o ccurred
with Ba2+ and Sr” at equimolar concentrations, but Fe“ and Sn2+ were usually
ineffective.

When tested against various gram-positive and gram-negative organisms, caprylic
acid was not inhibitory at concentrations up to 7.8 umol/mL (Kabara et al.1972; Doores,
1983). The antimicrobial performance of caprylic acid at pH 5.0 and 6.0 appears to
support the observation that, whereas acids with fewer than 7 carbons are more effective
at lower pH, acids with 8 to 12 carbons are more effective at neutrality or above (Doores,
1983)

Another fatty acid, oleic acid at concentrations of 2, 4, 6, 8 and 10% (w/v) was
reported to decrease the bacterial population on poultry skin (Hinton and Ingram, 2000).
Listeria monocytogenes was least resistant to the antibacterial activity in vitro while
Escherichia coli showed higher resistance. Salmonella Typhimurium was most resistant
to oleic acid (Hinton and Ingram, 2000).

When used at a concentration of 0.1%, hydrogen peroxide alone was reportedly
ineffective (V enkitanarayanan et al.,1999) against E. coli 0157:H7. However, Alasri et

al. (1992) reported a 5 log reduction in numbers of E. coli ATCC 8739 in an aqueous

77

model system afler 5 minutes of contact with 0.625 % hydrogen peroxide. A synergistic
biocidal effect (5 log reduction in 5 minutes) was also found between 391 ppm of
hydrogen peroxide and 2.5 ppm of peroxyacetic acid. The degree of synergistic action
was similar on the species studied : E. coli ATCC 8739, Pseudomonas aeruginosa ATCC
9027 and Staphylococcus aureus ATCC 6538 P (Alasri et al., 1992).

Optimum antimicrobial activity was found for fatty acids and their corresponding
monoglycerides when the chain length was C12 (Kabara et al. 1977), such as for lauric
acid (Nawar, 1996). Esterification of a fatty acid to glycerol to form monoacyl
derivatives generally results in greater biocidal activity. M onolaurin, the most active
ester is indeed more potent than flee lauric acid (Kabara et al., 1972; Conley and Kabara,
1973; Kabara et al., 1977). The minimal inhibitory concentration (MIC) is defined as the
lowest concentration of a compound at which no macroscopic evidence of growth is
observed after an appropriate period of incubation (Kabara et a1. 1972; Kabara et al.,
1977). Beuchat (1980) demonstrated that the MIC at which inhibition of V.
parahaemolyticus could be detected was lower for glycerol monolaurate (Sag/ml) than
for lauric (60 ,ug/ml), capric (60 ug/ml) or caprylic acid (100 rig/ml) in laboratory media
at pH 6.7.

According to Kabara (1979) and Oh and Marshall (1992), glycerol monolaurate
has a broad spectrum of antimicrobial activity in culture media against gram—positive
microorganisms. Kato and Shibasaki (1976) and Oh and Marshall (1992) showed that
glycerol monolaurate is also effective against Gram-negative bacteria in culture media

containing citric and polyphosphoric acid. Kabara (1979), Kato (1981) and Oh and

78

Marshall (1992) also reported on the antimicrobial effects 0 f glycerol fatty acid esters
against pathogenic and spoilage organisms.

Oh and Marshall (1992, 1993) demonstrated that the antimicrobial effect of
glycerol monolaurate on L. monocytogenes is strongly influenced by pH, temperature and
their interaction. However, differences in susceptibility between four different strains of
L. monocytogenes treated with glycerol monolaurate were negligible. By reducing the pH
flom 7.0 to 5.0, the minimum inhibitory concentration GVIIC) of glycerol monolaurate
decreased flom 10 to 3 ug/mL for three of four strains. The contribution of temperature
to glycerol monolaurate effectiveness showed that inactivation occurred more rapidly at
higher temperatures (Oh and Marshall, 1993). Listeria was rapidly inactivated by
glycerol monolaurate at lower pH values and higher temperatures (Oh and Marshall,
1993). These authors also assumed that, in addition to pH and temperature, other
environmental factors, such as initial inoculum level, competing microflora and food
composition may influence the effectiveness of glycerol monolaurate. Glycerol
monolaurate was more effective than sorbic acid and sodium benzoate against Vibrio
parahaemolyticus with MIC values of 5, 70 and 300 ug/mL, respectively (Beuchat,1980;
Oh and Marshall, 1992). At a concentration of 0.005%, glycerol monolaurate was unable
to reduce populations of E. coli 0157:H7 suspended in 0.1% peptone by 5 log CFU/ml
(V enkitanayaranan et al., 1999).

Lactic acid is inhibitory to a wide range of microorganisms. However
Venkitanarayanan et al. (1999) emphasized that E. coli 0157:H7 is unusually acid
tolerant. Conner and Kotrola (1995) and Venkitanarayanan et al.(1999) also reported that

E. coli 0157:H7 was able to survive up to 56 days in Tryptic Soy Broth acidified to pH

79

4.7 with lactic acid. Abdul-Raouf et a1. (1993) determined that E. coli 0157:H7 survived
well in b eef s lurries c ontaining lactic acid. L actic acid at l .5% w as unable to reduce
populations of E.coli 0157:H7 suspended in 0.1% peptone by 5 log CFU/mL
(Venkitanayaranan et al., 1999). Lactic acid showed marked inhibitory capacity against
Mycobacterium tuberculosis which increased as the pH decreased (Doores, 1983).

In a model system, evaluating the effectiveness of 1.5% lactic acid plus 0.005%
glycerol monolaurate against E. coli 0157:H7 resulted in a microbial reduction of > 5.0

log CFU/mL in 20 rrrinutes at 22 °C (V enkitanarayanan et al., 1999).

80

3.- SYNERGISTIC EFFECTS BETWEEN COMMERCIAL CHEMICAL SANITIZERS
(CLOROXTM, TSUNAMITM, VEGI—CLEANTM) AND SONICATION OR COPPER IONS FOR
REDUCTION OF ESCHERICHIA COLI 0157:H7, SALMONELLA TYPHIMURIUM DT104 AND

LISTERIA MONOC YT OGENES ON INOCULATED ALFALFA SEEDS AND SPROUTS.

ABSTRACT

Alfalfa seeds and sprouts were inoculated to contain 106 to 108 CFU/g
Escherichia coli 0157:H7, Salmonella Typhimurium DT104 or Listeria monocytogenes
and exposed to the following sanitizers for 30 seconds to 10 minutes : CloroxTM (sodium
hypochlorite, 200 to 20,000 ppm), TsunamiTM (peroxyacetic acid / hydrogen peroxide, 80
and 800 ppm) or Vegi-CleanTM (anionic surfactant, 1%, 2%, 5%). Appropriate dilutions
in 0.1% peptone were spiral plated on Sorbitol Mc Conkey Agar (SMAC) or Cefixime
Tellurite Sorbitol McConkey Agar (CT-SMAC) for E. coli 0157:H7, Xylose Lysine
Desoxycholate agar (XLD) for S. Typhimurium DT104 and Modified Oxford Agar
(MOX) for L. monocytogenes. Only sodium hypochlorite ( > 1,000 ppm) was able to
reduce pathogens on sprouts by > 5 logs. Ultrasound (20 kHz) generated by a sonicating
water bath or copper ions generated by an electrolytic process and dispersed into a
circulating water stream to a concentration of 1 ppm, were tested alone or in combination
with the previous sanitizers to assess possible synergistic effects. No appreciable
differences in pathogen reduction were observed using either sonication or copper ions in

combination with the previous sanitizers.

81

3.1.- INTRODUCTION

Alfalfa sprouts have been incriminated in several countries as the source of
numerous outbreaks involving E. coli 0157:H7 (Taormina and Beuchat, 1999a,b) and
Salmonella sp. (Mahon et al., 1997; Glynn, 1997; Taormina and Beuchat, 1999a,b).

Research efforts have focused on identifying various strategies that can reduce the
microbial load on alfalfa seeds by 5 logs while maintaining the germination rate at a
commercially acceptable level and being non toxic for consumers. Some of these methods
investigated include heat (J aquette et al., 1996), gamma irradiation (Rajkowski and
Thayer, 2000), volatile compounds (Park et al., 2000) and chemical sanitizers (Piemas
and Guiraud, 1997; Beuchat, 1997; Taormina and Beuchat, 1999a; Bari et al., 1999;
Weissinger and Beuchat, 2000). The antimicrobial efficacy of Clorox” (sodium
hypochlorite) for reducing pathogens on alfalfa seeds and sprouts has been investigated
by Jacquette et al. (1996) and Weissinger and Beuchat (2000). Similarly, Taormina and
Beuchat (1999b) have studied the effect of Tsunami” (peroxyacetic acid / hydrogen
peroxide) and Vegi-Clean” (acid-anionic surfactant) in controlling microbial growth
during alfalfa seed sprouting. Although, these studies have expanded our knowledge
concerning pathogen inactivation, none of these bactericidal treatments will effectively
guarantee the microbiological safety of organoleptically acceptable alfalfa sprouts.
Therefore, much further work still remains.

Sonication is a cell disruption technique involving the use of ultrasound. Large
stresses and strains produced during cavitation create a mechanical effect that ultimately
destroys bacterial cells. The magnitudes of ultrasonic waves are sufficiently high to cause

protein breakdown, protein hydrolysis, simple cell lysis, protein particulation and even

82

high temperature b iocatalysis if the enzymes are resistant to ultrasonic waves (Shukla,
1992; Lillard, 1993). In addition to causing bacterial cell wall damage, sonication may
also help to declurnp and dislodge bacteria flom the surface of alfalfa seeds and sprouts,
and more readily expose these weakened cells to the harmful effects of chemical
sanitizers, thus leading to greater microbial reduction.

Copper has been long known for its anti-microbial properties (Yeager 1991).
Riggle and Kunamoto (2000) reported that copper, as an essential cofactor for many
enzymes, is vital to a wide range of biological processes including respiration, destruction
of free radicals, iron homeostasis. N evertheless, excess levels 0 f c opper can induce a
high toxicity by generating reactive oxygen species via the Fenton reaction, thereby
disrupting metal ion binding and homeostasis and binding macromolecules, such as
proteins. Like other prokaryotes, bacteria resist copper toxicity by reducing the influx
and/or utilizing efflux mechanisms by way of ATPase transporters to control intracellular
copper levels. According to Yahya (1989) and Sanders (correspondence to Superior
Water Solutions, Inc.), the combination of copper ion and sodium hypochlorite showed
stronger antimicrobial activity than either copper ion or sodium hypochlorite alone. Thus
copper ions will exert a certain level of toxicity towards bacterial pathogens on alfalfa
seeds and sprouts that might be extentuated by chemical sanitizers. A synergistic
antimicrobial effect is expected flom the combination of copper ion with Clorox TM,
Tsunami TM or Vegi-Clean T“. It is thought that the formation of electrostatic bonds
between positively charged ions and negatively charged sites on the microbes (Superior
Water Solutions, Inc.) promotes greater sanitizer contact which leads to enhanced

antimicrobial activity.

83

The objective of this study is to carry out an exploratory assessment of the various
antimicrobial combinations previously mentioned in order to identify some promising

schemes toward the 5 log reduction targeted.

3.2.- MATERIALS AND METHODS
Bacterial strains

Three Escherichia coli 0157:H7 strains (AR, AD 305 and AD 317) and 3 Listeria
monocytogenes strains (LM Brick silage, LM T3BL, LM F5027) were obtained flom Dr.
Catherine W. Donnelly (Dept. of Nutrition and Food Sciences, University of Vermont,
Burlington, VT). Three Salmonella Typhimurium DT104 strains (G1601, G1074 and
G10931) were obtained flom Dr. Peggy Hayes (Centers for Disease Control and
Prevention). Laboratory stock cultures were maintained at — 80 0C in Tryptic Soy Broth
(TSB, pH 7.3) (Difco Laboratories, Detroit, MI) containing 10% (v/v) glycerol. Individual
strains were separately activated by transferring a loop of the flozen stock culture into 9
ml of sterile TSB containing 0.6% (w/v) yeast extract (YE) (Difco) then incubated at
35°C for 18-24 hours. Thereafter, for each strain, 5 tubes containing 35 ml each of TSB-

YE were inoculated and similarly incubated before use.

84

Preparation of inoculum

The aforementioned cultures were harvested by centrifugation (Sorvall Super T21 ,
Newtown, CT) at 10,000 rpm for 15 minutes at 4 ° C, resuspended in 0.1 % peptone
(Difco) and combined in equal volumes to prepare the 3-strain inoculum cocktails

containing ~ 109 CFU/ml.

Inoculation of alfalfa seeds and sprouts

Alfalfa seeds used in this study (originating flom Australia and identified by the
batch serial # AUS / S / DM / 71) were purchased in bags of 25 kg flom Dan Caudill
(Louisville, KY).

Seeds were sprouted at room temperature (22 — 25 °C), in a Kitchen Crop
sprouting device manufactured by NK Lawn and Garden (Chattanooga, TN). According
to the manufacturer’s instructions, 10 to 15 g of seeds were layered on the plastic trays
and watered twice daily using tap water. The overall process took approximately 4 to 5
days. Then, the sprouts were rinsed several times in tap water and stored at 5 °C prior to
inoculation.

Alfalfa seeds and sprouts were inoculated so as to contain 105 to 107 CFU/g and
108 to 109 CFU/g, respectively. Seeds (500 g) and sprouts (300 g) were separately
immersed in a 525 ml 3-strain cocktail of E. coli 0157:H7, S. Typhimurium DT104 or L.
monocytogenes and gently agitated in this mixture for approximately 10 minutes. The
seeds were dried for 4 hours under laminar flow in a biosafety cabinet and stored at room

temperature. The sprouts were strained and stored at 5 °C. Seeds and sprouts were stored

85

at least 24 hours before use in order to allow for adequate pathogen attachment. Unused

inoculated seeds and sprouts were discarded after 5 days.

Inactivation of pathogens on alfalfa seeds and sprouts
Chemical sanitizers

The following chemical sanitizers were investigated and compared to a water
control:

1.- Clorox” (The Clorox Company, Oakland, CA), (sodium hypochlorite as active
ingredient) at concentrations of 200, 1000, 2000, 10,000 and 20,000 ppm chlorine.

2.- Tsunami” (Ecolab, Mendota Heights, MN), ( peracetic acid and hydrogen peroxide
(PAH) as active ingredients) at concentrations of 80 and 800 ppm.

3.- Vegi-Clean” (Microcide, Inc., Detroit, MI, US. Patents # 5,143,720 and #
5,280,042), (citric acid, sodium acid phosphate and sodium dodecyl benzene sulfonate as
active ingredients) at concentrations of 1%, 2% and 5%.

Clorox” solutions were prepared by diluting the commercial concentrate with
sterile deionized water and adjusting the concentration with a chlorine test kit (La Motte
Chemical Products Co., Inc., Chestertown, M D). P reparation o f T sunami” s olutions,
according to label instructions, consisted of adding 47.8 III and 478 pl of sanitizer
concentrate to 100 ml of sterile deionized water to obtain concentrations of 80 and 800
ppm, respectively. To prepare the Vegi-Clean” 1, 2 and 5% experimental solutions, 1, 2
and 5 g of powdered product, respectively, were dissolved in 100 ml of sterile deionized

water. All sanitizer solutions were fleshly prepared just prior to use.

86

Sonication

A Fisher Scientific (Chicago, IL) FS 220 water bath sonicator was used to
generate ultrasound (20 kHz). Sterile 24 oz stomacher bags (Whirl—Pak, Nasco, USA)
containing 10 g of seeds or sprouts in 40 or 90 ml treatment solution, respectively, were
immersed in the water bath sonicator for 30 seconds to 10 minutes.
Copper ion water

Copper ions were generated through an electrolytic process (Superior Water
Solutions, Inc). A pilot plant-sized copper ion generator (Superior Water Systems, Inc.,
Fort Wayne, Indiana) dispersed copper ions into a recirculating water stream so as to
reach a concentration of 1 ppm. The copper ion concentration was determined prior to
treatment using a colorimetric copper ion test kit (model EC-20; La Motte Chemical
Products Co., Inc., Chestertown, MD), with this solution used as the disinfectant.
Hurdle approach treatment

Sonication and c0pper ion were combined with the aforementioned sanitizers in
order to increase the antimicrobial efficacy of the latter. When combined with copper
ion, the sanitizer working solutions were prepared using copper ion water (1 ppm) rather
than deionized water.
Experimental procedure

In order to assess the effect of treatment on microbial load of the pre-inoculated
seeds and sprouts, an experimental procedure, modified flom Taormina and Beuchat
(1999a) was adopted. Forty ml of sanitizer were added to a 24 oz. sterile stomacher bag
containing 10 g of inoculated seeds and manually agitated for 30 seconds, 1, 3, 5 or 10

minutes. When combined with sonication, the stomacher bag containing the seeds and

87

sanitizer was held in sonicating water bath for the same durations. After treatment, the
sanitizer was discarded and replaced by 40 ml of neutralizing buffer after which the
sample was homogenized at high speed in a stomacher (Model SD-45 Tekmar Co.,
Cincinnati, 0H)) for 2 minutes.

Alternatively, 90 ml of sanitizer were added to a sterile stomacher bag containing
10 g of inoculated sprouts and manually agitated during the chosen duration of treatment.
The sanitizer was discarded and replaced by 90 ml of appropriate neutralizing buffer with
the sample similarly homogenized at high speed for 2 minutes in a stomacher.

A water control was also prepared by replacing the sanitizer with a corresponding
amount of water and applying the procedure previously described for the seeds and the
sprouts. For calculating microbial load reduction, the inoculated seeds or sprouts were
analyzed by adding 40 ml or 90 ml of buffer, respectively, in a stomacher bag containing

a 10 g sample, followed by processing in a stomacher.

Microbial Analysis

In all cases, appropriate dilutions in 0.1% peptone were spiral-plated (Autoplate
4000, Spiral Biotech Inc., Bethesda, MD) on Sorbitol Mc Conkey Agar (SMAC) or
Cefixime Tellurite Sorbitol McConkey Agar (CT-SMAC) for E. coli 0157:H7, Xylose
Lysine Desoxycholate agar (XLD) for Salmonella Typhimurium DT 104, Modified
Oxford Agar (MOX) for Listeria monocytogenes and Plate Count Agar (PCA) for
Mesophilic Aerobic Bacteria with the latter counts being reported in Appendix A.

Neutralizing buffers were 0.1% peptone (Difco) for the inoculated seeds and the

water control; 0.1%, 1% and 5% sodium thiosulfate (JT Baker, Phillipsburg, NJ) for

88

Clorox ” 200 ppm, 1000 to 2000 ppm and 10,000 to 20,000 ppm, respectively, and 1%
and 5% peptone for Tsunami” 80 ppm and 800 ppm, respectively. The buffer for Vegi-
Clean” was prepared by dissolving lecithin (2,2g) (Sigma, St Louis, M 0), Tween 8 0
(15.5ml) (Sigma), and KH2P04 (4.6 g) (JT Baker, Phillipsburg, NJ) in 100 ml of sterile
deionized water. This stock solution was diluted 1:10, 1:5, 1:2 to neutralize
concentrations of 10,000, 20,000 and 50,000 ppm. The pH of these buffers were adjusted
to 7 .0 / 7.4 with sterile 0.1 N NaOH solution.

Reduction in the microbial load was calculated by subtracting the microbial count
after treatment flom the corresponding initial microbial count on the untreated inoculated

seeds. In absence of replications, statistical analysis was not applied to these results.

3.3.- RESULTS AND DISCUSSION
3.3.1.- WATER WASHING

A reduction in bacterial load on alfalfa seeds previously inoculated with
pathogens was always observed after rinsing with water. Reductions in E. coli 0157:H7
population averaged 0.32 logro CFU/ g on alfalfa seeds previously inoculated with E. coli
0157:H7 (Table 3.1). After washing alfalfa seeds previously inoculated with Salmonella,
the pathogen population fluctuated between an apparent increase of 0.23 log and a
reduction of 0.92 log 10 CFU/g (Table 3.2). These results are consistent with the work of
Piemas and Guiraud (1997) who reported that washing rice seeds with sterile water

decreased aerobic plate counts <1 log.

89

TABLE 3.1.- REDUCTION OF ESCHERICHIA COLI 0157:H7 (logro CFU/g) ON INOCULATED
ALFALFA SEEDS USING VARIOUS ANTIMICROBIAL TREATMENTS.

 

 

TREATMENT TIME
30 sec 1 min 3 min 5 min 10 min

Sonication alone 0.84 0.88 0.30
Water control "' 0.68 0.68 0.58
Clorox 200 ppm 1.36 1.57 0.60
Water control 0.68 0.68 0.58
Clorox 20,000 ppm 1.32 1.18 1.53 1.23 1.49
Water control 0.23 0.28 0.06 0.06 0.03
Clorox 200 ppm + sonication 1.91 1.56 1.15
Water control 0.68 0.68 0.58
Tsunami 80 ppm 0.91 0.61 1.11
Water control 0.68 0.68 0.58
Tsunami 800 ppm 0.46 0.50 0.62 0.73 0.63
Water control 0.23 0.28 0.06 0.06 0.03
Tsunami 80 ppm + sonication 1.91 1.79 1.28
Water control 0.68 0.68 0.58
Vegi-Clean 1% 1.60 1.41 1.24
Water control 0.68 0.68 0.58
Vegi-Clean 2% 0.44 0.66 0.71 0.57 1.06
Water control 0.23 0.28 0.06 0.06 0.03
Vegi-Clean 1% + sonication 1.51 1.70 1.60
Water control 0.68 0.68 0.58
Copper ion 1 ppm 0.52 0.29
Water control 0.68 0.68 0.58
Copper ion + sonication 1.07 0.86
Water control 0.68 0.68 0.58

 

"‘ All water control data in this table have to be compared to the treatment results reported on the row placed
above them

90

TABLE 3.2.- REDUCTION OF SALMONELLA TYPHIMURIUM DT104 (10810 CFU/g) ON
INOCULATED ALFALFA SEEDS USING VARIOUS ANTIMICROBIAL TREATMENTS.

 

TREATMENT

Sonication alone
Water control "

Clorox 200 ppm

Water control

Clorox 20,000 ppm

Water control

Clorox 200 ppm + sonication
Water control

Tsunami 80 ppm

Water control

Tsunami 800 ppm

Water control

Tsunami 80 ppm + sonication
Water control

Vegi-Clean 1%

Water control

Vegi-Clean 2%

Water control

Veg-Clean 1% + sonication
Water control

Copper ion 1 ppm
Water control

Copper ion + sonication
Water control

* All water control data in this table have to be compared to the treatment results reported on the row placed

above them.

 

TIME
30 sec 1 min 3 min 5 min 10 min
0.04 0.18 0.28 0.39 +0.06
0.32 +0.23 0.21 0.09 +0.09
1.42 >1.92 1.42 0.98 0.94
0.32 +0.23 0.21 0.09 +0.09
+ 0.07 +0.16 +0.16 +0.10 +0.10
0.19 1.11 0.60 0.92 0.49
>1.92 1.73 0.70 1.65 1.21
0.32 +0.23 0.21 0.09 +0.09
2.02 >3.32 >3.32 >3.32 >3.32
0.32 +0.23 0.21 0.09 +0.09
1.07 1.32 0.62 1.79 1.61
0.19 1.11 0.60 0.92 0.49
>1.92 >1.92 0.98 >1.92 1.94
0.32 +0.23 0.21 0.09 +0.09
1.32 1.00 1.09 0.96 0.34
0.32 +0.23 0.21 0.09 +0.09
1.07 1.43 1.79 1.94 1.60
0.19 1.11 0.60 0.92 0.49
0.72 0.72 1.15 1.02 0.54
0.32 +0.23 0.21 0.09 +0.09
+0.18 0.24 0.40 0.24 0.47
0.32 +0.23 0.21 0.09 +0.09
0.46 0.94 0.21 0.78 0.24
0.32 +0.23 0.21 0.09 +0.09

+ This sign placed before a data inside a colurrm refer to an increase instead of a decrease of the microbial

load.

91

When inoculated sprouts were washed in water, populations of E. coli 0157:H7,
Salmonella DT 104 and L. monocytogenes decreased by an average of 0.60, 0.76 and
0.45 logs, respectively (Tables 3.3, 3.4, 3.5).

The microbial reductions obtained after washing the alfalfa seeds or sprouts with
water, were not a function of treatment duration. Since water was assumed to have no
bactericidal effect, similar microbial reductions should have been expected independently
of the duration of treatment.

Using either seeds or sprouts showed that microbial reductions were generally
lower for the longest washing treatments. This observation is consistent with previous
reports flom Jaquette et al. (1996) and Taormina and Beuchat (1999a). In the case of a
longer water-seed contact time, the seed imbibes water and therefore releases bacterial
cells that were previously trapped or hidden in crevices or between the testae
and cotyledons. A similar explanation is also applicable for sprouts. Moreover, the
process of drying seeds after inoculation would protect bacteria that had entered the seeds
through cracks and crevices (Jaquette et al.,1996, Taormina and Beuchat 1999a; Mohle-
Boetani et al., 2002).

Water washing exerted a stronger effect on bacterial reduction in sprouts than in
seeds. Sprouts are usually kept in a very moist environment. Therefore, stems and leaves
are very turgid and, subsequently, offer a larger contact surface for water action. In
contrast, seeds are covered with a waxy material that repels water (Jaquette et al.,1996),
suggesting that the seed coat might be responsible for the lower microbial reductions

observed during washing.

92

TABLE 3.3.- REDUCTION OF ESCHERICHIA COLI 0157:H7 (logro CFU/g) ON INOCULATED
ALFALFA SPROUTS USING VARIOUS ANTIMICROBIAL TREATMENTS.

 

TREATMENT

Clorox 1,000 ppm

Water control"I

Clorox 2,000 ppm

Water control

Clorox 10,000 ppm

Water control
Clorox10,000 ppm+Cu ion
Water control

Vegi-Clean 5%

Water control

Vegi-Clean 5%+copper ion
Water control

 

TIME
30 sec 1 min 3 min 5 min 10 min
3.58 4.48 5.44 5.96
0.99 0.63 0.40
3.54 5.31 >7.92
0.99 0.63 0.40
4.79 4.57 5.44 >5.97 >7.37
0.52 0.39 0.39 0.45 0.55
4.44 4.07 5.69 >5.97 >7.37
0.52 0.39 0.39 0.45 0.55
2.24 2.99 2.60 3.00 3.20
0.60 0.78 0.76 0.68
2.35 2.52 2.73 2.74 3.12
0.60 0.78 0.76 0.68

 

* All water control data in this table have to be compared to the treatment results reported on the row placed

above them

93

TABLE 3.4.- REDUCTION OF SALMONELLA TYPHIMURIUM DT104 (logro CPU/g) ON
INOCULATED ALFALFA SPROUTS USING VARIOUS ANTIMICROBIAL TREATMENTS.

 

 

TREATMENT TIME

30 sec 1 rrrin 3 min 5 min 10 min
Sonication alone 0.62 0.49 0.98
Water control * 0.86 0.56 0.88 0.68 0.89
Clorox 200 ppm 0.51 1.13 1.68 2.34 2.34
Water control 0.86 0.56 0.88 0.68 0.89
Clorox 20,000 ppm 4.26 4.68 4.36 4.60 >6.36
Water control 0.68 0.73 0.88 0.66 0.75
Clorox 200 ppm + sonication 1.27 1.24 2.53 2.63 >3.98
Water control 0.86 0.56 0.88 0.68 0.89
Tsunami 80 ppm 1.00 1.27 1.34 1.46 1.48
Water control 0.86 0.56 0.88 0.68 0.89
Tsunami 800 ppm 2.11 2.75 3.44 3.27 3.62
Water control 0.68 0.73 0.88 0.66 0.75
Tsunami 80 ppm + sonication 1.21 1.10 2.38 1.68 1.86
Water control 0.86 0.56 0.88 0.68 0.89
Vegi-Clean 1% 0.74 1.24 1.47 1.76 2.08
Water control 0.86 0.56 0.88 0.68 0.89
Vegi-Clean 2% 1.81 1.75 2.13 2.20 2.43
Water control 0.68 0.73 0.88 0.66 0.75
Veg-Clean 1% + sonication 0.72 1.46 1.74 2.02 2.74
Water control 0.86 0.56 0.88 0.68 0.89
Copper ion 1 ppm 0.70 0.46 0.54 0.64 0.92
Water control 0.86 0.56 0.88 0.68 0.89
Copper ion + sonication 0.45 0.58 0.60 0.84
Water control 0.86 0.56 0.88 0.68 0.89

 

* All water control data in this table have to be compared to the treatment results reported on the row placed
above them.

94

TABLE 3.5.- REDUCTION OF LISTERIA MONOCYTOGENES (logro CFU/g) ON INOCULATED
ALFALFA SPROUTS USING VARIOUS ANTIMICROBIAL TREATMENTS.

 

 

TREATMENT TIME

30 sec 1 min 3 min 5 min 10 min
Clorox 20,000 ppm 2.42 2.87 3.1 1 4.66 5.46
Water control“ 0.46 0.42 0.51 0.49 0.38
Vegi-Clean 2% 0.91 1.1 1 1.44 1.64 ' 2.08
Water control 0.46 0.42 0.51 0.49 0.38

 

* All water control data in this table have to be compared to the treatment results reported on the row placed
above them.

95

The general trends of these results support the assertion of F ett (2000) who stated
that the common occurrence of natural biofilms on the surface of fluits and vegetables
may account for the fact that rinsing with water usually reduces total bacterial counts by

$1 log.

3.3.2 .- SONICATION

Sonicating seeds previously inoculated with E. coli 0157:H7 and S. Typhimurium
DT 104 did not noticeably alter the microbial populations. Alter sonication, E. coli
populations decreased an average of 0.67 log compared to reductions averaging 0.65 log,
after washing inoculated seeds in water. On alfalfa seeds previously inoculated with
Salmonella, microbial reductions averaged 0.16 log, after sonication, compared to 0.06
log for the water control.

Sonication alone was also applied to alfalfa sprouts previously inoculated with
Salmonella, with reductions of 0.49 to 0.98 log. The corresponding water control
decreased Salmonella population 0.56 to 0.89 log.

As for the seeds, sonicating sprouts did not offer any benefit over washing.
Microbial reductions were not a function of the duration of treatment. The phenomenon
described for water soaking was also seen using with sonication with the release of
previously imbedded bacteria giving the appearance of a lower bacterial reduction after
10 minutes of treatment.

Our results with sonication alone are consistent with the work of Burleson et al.
(1975) who reported that sonicating alone for 10 minutes did not inactivate Salmonella

Typhimurium or enteropathogenic E. coli 0126:B16 in phosphate saline buffer or

96

secondary effluent flom a wastewater treatment. While most bacteria present in milk
fihns on metal surfaces were removed using 80 kHz (Daufin and Saincliviert, 1967;
Lillard, 1993), our sonicating water bath which generated 20 kHz was likely too weak to

remove bacteria flom alfalfa seeds or sprouts.

3.3.3.- COPPER ION

A 1 ppm copper ion solution was generally unable to decrease populations of E.
coli 0157:H7 and S. Typhimurium DT104 > 0.50 logs on previously inoculated alfalfa
seeds, with these reductions usually no greater than water alone (Tables 3.1, 3.2).

As for alfalfa seeds, the microbial reduction resulting flom the action of copper
ion on alfalfa sprouts inoculated with S. Typhimurium DT104 was similar to that seen for
water. Salmonella reductions ranged flom 0.46 to 0.92 and 0.56 to 0.89 logs for copper
ion and water, respectively. Thus, copper ion alone did not result in any additional
antimicrobial activity against either pathogen.

III aqueous model systems, Yahya et al. (1989) reported an E. coli reduction of
0.12 longFU/ g following a 6 minute exposure to a 433 ug/L : 43 [1. g/L copper-silver ion
solution. These findings more closely reflect our results with seeds and sprouts than
those of Rodrick and Hultstrand (1998) who reported a 2 log reduction in total surface
bacteria on tomatoes after a 5 minute exposure to a 0.5 to 1.0 mg/L copper ion solution.
Similarly, our results are not in accordance with the 3.5 logs CFU/g reduction in the
microbial load of scallions claimed by Sanders (Superior Water Solutions, Inc., in a non-

dated correspondence) after a 1 ppm copper ion water treatment.

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When combined with sonication, 1 ppm copper ion reduced populations of E. coli
0157:H7 (Table 3.1) and S. Typhimurium DT104 (Table 3.2) 0.86 to 1.07 and 0.24 to
0.94 logs, respectively, on inoculated alfalfa seeds. Compared to copper ion alone, the

combined use of copper ion and sonication did not result in greater microbial reduction.

3.3.4.- CLOROX TM

When alfalfa seeds previously inoculated with E. coli 0157:H7 were treated with
200 ppm, populations of E. coli 0157:H7 decreased 0.60 to 1.57 logs (Table 3.1). Using
20,000 ppm NaClO, microbial reductions were more constant over the treatment times,
ranging flom 1.18 to 1.53 logs for E. coli 0157:H7 (Table 3.1).

Taormina and Beuchat (1999a) obtained a 2 to 3 log reduction in E. coli using
20,000 ppm of Ca(OCl)2. They reported that treatment with 20,000 ppm Ca(OCl)2
reduced E. coli 0157:H7 populations flom 2.33 to 3.46 loglo CFU/g to less than 0.3 logm
CFU/g, after 3 or 10 minutes. These data show that a higher bacterial reduction was
achieved with Ca(OCl)2 than what we observed with NaClO. However, their inability to
achieve > 3 log reduction with a concentration of 20,000 ppm of Ca(OCl)2 is consistent
with our findings.

Results obtained after treatment with .200 ppm and 20,000 ppm of NaClO Show
that bactericidal activity is not related to exposure time. For greater exposure times,
bacteria hidden in crevices located on the seed surface were released by the action of the
aqueous solution. This phenomenon led to bacterial reductions that appeared lower than
those obtained for shorter times. When active chlorine comes in contact with high levels

of organic matter, such as alfalfa seeds, its potency is quickly diminished (J aquette et al.,

98

1996). The comparatively lower bacterial reduction obtained for the 10 minute / 200 ppm
treatment could be due to insufficient levels of flee chlorine remaining available in
solution after 10 minutes of soaking, to destroy organisms previously hidden in crevices.
In contrast, sufficient flee chlorine likely remained available after 10 minutes using
20,000 ppm. This assumption is supported by the work of Taormina and Beuchat (1999a)
who showed that, although the amount of active chlorine decreased in the presence of
organic matter, 80% of the initial available chlorine remained in solution, after holding 10
g of alfalfa seeds in 40 ml of 20,000 ppm active chlorine for 12 minutes. When alfalfa
seeds were inoculated with S. Typhimurium DT104, exposure to 200 ppm NaClO
decreased populations 0.94 to at least 1.92 logs (Table 3.2). Hence, the sanitizer effect
resulted in higher bacterial reductions than those obtained by washing in water for the
same length of time.

In contrast, comparing counts flom all treatments to initial populations present on
inoculated seeds, Weissinger and B euchat (2000) calculated the reduction in microbial
load in reference to the water control. After plating on T SAN (Trypticase Soy Agar +
Nalidixic acid), Salmonella populations decreased 0.33 and 0.72 log after a 10 minute
exposure to 200 and 2,000 ppm NaClO, respectively. Their results for reduction of
Salmonella using 200 ppm NaClO were less impressive than ours (0.94 to 1.3 logs
compared with water control). The main conclusion remains that both their results and
ours are far flom the 5 log reduction targeted.

The aforementioned reductions in Salmonella populations reported by Weissinger
and Beuchat (2000) after 10 minute treatments with 200 and 2000 ppm NaClO were in a

similar range (0.33 versus 0.72 log). Our findings with Salmonella and E. coli are

99

consistent with these authors, in that none of these treatments completely destroyed the
pathogen on seeds. This observation explains why Jaquette et a1 (1996) needed to use
concentrations of 2,040 and 3,990 ppm flee chlorine to reduce Salmonella Stanley flom
65 CFU/g to undetectable levels when analyzed by direct plating. Beuchat (1997)
observed a < 3 .9 log reduction for Salmonella w hen s eeds w ere s oaked in l ,800 p pm
Ca(ClO); or 2,000 ppm NaClO for 30 seconds. Both our results and those of Weissinger
and Beuchat (2000) fell in the lower range compared to those of Beuchat (1997).

According to Piemas and Guiraud (1997), a 20 minute treatment in 1000 ppm,
NaClO was able to reduce the bacterial load 2 to 3 logs on rice seeds. Decontamination
efficacy was not improved by extending the contact time, nor by increasing the chlorine
concentration to 10,000 ppm as observed in our work. Piernas and Guiraud (1997) also
reported that the greater bactericidal activity using 10,000 ppm NaClO was due to the
higher pH. A buffer at the same pH had an equivalent effect on the aerobic plate count,
which was not seen using the 1,000 ppm concentration.

Clorox”, at a concentration of 200 ppm chlorine was also combined with
sonication with populations of E. coli 0157:H7 on inoculated seeds decreasing 1.15 to
1.91 logs (Table 3.1). Similarly, Salmonella counts decreased 0.70 to >1.92 logs on
Salmonella-inoculated seeds (Table 3.2). This combination did not bring the superior
microbial load reduction expected.

Using 200 ppm NaClO, populations of Salmonella on inoculated alfalfa sprouts
decreased 0.51 to 2.34 logs (Table 3.4). Slightly greater reductions (1.24 to >3.98) were

seen when 200 ppm NaClO was combined with sonication. In contrast, reductions of

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4.26 to > 6.36 logs in Salmonella were reached after a 10 minute exposure to 20,000 ppm
NaClO.

A progressive decrease of the microbial load, although not proportional with the
duration of treatment, was observed with alfalfa sprouts that were previously inoculated
with L. monocytogenes and then treated with 20,000 ppm NaClO (Table 3.5).
Populations of L. monocytogenes decreased 2.42 to 5.46 logs, with the targeted 5 log
reduction a gain b eing reached. H owever, exposing the sprouts to 20,000 ppm NaClO
resulted in producing an obviously inedible product.

Based on these results, the efficacy of 10,000 ppm NaClO against E. coli
0157:H7 was also assessed (Table 3 .3). T his treatment reduced population of E. coli
4.57 to >7.37 logs, with the greatest reductions observed after the longest exposure. A >
5 log reduction was again seen after 10 minutes of soaking; however, sprout quality was
undesirable.

Some researchers (Yahya, 1989; Sanders in a Superior Water Solutions, Inc.
correspondence) reported that the combination of copper ion and sodium hypochlorite
exhibited stronger antimicrobial activity than when either was used alone. Consequently,
10,000 ppm sodium hypochlorite was added to a 1 ppm copper ion solution. However,
no noticeable differences were observed in reductions of E. coli 0157:H7 using copper
ion / sodium hypochlorite compared to sodium hypochlorite alone (Table 3.3).
Populations of E. coli decreased 4.07 to > 7.37 logs after the combined copper ion /
sodium hypochlorite treatment, whereas reductions of 4.79 to > 7.37 logs were found

with sodirun hypochlorite alone.

101

Given that the sprouts were damaged following exposure to 10,000 ppm NaClO,
the concentration was lowered to 2,000 and 1,000 ppm with both of these concentrations
proving effective against E. coli 0157:H7 (Table 3.5). The 2,000 ppm and 1,000 ppm
treatments decreased the E. coli load by 3.54 to >7.92 and 3.58 to 5.96 logs, respectively.
However, the sprouts’ appearance remained unsatisfying.

When NaClO was used in combination with either sonication or copper ion, the
reduction in microbial populations present on seeds and sprouts was primarily due to the

effect of NaClO rather that of the other treatments.

3.3.5 .- TSUNAMI”

After inoculated seeds were treated with 80 and 800 ppm Tsunami”, populations
of E. coli 0157:H7 decreased 0.61 to 1.11 and 0.46 to 0.73 logs, respectively (Table 3.1).
Using Tsunami” at 80 ppm, Taormina and Beuchat (1999a) reportedly obtained a >1.70
log reduction in microbial load with their findings similar to ours. In combination with
sonication, Tsunami” 80 ppm yielded reductions of 1.28 to 1.91 logs for E. coli
0157:H7 on inoculated seeds (Table 3.1). Hence, sonication did not markedly increase
the effectiveness of Tsunami”.

Tsunami” 80 and 800 ppm reduced populations of S. Typhimurium 2.02 to
>3.32 and 0.62 to 1.79 logs, respectively (Table 3.2), on previously inoculated seeds.
When Weissinger and Beuchat (2000) exposed Salmonella-inoculated alfalfa seeds to
Tsunami” concentrations of 270 ppm, 530 ppm and 1,060 ppm, Salmonella populations
decreased 0.79, 1.12 and 1.50 logs, respectively (water control taken as basis for the

calculations). Their results for reductions in Salmonella were similar to ours. Our

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observations flom both Tables 3.1 and 3.2 agree with Weissinger and Beuchat’s (2000)
findings in that a higher concentration of Tsunami” did not lead to a proportional
decrease in microbial load. When sonication was combined with 80 ppm Tsunami”
(Table 3.2), populations of Salmonella decreased 0.98 to > 1.94 logs with sonication
failing to enhance the effectiveness of Tsunami”. Reductions in E. coli populations
were usually less than those noticed for Salmonella.

When alfalfa sprouts were inoculated with S. Typhimurium D T104 and treated
with 80 ppm Tsunami”, populations of Salmonella decreased 1.00 to 1.48 logs (Table
3.4). Taormina and Beuchat (1999b) did not soak their inoculated sprouts in Tsunami”
80 ppm s olution, as w as d one in 0 ur s tudy. I nstead, these authors sprayed inoculated
seeds, at various steps throughout the sprouting process, with Tsunami” 80 ppm.
Despite these spray applications, E. coli populations increased flom 2.62 loglo CFU/ g on
seeds to 6.61 loglo CFU/g in the mature sprouts. When combined with sonication, 80
ppm Tsunami” decreased populations of Salmonella 1.10 to 2.38 logs (Table 3.4), with

sonication failing to enhance the effectiveness of Tsunami”.

3.3.6.- VEGI-CLEANTM

Use of 1 and 2% Vegi-Clean” generally reduced bacterial populations < 1.50
logs (Table 3.1). A concentration of 1%, as recommended by the manufacturer for
washing fluit and v egetables, d ecreased p opulations o f E . coli 0 157:H7 o n inoculated
seeds only 1.24 to 1.60 logs. Results obtained with the 2% concentration were inferior,

with reductions of 0.44 to 1.06 logs for E. coli 0157:H7. When sonication was

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combined with 1% Vegi-Clean”, E. coli 0157:H7 decreased 1.51 to 1.60 logs. Hence,
sonication also failed to enhance the effectiveness of Vegi-Clean”.

Taormina and Beuchat (1999a) studied the effect of 1 and 2 % Vegi-Clean” on
alfalfa seeds inoculated with E. coli 0157:H7. These authors obtained slightly greater
microbial reductions with 1.77 to 2.10 logs for 1% and 1.50 to 1.72 logs for 2%
(compared to the water control). These microbial reductions were not a function of
treatment time with 0.10 log and 0.26 log remaining on the seeds, respectively, after
treaments of 3 and 10 minutes with 1% Vegi-Clean”. The same trend was observed
using 2% Vegi-Clean” (0.48 log present after 3 minutes versus < 0.30 log after 10
minutes). As in our study, microbial reductions were usually greater using 1% rather than
2% Vegi-Clean”.

Reductions in numbers of S. Typhimurium DT104 on inoculated alfalfa seeds
were also minimal using 1 and 2% Vegi-Clean” (Table 3.2). With a 1% solution, S.
Typhimurium DT104 decreased 0.34 to 1.32 logs. Increasing the concentration to 2%
resulted in decreases of 1.07 to 1.94 logs. Thus, somewhat greater reductions were seen
at the higher concentration. Length of treatment exposure had a greater impact using 2%
as compared to 1% Vegi-Clean”. When combined with sonication, 1% Vegi-Clean”
reduced Salmonella population 0.72 to 1.15 logs (Table 3.2). As for the other sanitizers,
sonication also failed to enhance the efficacy of Vegi-Clean”.

Two concentrations of Vegi-Clean” (1% and 2%) were applied to alfalfa sprouts
inoculated with S. Typhimurium DT104 (Table 3.4). Results for both concentrations

were generally similar, with populations of S. Typhimurium decreasing 0.74 to 2.08 logs

104

for the 1% solution, and 1.81 to 2.43 logs for the 2% application. The combination of 1%
Vegi—Clean” and sonication failed to enhance the reduction in Salmonella, with
decreases of 0.72 to 2.58 logs. For all Vegi-Clean” treatments with sprouts, a trend
toward greater microbial reduction was observed as the duration of treatments increased.

The results obtained after treating Listeria-inoculated sprouts with 2% Vegi-
Clean” were similar to those previously recorded for Salmonella with reductions of 0.91
to 2.08 logs (Table 3.5).

Sprouts previously inoculated with E. coli 0157:H7 were treated with a higher
concentration of Vegi-Clean” (5%) in order to maximize the chance for obtaining a 5 log
reduction. Under these conditions, E. coli 0157:H7 reductions of 2.24 to 3.20 logs were
observed with these decreases only slightly higher than those seen using 2% Vegi-
Clean” against Salmonella and Listeria (Tables 3.4 and 3.5). Adding 5% Vegi-Clean”
to 1 ppm copper ion water did not enhance antimicrobial activity, as reductions ranged
flom 2.35 to 3.12 logs (Table 3.3).

Considering the effect of the various Vegi-Clean” concentrations applied alone
or in combination with sonication or copper ion, reductions in microbial populations on
seeds and sprouts were mainly due to Vegi-Clean”. Although the mechanism of action
of acid-anionic surfactants has not been firlly elucidated, it is assumed that acid-anionic
surfactants play a role in inhibiting key enzyme activities, disrupting the cell membrane,
interrupting cellular transport and / or denaturating cellular proteins (Dychdala and Lopes,
1991). In most cases, these microbial reductions were usually greater than those obtained

using water alone.

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3.4.- CONCLUSIONS

When used alone, sonication or copper ion were no better than water for
decreasing pathogen populations on inoculated alfalfa seeds and sprouts. In addition,
sonication or copper ion failed to enhance the efficacy of Clorox”, Tsunami” or Vegi-
Clean”. Only NaClO at concentrations higher than 1,000 ppm decreased the microbial
load on alfalfa sprouts by more than 5 logs. However, this treatment produced sprouts
that were unacceptable due to osmotic dehydration and bleaching. None of the other
treatments tested were able to reduce microbial populations to commercially acceptable
levels. Therefore, replications of the individual experiments were not carried out.

Fett (2000) documented the presence of natural biofilms on the surface of
fluits and vegetables. Moreover, bacterial pathogens can enter plant tissues with
organisms remaining protected in seeds and sprouts. Itoh et al.(1998), using
immunofluorescence microscopy and scanning electron microscopy, demonstrated the
presence of viable and culturable E. coli 0157:H7 cells in the inner tissues and stomata
of cotyledons of radish sprouts grown flom experimentally contaminated seeds. These
observations help to explain the inability of sonication to efficiently detach bacteria
flom seeds and sprouts. The ionization state of the copper ion solution was expected to
increase contact between surface pathogens and the chemical sanitizers. Biofilm
formation and internalization within seeds and sprouts likely played a role in protecting
these organisms flom the harmful effects of these sanitizers. In contrast, NaClO at
concentrations higher than 1,000 ppm were able to enter the sprout tissues by means of
a strong osmotic pressure. This was inferred by the fact that, after soaking, the sprouts

appeared thoroughly dehydrated and as thin as a strand of hair. This observation

106

combined with complete bleaching may explain how this sanitizer was able to enter the

tissues to destroy these pathogens in sprouts as opposed to seeds.

107

4.- EFFICACY OF A FATTY ACID-BASED SANITIZER TO INACTIVATE ESCHERICHIA COLI
0157:H7, SALMONELLA TYPHIMURIUM DT104 AND LISTERIA MONOCYTOGENES ON
ARTIFICIALLY CONTAMINATED ALFALFA SEEDS.
ABSTRACT

Alfalfa seeds were inoculated with a 3-strain cocktail of Escherichia coli
0157:H7, Salmonella Typhimurium DT104 or Listeria monocytogenes by immersion so
as to contain ~ 6 to 8 log CFU/g, then sanitized with an FDA-approved fatty acid based-
sanitizer containing 250 ppm peroxyacid (PA), 1,000 ppm caprylic and capric acid
(Emery 658), 1,000 ppm lactic acid (LA) and 500 ppm glycerol monolaurate (GM) for a
reference concentration of 1x. Concentrations of 5x, 10x and 15x were used for 1, 3, 5
and 10 minutes. The lowest concentration to decrease all three 3 pathogens > 5 logs was
15x. Following a 3 minute immersion, populations of E. coli 0157:H7, S. Typhimurium
DT 104 and L. monocytogenes decreased >5 .45, >5.62 and >6.92 logs, respectively, with
no injury and no significant loss in seed germination rate or sprout yield. The components
of this 15 x concentration (Treatment A) were assessed alone and in various combinations
to optimize inactivation o f the three pathogens. D uring 3 and 5 minutes of exposure,
Treatment C (15,000 ppm B 658, 15,000 ppm LA and 7,500 ppm GM) decreased
Salmonella 6.23 and 5.57 logs, respectively, and E. coli 4.77 and 6.29 logs, respectively.
Treatment D (15,000 ppm E 658 and 15,000 ppm LA) reduced Salmonella > 6.90 logs
after all exposures and E. coli 4.60 and > 5.18 logs after 3 and 5 minutes, respectively.
No significant differences were found between Treatments A, C and D. Overall,
Treatment D was most effective in reducing E. coli and Salmonella populations ~ 5 lOgs

and may provide a Viable alternative to the recommended 20,000 ppm chlorine.

108

4.1.- INTRODUCTION

Alfalfa seeds and sprouts have been incriminated as the source of at least 20
Escherichia coli 0157:H7 and Salmonella Sp. outbreaks worldwide and the target of at
least 19 Class I recalls (POnka et al., 1995; Jaquette et al., 1996; Tauxe et al., 1997;
Mahon et al., 1997; Glynn, 1997; Gutierrez, 1997; Como-Sabetti et al., 1997; Hara-Kudo
et al., 1997; Itoh et al., 1998, Van Beneden et al., 1999; Watanabe et al., 1999; Taormina,
1999; Taormina et al., 1999; Taormina and Beuchat, 1999a; FDA/NACMCF, 1999;
Proctor et al., 2001; Mohle-Boetani et al., 2001 and 2002; Huff, 2002a,b,c; FDA, 2004).
111 one such E. coli 0157:H7 outbreak involving 108 cases, 30,000 pounds of sprouts
were recalled by a Michigan grower. In a similar outbreak involving Salmonella
Muenchen in Wisconsin and six other states that was traced to 157 cases, over 12,500
pounds of alfalfa seeds and 2,700 pounds of sprouts were recalled. Thus far, Listeria
monocytogenes has not been linked to any outbreaks involving the consumption of raw
alfalfa sprouts. However, the pathogen has been detected on various vegetable sprouts,
leading to two Class I recalls, in 1998 and 2002 as recorded in FDA Enforcement
Reports (FDA, 2004). Since 1998, the US has maintained its policy of “zero tolerance”
for L. monocytogenes in all ready-to-eat and cooked foods (Ryser, 1998).

Numerous microbial reduction strategies including heat (J aquette et al., 1996),
gamma irradiation (Rajkowski and Thayer, 2000), 20,000 ppm calcium hypochlorite
(Taormina and Beuchat, 1999a), acidified sodium hypochlorite (Weissinger and Beuchat,
2000), hydrogen peroxide (Beuchat, 1997; Taormina and Beuchat, 1999a; Weissinger
and Beuchat, 2000), trisodium phosphate ( Taormina and Beuchat, 1999a; Weissinger and

Beuchat, 2000), calcium hydroxide (Weissinger and Beuchat, 2000), calcinated calcium

109

(Weissinger and Beuchat, 2000), active oxygen (Taormina and Beuchat, 1999a;
Weissinger and Beuchat, 2000), organic acids (Weissinger and Beuchat, 2000) and allyl
isothiocyanate (Park et al., 2000) have been assessed for their ability to decrease bacterial
populations >5 logs on alfalfa seeds while still maintaining a commercially acceptable
seed germination rate. However, none of those treatments have proven to be completely
successful.

A novel fatty acid-based sanitizer containing peroxyacid, fatty acids (caprylic and
capric acids), lactic acid and glycerol monolaurate was recently developed as an
antimicrobial dip for ready-to-eat foods. When caprylic, capric and acetic acids are
reacted with hydrogen peroxide overnight, peroxycarboxylic acids possessing particularly
strong antimicrobial activity are formed (Guthery, 2001, personal communication).
Peracetic acid is bactericidal at fairly high concentrations, generally greater than 100 ppm.
At pH 3.5, 5 and 10 ppm of peroxycaprylic and peroxycapric acid, respectively, decreased
E. coli populations 5 logs, in an aqueous model system (0akes et al., 1993). However, at
pH 5, 5 ppm peroxycaprylic and 6 ppm peroxycapric acid, respectively, were
noninhibitory to E. coli when suspended in distilled water (0akes et al., 1993). The
combination of these acids produced a much more potent biocide than when used
individually with antimicrobial activity observed at far lower concentrations (0akes et al.,
1993)

Lactic acid is inhibitory to a wide range of bacterial pathogens (Anderson and
Marshall, 1990; Greer and Dilts, 1992). Glycerol monolaurate also possesses
antimicrobial activity which has been extensively investigated (Oh and Marshall

1992,1993; Kabara, 1972, 1975, 1979; Kabara et al., 1977). More importantly, the

110

biocidal activity of these individual antimicrobial agents is greatly enhanced when
combined with other food preservatives (Shibasaki and Kato, 197 8; Kabara, 1984; Oakes
et al., 1993; Bogh-Sorensen 1994; Marshall and Kim, 1996; Venkitanarayanan et
al.,1999).

All components of this novel fatty acid-based sanitizer are GRAS with these non-
toxic fatty acids and their esters occurring naturally in foods (Kabara, 1984; Oh and
Marshall, 1993; Andrews, 1996). T herefore, they carry a considerable advantage over
other types of chemical sanitizers developed to control microorganisms.

The two-fold objective of this study is to ( 1) determine the optimal concentration
and length of exposure to this fatty acid-based sanitizer for decreasing E. coli 0157:H7,
Salmonella Typhimurium DT104 and Listeria monocytogenes populations 5 logs on
alfalfa seeds while maintaining an acceptable germination rate and sprout yield, and (2)
optimize the sanitizer formulation and exposure time, by testing its individual
components alone and in combination, for inactivation of the aforementioned pathogens
with the end-result being a custom-designed antimicrobial formulation for alfalfa seeds
that will reduce pathogen populations at least 5 logs without negatively impacting

germination rate and sprout yield.

111

4.2.- MATERIAL AND METHODS
Bacterial strains

Three Escherichia coli 0157:H7 strains (AR, AD 305 and AD 317) and three
Listeria monocytogenes strains (LM Brick silage, LM T3BL, LM F5027) were Obtained
flom Dr. Catherine W. Donnelly (Department of Nutrition and Food Sciences, University
of Vermont, Burlington, VT). Three Salmonella Typhimurium DT104 strains (G1601,
G1074 and G10931) were obtained flom Dr. Peggy Hayes (Centers for Disease Control
and Prevention). Laboratory stock cultures were maintained at —- 80 °C in Tryptic Soy
Broth (T SB, pH 7.3) (Difco Laboratories, Detroit, MI) containing 10% (v/v) glycerol.
Individual strains were separately activated by transferring a loop of the frozen stock
culture into 9 m1 of sterile TSB containing 0.6% (w/v) yeast extract (YE) (Difco) then
incubated at 35°C for 18-24 hours. Thereafter, for each strain, 5 tubes containing 35 ml

each of TSB-YE were inoculated and similarly incubated before use.

Preparation of inoculum

The aforementioned cultures were harvested by centrifugation (Sorvall Super T21,
Newtown, CT) at 10,000 rpm for 15 min at 4 °C, resuspended in 0.1% peptone (Difco)
and combined in equal volumes to prepare three 3 eparate 3 -strain c ocktails c ontaining

approximately 109 CFU/ml of each pathogen.

Inoculation of alfalfa seeds
Alfalfa seeds originating flom Australia were purchased in 25 kg-bags (batch

serial # AUS / S / X / 2017) flom Dan Caudill (Louisville, KY).

112

The seeds (500 g) were separately immersed in 525 ml of the 3-strain cocktail and
gently agitated for approximately 10 minutes so as to contain 106 to 108 CFU/ g of E. coli
0157:H7, S. Typhimurium DT104 or L. monocytogenes. Thereafter, the seeds were dried
for 4 h in a laminar flow biosafety cabinet and stored at 22 — 24 0C for at least 24 h before

use. All seeds were used within 5 days of inoculation.

Inactivation of pathogens on alfalfa seeds
_S__anitizer formulations

Two separate solutions, Asep I and Asep H, were obtained flom Dr. B. Eugene
Guthery (Broken Bow, OK). Asep I contained the following ingredients (v/v) : 49%
propylene glycol (V WR, Chicago, IL), 15% Emery 658 (a mixture of 60% w/w caprylic
(octanoic,Cg) and 40% w/w capric (decanoic,C 10) acids) (Cognis Corporation, Cincinnati,
OH), 10% glacial acetic acid (VWR), 1% sulfuric acid and, added just before use, 25%
hydrogen peroxide (50% v/V) (Solvay-Interox, Houston, TX) to activate the solution
overnight, in the dark. After 24 h, activated ASEP 1 contained 10% peroxycarboxilic acid
(PA) (a combination of p eroxyacetic acid, p eroxycaprylic acid and p eroxycapric acid).
Asep 11 contained the following ingredients (v/v) : 40% Emery 658, 40% lactic acid (88%
v/v) (LA) (JT Baker, Phillipsburg, NJ) and 20% glycerol monolaurate (GM) (Lauricidin,
Inc., 0kemos, MI).

To obtain 100 ml of the various working solutions, ASEP I and II were diluted as

shown in Table 4.1, with concentrations of active components in these solutions provided

113

in Table 4.2.1. Asep I and Asep H were added to rapidly stirring sterile deionized water to
produce an emulsion. Aliquots of the sanitizer were taken flom the stirring solution and

immediately used for inactivation experiments.

TABLE 4.1 .- COMPOSITION (ML) OF THE VARIOUS SANITIZER WORKING SOLUTIONS.

 

 

SANITIZER ASEP I ASEP 11 WATER

CONCENTRATION

1 X (reference cone.) 0.25 0.25 99.50
5 X 1.25 1.25 97.50

10 X 2.50 2.50 95.00

15 X 3.75 3.75 92.50

 

TABLE 4.2.- ACTIVE ANTIMICROBIAL COMPONENTS (PPM) OF VARIOUS CONCENTRATION OF
THE DILUTED FATTY ACID-BASED SANITIZER WORKING SOLUTIONS

 

 

SANITIZER ASEP I ASEP II
CONC.
(PA) (13 658) (LA) (GM)
1 X (ref. conc.) 250 1,000 1,000 500
5 X 1,250 5,000 5,000 1,000
10 X 2,500 10,000 10,000 5,000
15 X 3,750 15,000 15,000 7,500

 

After identifying the minimal sanitizer concentration needed to decrease any of
the three pathogens 2 5 logs on inoculated alfalfa seeds, PA, E 658, LA and GM were

assessed individually and in various combinations to determine the most efficacious

 

' Some components of this sanitizer theoretically do not possess antimicrobial activity in the concentration
given. After activation of ASEP I, the concentration of hydrogen peroxide decreases to non-inhibitory
levels. Propylene glycol is used as a solvent in the sanitizer. Although propylene glycol can exhibit
antimicrobial activity at concentrations > 70%, the concentration used in this sanitizer (40%) is too low for
this purpose. Moreover, a small amount of flee fatty acid remains unreacted after ASEP I activation and is
not taken in account in Table 4.2 (Guthery, 2002).

114

formulation for inactivating E. coli 0157:H7, S. Typhimurium DT104 and L.
monocytogenes 2 5 logs. Formulations for these nine antimicrobial solutions tested,
designated A through I, are s hown in T able 4 .3. A ll nine 8 olutions w ere p repared by
adding the component(s) to stirring sterile deionized water as previously described. GM
was melted by heating to approximately 70 °C and was added to stirring water at the same
temperature, after which the stirring solution was allowed to return to ambient

temperature before use.

TABLE 4.3.- ANTIMICROBIAL SOLUTIONS FORMULATIONS (PPM) INCLUDING PEROXY ACID
(PA), EMERY 658 (E658), LACTIC ACID (LA), GLYCEROL MONOLAURATE (GM) ALONE OR
IN VARIOUS COMBINATIONS.

 

TREATMENT PA E 658 LA GM pH
A (Asep 1+2) 3,750 15,000 15,000 7,500 2.30
B (Asep 1) 3,750 - - - 2.30
C (Asep 2) - 15,000 15,000 7,500 2.52
D (E658+LA) - 15,000 15,000 - 2.50
E (E658+GM) - 15,000 - 7,500 3.40
F (GM+LA) - - 15,000 7,500 2.50
G (E658) - 15,000 - - 3.22
H (LA) - - 15,000 - 2.19
I (GM) - - - 7,500 4.60

 

Experimental procedure

In order to assess the treatment effect on microbial load of pre-inoculated seeds,
an experimental procedure, modified flom Taormina and Beuchat (1999a) was adopted.
Forty ml of any given sanitizer formula, or sterile deionized water as a control, were

added to a 24 oz. sterile stomacher bag (Whirl-pack, Nasco, USA) containing 10 g of

 

115

inoculated seeds and manually agitated for 1, 3, 5 and/or 10 minutes. The treatment
solution was then discarded and replaced by 40 m1 of an appropriate neutralizing buffer
afler which the sample was homogenized at high speed in a stomacher (Model SD-45
Tekmar Co., Cincinnati, 0H) for 2 min. Inoculated control seeds were analyzed by
adding 40 ml of buffer to a stomacher bag containing 10 g of seeds, followed by
processing in a stomacher.

The neutralizing buffer solution (Guthery, 2001) was prepared by adding 5 g
peptone (Difco Laboratories, Detroit, MI), 1 g sodium thiosulfate (JT Baker, Phillipsburg,
NJ), 0.25 g mono-potassium phosphate (JT Baker), 0.25 g catalase (Sigma, St Louis,

MO), 30 g tween 80 (Sigma) , 10 g lecithin (Sigma) to 1 liter of Letheen broth (Difco).

Microbial Analysis
Determination of bacterial counts

Appropriate dilutions in 0.1% peptone were spiral-plated (Autoplate 4000, Spiral
Biotech Inc., Bethesda, MD) in duplicate on Cefixime Tellurite Sorbitol McConkey Agar
(CT-SMAC) (SMAC Difco) (CT supplement Dynal, Lake Success, NY) for enumeration
of E. coli 0157:H7, Xylose Lysine Desoxycholate Agar (XLD) (Difco) for S.
Typhimurium DT104 and Modified Oxford Agar (MOX) (Difco) for L. monocytogenes.
and Tryptic Soy Agar (TSA) for Mesophilic Aerobic Bacteria with the latter counts being

reported in Appendix B.

116

Reduction in microbial load was calculated by subtracting the microbial count
after treatment flom the corresponding initial microbial count obtained flom the untreated
inoculated seed control.

Detection of iniured cells

After homogenization in the stomacher, aliquots of the supernatant were also
spiral-plated in q uadriplicate o n T ryptic S oy A gar c ontaining 0 .6% (w/v) yeast e xtract
(TSA-YE) (Difco), incubated 4 to 6 h and then overlaid with CT-SMAC for E. coli
0157:H7, XLD for S. Typhimurium DT104 and MOX for L. monocytogenes. Numbers
of injured cells were determined by subtracting the number of colonies on the selective
media (non-injured cells)(S) flom those enumerated on overlaid TSA—YE (total pathogen

population composed of repaired and non-inj ured cells)(O).

Germination and Yield Tests

Forty m1 of the aforementioned sanitizer formulas were added to a stomacher bag
containing 10 g of inoculated seeds. The bag contents were agitated for 1, 3, 5 and / or 10
min after which the sanitizer was discarded and the seeds washed three times by agitating
in 150 ml of tap water. The washed alfalfa seeds (treated samples and untreated controls)
were placed on moistened 5 x 5 cm cotton squares (Meijer Distribution, Inc, Grand
Rapids, MI) in 150 mm-diameter Petri dishes (40 seeds per Petri dish x 3 dishes per
replicate x 3 replicates per treatment) and kept moist for 6 days at 22 - 24 oC. Percent
germination and sprout yield were then calculated as followed:

% Germination = Number of sprouts Der fleflent x 100
Number of seeds per treatment

117

Sprout yield = Weight of sprouts per treatment
Weight of seeds per treatment

Statistical analysis

Two-way Analysis of Variance (ANOVA) was done on microbial, germination
and yield data using the Statistical Analysis system (Proc Anova, SAS, Version 8, SAS©
Institute Inc., Cary, NC) to determine the effect of time and concentration. Data in the
tables are means flom duplicate or quadriplicate samples flom three replicates and were

compared using the Tukey-Kramer adjustement at the 95% confidence level (P = 0.05).

118

4.3.- RESULTS
Minimal c oncentration f or r educing o f E . coli 0157:H7, S. Typhimurium DT104
and L. monocytogenes 2 S logs on alfalfa seeds.

E. coli 0157:H7. The effect of three concentrations of this novel fatty acid-based
sanitizer (5x, 10x and 15x) on the microbial load of alfalfa seeds previously inoculated
with E. coli 0157:H7 was assessed (Tables 4.4 and 4.5). No bacterial survivors were
observed after seeds inoculated with E. coli 0157:H7 were exposed to the 10x and 15x
concentrations for 1 to 10 minutes. Both concentrations decreased E. coli populations
> 4.90 logs, in a first set of experiments (Table 4.4) and > 5.45 logs (Table 4.5), in a
second set of experiments. When the concentration was reduced to 5x, 3.23 and 2.68 log
reductions were observed in the E. coli 0157:H7 population after 5 and 10 minutes of
exposure, respectively (Table 4.4). Significant differences (p < 0.05) were found between
the 5x concentration and the other concentrations and controls. The interaction between
concentration and time was not significant for any of the treatments applied to seeds
inoculated with E. coli 0157:H7.

S. Typhimurium DT104. Populations of S. Typhimurium DT104 also decreased
> 5 logs following 1 to 5 minutes of exposure to the 15x concentration (Table 4.6).
However, in contrast to E. coli 0157:H7, the 10x concentration was significantly less
effective against S. Typhimurium DT104 (p < 0.05) with a maximum reduction of 4.28
logs achieved after a 5 minute exposure. Inactivation of this bacterial population was a
fimction of treatment duration as expressed by a significant concentration-time

interaction.

119

TABLE 4.4.- INACTIVATION OF E. COLI 0157:H7 ON ARTIFICIALLY CONTAMINATED ALFALFA

SEEDS INOCULATED TO CONTAIN 6.20 i
CONCENTRATIONS OF A FATTY ACID-BASED SANITIZER.

0.11‘il

LOGS USING 5X, 10X AND 15X

 

E. coli 0157:H7 (log CFU/g)

 

 

Sanitizer Exposure Time After Treatment Reduction
Concentration (min)

0x 5 6.24 i 0.17a - 0.04 :1: 0.17at
(Water control) 10 6.32 :t 0.04a - 0.12 :1: 0.04a

5x 5 2.97 i- 0.17b 3.23 :1: 0.17b

10 3.52 i 0.24b 2.68 :1: 0.24b
10x 5 < 1.30 :1: 000° > 4.90 :1: 000°
10 < 1.30 :1.- 000° > 4.90 i 0.00c
15x 5 < 1.30 i 000° > 4.90 i 000°
10 < 1.30 :1: 000° > 4.90 :1: 000°

 

- Stands for an increase of the microbial load compared to the unwashed inoculated seeds control.
Means i standard deviation (n = 3)
Means with different letters are significantly diflerent (p < 0.05)

120

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122

L. monocytogenes. Results observed after disinfecting Listeria-inoculated alfalfa
seeds with a 15x concentration of the fatty-acid based sanitizer (Table 4.7) were similar to
E. coli 0157:H7 and S. Typhimurium DT104 (Tables 4.4, 4.5 and 4.6) with populations
decreasing > 6.92 logs. The 10x concentration yielded significantly lower (p < 0.05)
Listeria reductions of 2.16, 3.48 and 3.79 logs after 1, 3 and 5 min of exposure,
respectively. The concentration-time interaction was not significant.

Extent of cell injury. Regardless of exposure time, no injured cells were
detected after applying the 15x concentration to seeds inoculated with the three
pathogens. S imilar results were obtained using the 1 0x concentration on alfalfa seeds

inoculated with E. coli 0157:H7.

Optimization of the 15x fatty acid-based sanitizer for inactivation of E. coli
0157:H7, S. Typhimurium DT104 and L. monocytogenes on alfalfa seeds.

Treatment A. After identifying 15x (Treament A) as the minimum sanitizer
concentration needed to reduce the populations of all three pathogens at least 5 logs on
inoculated alfalfa seeds, the sanitizer components (PA, E 658, LA and GM) were
assessed, at their 15x concentration, individually and in various combinations to optimize
this sanitizer for inactivation of S. Typhimurium DT104, E. coli 0157:H7 and L.
monocytogenes.

Treatment B. When Treatment B (3,750 ppm PA) was applied for 3 and-5 min,
populations of S. Typhimurium DT104 and E. coli 0157:H7 (Tables 4.8 and 4.9) were
reduced similarly to the water controls with decreases ranging from 1.01 to 1.91 logs for

both pathogens.

123

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124

TABLE 4.8.- INACTIVATION OF s. TYPHIMURIUM DT 104 ON ALFALFA SEEDS INOCULATED TO
CONTAIN 7.60 i 0.50 ‘ LOGS USING PEROXYACID (PA), CAPRIC/CAPRYLIC ACID (EMERY
65 8), LACTIC ACID (LA), GLYCEROL MONOLAURATE (GM) ALONE OR IN COMBINATION.

 

 

 

Salmonella (log CFU/ g)
Sanitizer Exposure Time Afier Treatment Reduction
Treatment (min) (log CPU/g) (S) (log CF U/g)
Water control 3 6.80 i 0.17 ab 0.80 i 0.17 ab
5 6.91 21:0.15 3" 0.69:0.15ab
A-- (ASEP 1+2) 3 < 0.70 :t 0.00 ° > 6.90 i 0.00 °
5 < 0.70 i 0.00 c >6.90 i 0.00 °
B-- (ASEP 1) 5.87 :1: 0.38 b 1.73 i 0.38 b
5 6.00:0.17 b 1.80i0.17b
C--(ASEP 2) 3 1.37:1.15c 6.23:1.15c
5 2035115° 5.57:1.15C
D-- (E658 + LA) 3 < 0.70 :1: 0.00 ° > 6.90 :t 0.00 c
< 0.70 i 0.00 ° > 6.90 :1: 0.00 °
E-- (E658 + GM) 3 6.78 i: 0.69 ab 0.82 i 0.69 ab
5 6.32 i 0.28 ab 1.28 :1: 0.28 ab
F-- (GM + LA) 6.41 i 0.20 ab 1.19 i 0.20 ab
5 65010.13ab 1.10:0;13ab
(1413658) 3 7.16 i 0.14 ab 0.54 i 0.14 ab
5 7.15 :1; 0.07 ab 0.55 x 0.07 ab
H--(LA) 3 6.48:1:0.17 ab l.12:t0.17ab
6.26 i 0.23 ab 1.34 i 0.23 ab
1-- (GM) 3 7.16 :t 0.14 ab 0.44 i 0.14 ab
7.15 i 0.07 “b 0.45 i 0.07 ab

 

Means i standard deviation (n = 3).
Means with different letters are significantly different ( p< 0.05)

TABLE 4.9.- INACTIVATION OF E. COLI 0157:H7 ON ALFALFA SEEDS INOCULATED TO
CONTAIN 7.66 i 0.42 “ LOGS USING PEROXYACID (PA), CAPRIC/CAPRYLIC ACID (EMERY
65 8), LACTIC ACID (LA), GLYCEROL MONOLAURATE (GM) ALONE OR IN COMBINATION.

 

E. coli 0157:H7 (10g CFU/g)

 

 

Sanitizer Exposure Time After Treatment Reduction
Treatment (min)
Water control 3 7.38 :t 0.51 ab 0.28 a: 0.51 ab
5 7.09 i 0.11 “b 0.57 i 0.11ab
A-- (ASEP 1+3) 3 < 0.70 1: 0.00 f > 6.96 i 0.00 f
5 < 0.70 i 0.00 f >6.96 i 0.00 f
B-- (ASEP 1) 5.75 i 0.60 bc 1.91 a: 0.60 b°
5 6.65 :1: 0.27 ab 1.01 :t 0.27 ab
C-- (ASEP 2) 3 2.89 i 0.21 d" 4.77 i 0.21 d"
5 137281.15cf 62911.15cf
D-- (E658 + LA) 3 3.06 i 0.42 d° 4.60 i 0.42 de
5 <2.48:t 1.68 def >5.1811.68def

 

Means i standard deviation (n = 3).

Means with different letters are significantly different (p < 0.05)

126

Treatment C. In contrast, Treatment C (15,000 ppm B 658, 15,000 ppm LA and
7,500 ppm GM) drastically reduced the microbial load on inoculated alfalfa seeds (Tables
4.8, 4.9 and 4.10). On seeds previously inoculated with S. Typhimurium DT104,
populations decreased 6.23 and 5.57 logs after 3 and 5 min of exposure, respectively,
with these reductions not significantly different (p < 0.05) from that Observed using
Treatment A (Table 4.8). Three and 5 min Of exposure to Treatment C led to 4.77 and
6.29 log reductions in E. coli 0157:H7. Pathogen reductions Observed after 5 min Of
exposure to Treatments C and A were not significantly different (Table 4.9). Exposing
Listeria-inoculated seeds to Treatment C for 3 and 5 min reduced the pathogen
populations 3.86 and 4.21 logs, respectively. These reductions were Significantly
different (p < 0.05) from both Treatment A and the controls (Table 4.10).

Treatment D. Treatment D (15,000 ppm B 658 and 15,000 ppm LA) produced
microbial reductions that were similar to or higher than those observed using Treatment
C. Treatment D reduced Salmonella populations > 6 .90 logs, regardless O f e xposure
time, with those reductions similar to those Obtained using Treatments A and C (Table
4.8). When E. coli 0157:H7-inoculated seeds were exposed to Treatment D for 3 and 5
min, reductions of 4.60 and > 5.18 logs, respectively, were Observed (Table 4.9). These
results were statistically similar to those Obtained using Treatment C on E. coli 0157:H7-
inoculated seeds with no statistical differences seen between the 5 minute exposure for
Treatments A, C and D. Given the 3.55 and 3.17 log reduction Observed after 3 and 5
min exposures, respectively, the effect Of Treatment D on alfalfa seeds previously
inoculated with L. monocytogenes w as statistically similar to that O f T reatment C , b ut

significantly different from Treatment A (Table 4.10).

127

TABLE 4.10.- INACTIVATION OF L. MONOCYTOGENES ON ALFALFA SEEDS INOCULATED TO
CONTAIN 7 .31 i 0.098 LOGS USING PEROXYACID (PA), CAPRIC/CAPRYLIC ACID (EMERY 65 8),
LACTIC ACID (LA), GLYCEROL MONOLAURATE (GM) ALONE OR IN COMBINATION.

 

L. monocytogenes (log CFU/g)

 

 

Sanitizer Exposure Time Afier Treatment Reduction
Treatment (min)
Water control 3 6.94 1 0.28a 0.36 1 0.283
7.03 1 0.12‘al 0.27 1 0.12al
1’" (ASEP 1+2) 3 < 0.70 1 000“ > 6.61 1 0.00c
< 0.70 1 0.00c > 6.61 1 0.00c
C- (ASEP 2) 3 3.45 1 0.66b 3.86 1 0.66b
5 3.101041b 42110.41b
9' (E653 + LA) 3 3.76 1 0.58b 3.55 1 0.58b
5 4.13 1 0.74b 3.17 1 0.74b

 

Means i standard deviation (n = 3).
Means with different letters are significantly different (p < 0.05)

128

Other Treatments. The remaining treatments (B through I) were generally
ineffective, yielding results that were statistically similar to the water control (Table 4.8).
In addition, Treatment B and Treatments B through I were equally ineffective and

significantly different from Treatments A, C and D.

Washing effect of water

For alfalfa seeds inoculated with the three pathogens, microbial reductions
resulting from w ashing the s eeds in water w ere significantly different (p < 0.05) from
those Obtained using the 5x, 10x and 15x sanitizer concentrations and also from those
produced by Treatments A, C and D. No statistical differences were found between the

water controls and the corresponding inoculated seeds.

Effect of the fatty acid-based sanitizer on alfalfa seed germination

The impact Of the 15x, 10x and 5x sanitizer concentrations on seed germination
rate is presented in Tables 4.11 and 4.12. For each treatment time, germination rate
decreased as the sanitizer concentration increased; however, significant differences were
only seen when seeds were exposed to the 15x concentration for 5 and 10 minutes. NO
statistically significant differences were found between the treatments after 1 and 3
minutes Of soaking; whereas significant differences were Observed between some
treatments after 5 and 10 minutes. When Treatment A was applied for 3 minutes, a non-
statistically Significant germination loss of 15.98% was Observed (Tables 4.11 and 4.12)

indicating that this treatment will ensure a > 5 log reduction in pathogens and an

129

acceptable germination rate. In addition, a 3 minute exposure to Treatment C reduced the

germination rate by only 11.0% (Table 4.12).

Effect of the fatty acid-based sanitizer on alfalfa sprout yield

Sprout yields were usually acceptable following treatment as they did not
significantly differ from the untreated controls (Table 4.13). A 3 minute exposure tO
Treatment A led to a non-significant yield loss Of 8.5% (p < 0.05). Use of the 15x
concentration for 10 minutes was the only treatment that decreased sprout yield greater

than 10% with this treatment significantly different from the others (4.13).

130

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131

TABLE 4.12.- GERMINATION RATE (%) FOR ALFALFA SEEDS AFTER A 3-MINUTE
EXPOSURE TO TREATMENTS A AND C.

 

 

Treatment Untreated Treated Gerrmna' tion
Seeds seeds loss

A 95.92 1 0.55 a 79.94 11.81 a 15-98 at 1.81

C 90.41 1 0.72 a 79.44 1 1.54 a 10.97 i 1.54

 

Means at standard deviation (n = 4)
Means in the same row with similar letters are not significantly different (p<0.05)

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4.4.- DISCUSSION

This novel FDA-approved fatty acid-based sanitizer was evaluated, for the first
time, for inactivating bacterial pathogens on alfalfa seeds. Therefore, scientific
publications comparing the p erformance O f this p roduce s anitizer to others are 11 ot yet
available.

Overall, the bactericidal performance of the 15x concentration, against the 3
pathogens studied, on inoculated alfalfa seeds, was far superior compared to chlorine and
other commonly used chemical sanitizers. NO other chemical treatment including 20,000
ppm chlorine (as currently recommended by FDA for the sprout growing industry), Vegi-
Cleanm, Tsunami“, CloroxTM or VortexxTM are currently able to achieve a 5 log
reduction for the pathogens studied. The 10x concentration demonstrated superior
efficacy with E. coli 0157:H7 populations decreasing > 4.90 logs. Treatments C and D
were also exceptional with E. coli 0157:H7 and Salmonella reductions ranging from 4.60
tO 6.29 logs and 5.57 tO 6.90 logs, respectively.

As presented in Table 4.14, the efficacy Of the 5x concentration against E. coli
0157:H7 is comparable to that seen using calcium hypochlorite (20,000 ppm), hydrogen
peroxide (1%), trisodium phosphate (4%), VortexxTM (40 and 80 ppm) and allyl
isothiocyanate (AIT) (2 g wet seeds exposed to an atmosphere containing 53 ppm AIT).
The 5x concentration also led tO higher microbial reductions than those seen with NaClO

(200 and 20,000 ppm), TsunamiTM (80 ppm) and Vegi-CleanTM (1 and 2%) (Table 4.15).

134

TABLE 4.14.- INACTIVATION OF 2 TO 3 LOGS E. COLI 0157:H7 ON ARTIFICIALLY
CONTAMINATED ALFALFA SEEDS USING CHEMICAL SANITIZERS.

 

REFERENCE

Taormina and Beuchat (1999a)
Taormina and Beuchat (1999a)
Taormina and Beuchat (1999a)
Taormina and Beuchat (1999a)
Park et al. (2000)

SANITIZERS

Ca (0C1); (20,000 ppm)
Hydrogen peroxide (1%)
Trisodium phosphate (4%)
VortexxTM (40 and 80 ppm)
AIT (2g wet seeds / 53 ppm)

REDUCTION (Logs)
~2to3in3and10minutes
~ 3 in 3 anle minutes
~ 2 in 0.5 and 2 minutes
~ 2 in 3 and 10 minutes
~ 2 in 24 hours

TABLE 4.15.- INACTIVATION OF 5 1.70 LOGS E. COLI 0157:H7 ON ARTIFICIALLY
CONTMINATED ALFALFA SEEDS USING CHEMICAL SANITIZERS.

 

 

SANITIZERS REDUCTION (Logs) REFERENCE

NaClO (200 and 20,000 ppm) < 1.50 in 0.5 to 10 minutes Ch 3 section 3.3.4.- Table 3.1
TsunamiTM (80 ppm) > 1.70 in 3 and 10 minutes Taormina and Beuchat (1999a)
TsunamiTM (80 ppm) ~ 0.61 to 1.1 in 0.5 to 10 minutes Ch 3 section 3.3.5.- Table 3.1
TsunamiTM (800 ppm) ~ 0.46 to 0.7 in 0.5 to 10 minutes Ch 3 section 3.3.5.— Table 3.1

~ 1.50 to 2.1 in 3 and 10 minutes
< 1.50 in 0.5 to 10 minutes

Vegi-CleanTM (1 and 2%)
Ve i-CleanTM (l and 2%)

Taormina and Beuchat (1999a)
Ch 3 section 3.3.6.- Table 3.1

TABLE 4.16.- INACTIVATION OF ~1 TO 2 LOGS SALMONELLA ON ARTIFICIALLY
CONTAMINATED ALFALFA SEEDS USING CHEMICAL SANITIZERS.

 

SANITIZERS
Ca (0C1); (20,000 ppm)

TsunamiTM (530 and 1,060 ppm)

TsunamiTM (800 ppm)

Trisodium phosphate (2 and 5%)
Acid. NaClO (500 & 1,200 ppm)

VortexxTM (530 and 1,060 ppm)
Lactic acid (2%)
Acetic acid (5%)

REDUCTTON (Logs)
1.95 in 10 minutes
1.12 and 1.50, respec./10 min.
1.61 in 10 minutes
0.90 and 1.99, respec. / 10 min.
1.26 and 1.43, respec. / 10 min.
1.29 and 1.62, respec. / 10 min.
1.19 in 10 minutes
1.74 in 10 minutes

REFERENCE

Weissinger and Beuchat (2000)
Weissinger and Beuchat (2000)
Ch 3 section 3.3.5.— Table 3.2
Weissinger and Beuchat (2000)
Weissinger and Beuchat (2000)
Weissinger and Beuchat (2000)
Weissinger and Beuchat (2000)
Weissinger and Beuchat (2000)

 

TABLE 4.17.- INACTTVATION OF ~ 2 .8 TO 3.2 LOGS SALMONELLA ON ARTIFICIALLY
CONTAMINATED ALFALFA SEEDS USING CHEMICAL SANITIZERS.

 

 

SANITIZERS REDUCTION (Logs) REFERENCE
Hydrogen peroxide (8%) 3.22 in 10 minutes Weissinger and Beuchat (2000)
Calcium hydroxide (1%) 2.84 in 10 minutes Weissinger and Beuchat (2000)

Calcinated calcium (1%)
Lactic (5%)
Citric acid (5%)

2.88 in 10 minutes
2.98 in 10 minutes
2.98 in 10 minutes

Weissinger and Beuchat (2000)
Weissinger and Beuchat (2000)
Weissinger and Beuchat (2000)

 

135

Reductions for E. coli 0157:H7 on alfalfa seeds using Treatment B (3,750 ppm
peroxyacid) are similar to those Obtained by Taormina and Beuchat (1999a) using
peroxyacid-based sanitizers such as TsunamiTM (80 ppm) and VortexxTM (40 and 80 ppm)
(Tables 4.14 and 4.15). However, as evident from Table 4.15, increasing the peroxyacid
concentration exponentially does not result in higher reductions.

The bactericidal efficacy of the 10x sanitizer concentration against Salmonella on
alfalfa seeds increased progressively with duration of exposure. A 1 -minute exposure
yielded a 1.50 log reduction in Salmonella which is Similar to that seen using calcium
hypochlorite (20,000 ppm), trisodium phosphate (2 and 5%), acidified NaClO (500 and
1,200 ppm), VortexxTM (530 and 1,060 ppm), acetic acid (5%) and lactic acid (2%)
(Table 4 .16). S imilar efficacy was also demonstrated by Treatment B with reductions
comparable to those seen using TsunamiTM (530 and 1,060 ppm) and VortexxTM (530 and
1,060 ppm) (Table 4.16). Reductions Obtained after 3 and 5 minutes Of exposure to
Treatment H (1.5% lactic acid) agree with those obtained by Weissinger and Beuchat
(2000) after applying 2% lactic acid to alfalfa seeds contaminated with Salmonella (Table
4.16).

When the 10x concentration of this fatty-acid based sanitizer was applied for 3
minutes, Salmonella populations decreased 3.71 logs which is equal to or greater than the
reductions obtained after treatment with hydrogen peroxide (8%), calcium hydroxide
(1%), c alcinated c alcium (1%), lactic (5%) and citric acid (5%) (Table 4.17). Similar
reductions were Obtained using heat (57 to 60 °C) on alfalfa seeds, with Salmonella

populations decreasing < 3 logs, afier 5 minutes (J aquette et al., 1996).

136

Given the 4.28 log reduction observed for Salmonella, efficacy Of the 10x
concentration, when applied for 5 minutes, exceeded that of the other treatments in Table
4.17.

J aquette et al. (1996) stated that seeds are covered with a waxy material that repels
water. This waxy covering limits the efficacy Of the aforementioned chemical sanitizers
(Tables 4 .14 to 4 .17) w hen c ompared to the p otency O f this fatty acid-based s anitizer.
While readily soluble in water, bactericidal potency of these other chemical sanitizers is
partly based on the extent of contact with surface and internalized bacteria. This may
explain why, after a certain dose, higher sanitizer concentrations do not lead to higher
microbial reductions. Fatty acids are non-polar and, as such, have more affinity with the
waxy m aterial c overing s eeds. T he c omponents O f t his s anitizer are insoluble in water
and, upon rapid agitation, form an emulsion, with microscopic droplets adhering to the
seed surface. Thus, this more intimate contact allows the active ingredients to more
easily penetrate the seed and disrupt the bacterial cell membranes.

Peroxyfatty acids form peroxides and other free radicals which have the ability to
inhibit bacterial growth (Kabara, 1979; Hinton and Ingram, 2000). P eroxyacetic acid,
like other peroxides and oxidizing agents, is assumed to oxidize sensitive sulfhydryl and
sulfur bonds in proteins, enzymes and other metabolites. Peroxyacetic acid may also
disrupt the chemiosmotic Motion of the lipoprotein cytoplasmic membrane and transport
through dislocation or rupture Of cell walls (Baldry and Fraser, 1988; Block, 1991).

Fatty acids typically function as surface-active anionic detergents (Kabara et al.,
1972) and are capable of uncoupling both substrate transport and oxidative

phosphorylation from the electron transport system (Freese et al., 1973). T hey act by

137

disrupting the bacterial cell membrane and lysing protoplasts as evidenced by the leakage
of 260 nm-absorbing material, protein and other internal metabolites. Thus, fatty acids
prevent bacterial growth by modifying cell membrane permeability which leads to uptake
changes and/or inhibition of oxygen, amino-acids, nucleic acids, organic acids,
phosphates and other substrate molecules (Freese et al., 1973; Kabara, 1979; Oh and
Marshall, 1992, 1993; Galbraith and Miller, 1973 a,b,c; Doores, 1983, 1993; Hinton and
Ingram, 2000). Although saturated fatty acids inhibit cellular oxygen consumption, they
do not inhibit NADH oxidation by isolated membranes which is controlled by the
cytochrome-linked electron transport system. Therefore, inhibition Of oxygen
consumption in whole cells must result fi'om the deficiency of compounds that yield
electrons entering the electron transport chain (Freese et al., 1973). Inhibition of cellular
uptake, however, does not necessarily prove that transport itself is inhibited; it may
merely reflect inhibition of metabolism with the unmetabolized compound preventing its
own uptake (Freese et al., 1973).

Only certain short chains fatty acids generally affect gram-negative organisms
(Sheu and Freese, 1973; Kabara et al., 1977). Fatty acids, up to eight carbon in length
which completely inhibit E. coli do not reduce the concentration Of ATP/A600 measured
after 20 or 40 minutes of incubation (Freese et al., 1973). Apparently, in E. coli, some
other compound is exhausted before ATP (Freese et al., 1973). When compared to C6
fatty acids, twice the concentration of C3 and about 50 times the concentration of C10 is
required to inhibit E. coli with no inhibition Observed with longer chain fatty acids
(Freese et al., 1973). As a possible explanation, the lipopolysaccharide (LPS) layer that

typically surrounds the cell wall Of gram-negative organisms may screen out the larger

138

compounds (Freese et al.,l973, Doores, 1983). The short chain fatty acids (caprylic C3
and capric C10) used in this sanitizer are within the lethal range for gram-negative bacteria
such as E. coli 0157:H7 and Salmonella. However, application of 15,000 ppm B 658 on
alfalfa seeds artificially contaminated with S. Typhimurium DT104 did not result in
statistically significant reductions when compared to the water control (Table 4.8). These
Observations appear more in accordance with another theory proposing that gram-
negative organisms can rapidly metabolize fatty acids with little or no accumulation
within the cell (Kabara et al. 1972; Freese etal., 1973; Doores, 1983 and 1993).

Most components of this antimicrobial sanitizer including the fatty acids are
organic acids, and as such, are weak acids. In aqueous solution, weak acids are only
slightly ionized and do not readily give up their proton(s) to water (Doores, 1983 and
1993). At a pH lower than the pkg, the equilibrium shifts toward the undissociated state.
Freese et al. (1973), Woolford (1975) and Doores (1983 and 1993) reported that the
antibacterial efficacy of weak acids was a fimction of the undissociated molecule.
Membranes are less permeable to charged as compared to uncharged molecules (Doores,
1983 and 1993). Therefore, bacterial inhibition by organic acids increases with decreasing
pH, in agreement with the pk, values (Freese et al., 197 3; Doores, 1983 and 1993).

According to Galbraith and Miller (1973) and Oh and Marshall (1992, 1993),
lowering the pH Of the suspending medium increases fatty acid uptake and reduces the
interfacial tension at the bacterial lipid membrane/aqueous medium interface. This
Observation s uggests that addition 0 f lactic acid to E mery 6 58 w hich resulted in a pH

decrease from 3.2 tO 2.5 (Table 4.3), may partially account for the dramatic reduction in

139

microbial load on alfalfa seeds previously inoculated with S. Typhimurium DT104 (Table
4.8).

Hunter and Segel (1973) and Doores, (1983 and 1993) reported that weak acids at
or below their pka could discharge the proton gradient and ionize within the cell to acidify
the interior. It was postulated that the rate Of proton leakage into the cell versus proton
ejection would determine the extent of inhibition (Freese et al., 1973; Doores, 1983).
Peracetic acid, upon contact with organic substrates, decomposes tO yield oxygen and
acetic acid (Doores, 1983). Antibacterial action Of acetic acid is partially due to lowering
of the intracellular pH below that which is optimal for growth. The same is also true for
lactic acid used alone as a microbicide. Indeed, cellular proteins, nucleic acids and
phospholipids can be structurally altered by pH changes (Doores, 1983 and 1993).

Lactic acid also acts as a chelating agent in this fatty acid-based sanitizer. The
cation chelating properties Of lactic acid are comparable to that of other metal chelators
such as ethylenediaminetetraacetic acid (EDTA), citric acid, sodium acid pyrophosphate
and polyphosphoric acid (Guthery, 1993 and 1994; Andrews, 1996).

Antimicrobial activity Of the fatty acids and fatty acid esters increases
dramatically when calcium or magnesium chelators are used to lower the pH to < 4.0
(Andrews, 1996; Guthery, 1993 and 1994). Although active against gram-positive
bacteria, fatty acids are not effective by themselves against gram-negative bacteria at a
neutral pH (Guthery, 1993). However, according to Guthery (1993), the effectiveness of
fatty acids against gram-negative bacteria, such as Salmonella, is enhanced in the

presence of citric acid.

140

Gram-negative bacteria have a lipopolysaccharide (LPS) layer which acts as a
permeability barrier to prevent some compounds from penetrating the cell membrane.
When added to foods, chelating compounds can sequester trace metals such as
magnesium, calcium, iron, sodium and potassium cations present in the LPS layer of
gram-negative bacteria (Boland et al., 2003). Chelation of metal ions from
microorganisms, specifically gram-negative bacteria, increases the antimicrobial spectrum
Of bactericidal compounds by apparently destabilizing the LPS layer and increasing cell
sensitivity by allowing these aforementioned compounds to penetrate the LPS layer,
resulting in cell lysis (Boland et al., 2003). Magnesium assists in cell division and many
membrane-bound enzyme reactions (Knabel et al., 1991; Boland et al., 2003).
Magnesium aids calcium in cross-linking negatively charged groups to form salt bridges
to bind polysaccharides on the s urface O f gram-negative b acteria. F ormation O f m etal
complexes with magnesium and calcium can result in leakage Of cell solutes and loss Of
viability. Iron is essential for growth, replication, respiration and DNA synthesis in
bacterial cells. Chelation of such essential cations from the LPS layer may disrupt the
structural or functional integrity Of the LPS, thus allowing other antimicrobial compounds
to penetrate the cell wall (Boland et al., 2003).

The theory Of lactic acid acting as a chelator of metal cations, increasing
synergistically the antimicrobial effect Of short chain fatty acids (Emery 658) against
gram-negative bacteria is supported by the dramatic reduction Observed when alfalfa
seeds inoculated with S. Typhimurium DT104 and E. coli 0157:H7 were exposed to
Treatment D (Tables 4.8 and 4 .9). T his Chelation m echanism appears to b e primarily

responsible for antimicrobial activity since the outcome of Treatment D was not

141

significantly different fi'om Treatments A and C for S. Typhimurium and, generally, not
significanly different from those of Treatments A and C for E. coli 0157:H7. Knabel et
al., (1991) Observed that chelators, such as polyphosphates, can inhibit gram-positive
bacteria by removing essential metals from cation-binding sites within their walls. This
explaination may also account for the reduction, although more modest, in the bacterial
load present on alfalfa seeds artificially contaminated with L. monocytogenes (Table
4.10).

Glycerol monolaurate was reported by Vadehra et al. (1985) and Oh and Marshall
(1992) to cause extensive leakage of 260 nm-absorbing intracellular proteins from
bacterial cells. However, in our study, 7,500 ppm GM did not result in statistically
Significant reductions in S. Typhimurium DT104 when compared to the inoculated seed
and water controls. These results agree with those of Venkitanayaranan et al. (1999) in
that 50 ppm GM did not yield a 5 log reduction for E. coli 0157:H7 when suspended in
0.1% peptone. Although a lower pH may increase the uptake of glycerol monolaurate
(Oh and Marshall, 1993), efficacy of GM was not increased by adding 15,000 ppm lactic
acid to 7,500 ppm GM or by lowering the pH of the treatment solution from 4.6 to 2.5.

According to Kabara (1979) and Oh and Marshall (1992), GM has broad spectrum
antimicrobial activity in culture media against gram-positive microorganisms. Therefore,
the poor performance Of GM (ester of C12 fatty acid) against gram-negative bacteria,
including S. Typhimurium DT104 and E. coli 0157:H7, agrees with the aforementioned
observation in that inhibition of gram-negative bacteria is increasingly inefficient as the
fatty acid chain length increases from C6 to Cm and higher (Freese et al., 1973). In

contrast, Kato and Shibasaki (1976) and Oh and Marshall (1992) showed that GM is also

142

effective against gram-negative bacteria in culture media containing citric and
polyphosphoric acid, both of which are chelators Of metallic ions (Andrews, 1996).
Given that the combination GM and lactic acid (Treatment F) was less than Optimal
against Salmonella on alfalfa seeds (Table 4.8), being an ester Of a C12 chain fatty acid, it
may have been less effective against this gram-negative pathogen due to its longer chain
length compared to Emery 658. Moreover, organic components of the seed itself may
have neutralized the antimicrobial potency of GM since Shibasaki and Kato (1978) stated
that the activity of GM was neutralized by addition of starch and gelatin.

Fatty acids and their related compounds disrupt bacterial respiration (Freese et al.,
1973). However, they may also adversely affect the respiratory mechanism in seeds with
the low pH of the sanitizer solution (Table 4.3) perhaps accounting for the loss in
germination.

Chemical sanitizing Of alfalfa seeds usually results in a lower germination rate.
Salmonella populations decreased 1.62 logs using 1,060 ppm VortexxTM (Table 4.16) and
3.22 logs using 8% hydrogen peroxide (Table 4.17) with no effect on germination
(Weissinger and Beuchat, 2000). The same authors experienced germination losses of
45.6%, 35.5% and 10.9% after application of 5% of acetic acid, lactic acid or citric acid,
respectively, for corresponding Salmonella reductions Of 1.74, 2.98 and 2.98 logs.
Weissinger and Beuchat (2000) also reported a 1.95 log decrease in Salmonella using
20,000 ppm Ca(ClO); (Table 4.4.3) with a significantly lower germination rate Of 91.6%
compared to the control. Beuchat et a1. (2001) observed a germination loss of 10 to 12%
using FitTM or 20,000 ppm chlorine for 30 minutes which led to a 2.3 log reduction in

Salmonella. Afier treating alfalfa seeds with 20,000 ppm Ca(ClO)2, Taormina and

143

Beuchat (1999a) reported a 2.93 log reduction in E. coli after 3 minutes (Table 4.14)
along with a 70.3 % germination rate compared to an initial rate Of 78.3%. A 10 minute
exposure to 70% ethanol reduced the numbers of mesophilic aerobic bacteria 2 to 4 logs
on rice, but only 11.5 % of rice seeds germinated afier treatment, producing abnormal
seedlings (Piemas and Guiraud, 1997).

These Observations lead to the conclusion that the non statistically significant
15.98% germination loss Observed after a 3 minute exposure to the 15x concentration Of
our fatty acid-based sanitizer (Treatment A) was acceptable considering that this
treatment reduced populations of all three pathogens ~ 5 to 7 logs. However, the 3
minute exposure to Treatment C, resulting in a non statistically Significant 10.97%
germination loss, led to reductions Of 6.23, 4.77 and 3.86 logs for S. Typhimurium
DT104, E. coli 0157:H7 and L. moncytogenes, respectively. Hence, Treatment C may be
a reasonable compromise between pathogen inactivation and germination rate
preservation, since a pathogen population of 2.5 logs on alfalfa seeds would be
considered large in a commercial setting and, in reality, would be unlikely to occur. On
alfalfa seeds, pathogen populations would likely be at least lOO-fold lower (Jaquette et
al., 1996). C onsidering that microbial p opulation O n alfalfa s eeds are u sually < 2 log
CFU/g and that Listeria has never been involved in any alfalfa sprout related outbreaks,
the 3 minute exposure to Treatment D which decreased S. Typhimurium DT104, E. coli
0157:H7 and L. monocytogenes populations > 6.90, 4.60 and 3.55 logs, respectively, may
be the best option for sprout growers.

Given that no statistical difference in pathogen reduction was seen for Treatments

A, C and D, the combination Of Emery 658 and lactic acid is primarily responsible for

144

inactivating S. Typhimurium DT104 and E. coli 0157:H7. Therefore, further
investigations should be done to determine the Optimum ratio between the
aforementioned compounds for attaining a > 5 log reduction in S. Typhimurium DT 104
and E. coli 0 157:H7 on alfalfa seeds while maintaining a germination loss Of < 10%.
Moreover, it is surmised that applications of this novel FDA-approved fatty acid-based
sanitizer, as a whole, and the combination Of Emery 658 and lactic acid, in particular,

should prove to be Of great interest to sprout growers.

145

5.- CONCLUSION

Exposing alfalfa seeds and sprouts previously inoculated with Escherichia coli
0157:H7, Salmonella Typhimurium DT104 and Listeria monocytogenes to CloroxTM
(sodium hypochlorite, 200 to 20,000 ppm), TsunamiTM (peroxyacetic acid / hydrogen
peroxide, 80 and 800 ppm) or Vegi-CleanTM (anionic surfactant, 1%, 2%, 5%), applied for
30 seconds to 10 minutes did not 0 ffer a c ommercially desirable altemative( > 5 log
reduction) tO the currently FDA-recommended 20,000 ppm calcium hypochlorite.
Maximum reductions in E. coli 0157:H7 and S. Typhimurium populations on the seeds
were, respectively, 1.57 and 1.92 logs afier treatment with Cloroxm, 1.11 and 3.32 logs,
after treatment with TsunamiTM and 1.60 and 1.32 logs after treatment with Vegi-CleanTM
compared to ~ 2 to 3 and 1.95 logs after treatment with 20,000 ppm chlorine. Only
sodium hypochlorite ( > 1,000 ppm) was able to reduce the three pathogens on sprouts >5
logs, however, these sprouts were deemed to be organoleptically unacceptable due to
osmotic dehydration and bleaching.

When used alone, ultrasound (20 kHz) generated by a sonicating water bath or
copper ions(1ppm) generated by an electrolytic process and dispersed into a circulating
water stream were no better than water for decreasing pathogen populations on inoculated
alfalfa seeds and sprouts. In addition, sonication or copper ion failed to enhance the
efficacy of Clorox”, TsunamiTM or Vegi-Cleanm.

Presence of hidden bacteria in crevices and internalization Of bacterial
pathogens in inner tissues with organisms remaining protected in seeds and sprouts
likely played a role in protecting these organisms fiom the harmfiil effects Of these

sanitizers. These Observations help to explain the inability Of sonication to efficiently

146

detach bacteria from seeds and sprouts, as well as the failure of the ionization state Of
the copper ion solution to increase contact between surface pathogens and the chemical
sanitizers. In contrast, NaClO at concentrations higher than 1,000 ppm was able to
enter the sprout tissues by means Of a strong osmotic pressure. This was inferred by the
fact that, after soaking, the sprouts appeared thoroughly dehydrated and completely
bleached. This Observation may explain how this sanitizer was able to enter the tissues
to destroy these pathogens in sprouts as Opposed to seeds.

Further research indicated that a novel FDA-approved fatty acid based-sanitizer
containing a combination Of 3,750 ppm peroxyacid (PA), 15,000 ppm caprylic and
capric acid (Emery 658), 15,000 ppm lactic acid (LA) and 7,500 ppm glycerol
monolaurate (GM) provided a successful alternative to the currently recommended
20,000 ppm calcium hypochlorite with populations Of E. coli 0157:H7, S.
Typhimurium DT 104 and L. monocytogenes decreasing >5.45, >562 and >6.92 logs,
respectively, after 3 minutes Of exposure. Additionally no injury and no significant loss
in seed germination rate or sprout yield was seen.

When the components of this sanitizer were assessed alone and in various
combinations to Optimize inactivation Of the three pathogens, the combination Of 15,000
ppm Emery 658, 15,000 ppm LA and 7,500 ppm GM, applied for 3 and 5 minutes,
decreased S. Typhimurium DT104 population 6.23 and 5.57 logs, respectively, and E. coli
0157:H7 population 4.77 and 6.29 logs, respectively, on inoculated alfalfa seeds. The
combination 0 f 1 5,000 p pm E mery 6 58 and 1 5,000 p pm LA r educed S. T yphimurium
DT104 > 6.90 logs after all exposures and E. coli 0157:H7 4.60 and > 5.18 logs after 3

and 5 minutes, respectively. NO significant differences were found between these 3

147

sanitizer combinations. Overall, the E 658 and LA combination was most effective in

reducing E. coli and Salmonella populations ~ 5 logs and represents the most viable

alternative to the recommended 20,000 ppm chlorine. Lactic acid likely acts as a chelator

Of metal cations, increasing 5 ynergistically the antimicrobial e ffect O f s hort c hain fatty

acids (E 658) by destabilizing the bacterial cell lipopolysaccharide layer and allowing the

fatty acid to penetrate the wall.

Based on these findings, this novel FDA-approved fatty acid-based sanitizer and
the combination Of Emery 658 and lactic acid, in particular, should be Of great interest to
sprout growers, in particular and to the produce industry, in general.

Future research g oals focusing O n the microbial safety 0 f alfalfa S prouts w ould
include the following:

0 Determination of the most efficacious ratio between lactic acid and Emery 658 to
improve inactivation Of E. coli 0157:H7, S. Typhimurium DT104 and L.
monocytogenes while maintaining an acceptable germination rate for alfalfa seeds.

0 Evaluation of the microbial load in alfalfa Sprouts grown from seeds previously
inoculated to several levels of pathogen and disinfected with treatment the Optimized
lactic acid and Emery 658 mixture.

- Scale-up Of the FDA-approved sanitizer treatment for commercial Sprout growers.

0 Assessment of pathogen penetration into alfalfa seeds and subsequent inactivation by

the FDA-approved sanitizer using confocal microscopy.

148

APPENDIX A

149

REDUCTION OF MESOPHILIC AEROBIC BACTERIA ON ALFALFA SEEDS AND SPROUTS
PREVIOUSLY INOCULATED WITH ESCHERICHIA coLI 0157:H7, SALMONELLA
TYPHIMURIUM DT104 AND LISTERIA MONOCYTOGENES USING COMMERCIAL CHEMICAL
SANITIZERS (CLOROXTM, TSUNAMITM, VEGI—CLEANT") AND SONICATION OR COPPER
IONS.

TABLE A.1.- REDUCTION OF MESOPHILIC AEROBIC BACTERIA (loglo CFU/g) ON ALFALFA
SEEDS PREVIOUSLY INOCULATED WITH ESCHERICHIA COLI 0157:H7 USING VARIOUS
ANTIMICROBIAL TREATMENTS.

 

 

TREATMENT TIME
30 sec 1 min 3 min 5 min 10 min

Sonication alone 0.71 0.80 0.28
Water control * 0.63 0.68 0.30
Clorox 200 ppm 1.15 1.17 0.51
Water control 0.63 0.68 0.30
Clorox 20,000 ppm 1.41 1.15 1.41 1.45 1.67
Water control 0.17 0.32 0.21 0.24 0.19
Clorox 200 ppm + sonication 1.59 1.33 0.94
Water control 0.63 0.68 0.30
Tsunami 80 ppm 1.75 0.57 0.52
Water control 0.63 0.68 0.30
Tsunami 800 ppm 0.71 0.67 0.72 0.82 0.78
Water control 0.17 0.32 0.21 0.24 0.19
Tsunami 80 ppm + sonication 1.68 1.39 1.07
Water control 0.63 0.68 0.30
Vegi-Clean 1% 1.41 1.31 0.97
Water control 0.63 0.68 0.30
Vegi-Clean 2% 0.71 0.78 0.87 0.74 1.01
Water control 0.17 0.32 0.21 0.24 0.19
Vegi-Clean 1% + sonication 1.23 1.57 1.50
Water control 0.63 0.68 0.30
Copper ion 1 ppm 0.43 0.17
Water control 0.63 0.68 0.30
Copper ion + sonication 1.07 0.75
Water control 0.63 0.68 0.30

 

"‘ All water control data in this table have to be compared to the treatment results reported on the row placed
above them.

150

TABLE A.2.- REDUCTION OF MESOPHILIC AEROBIC BACTERIA (logIo CFU/g) ON ALFALFA
SEEDS PREVIOUSLY INOCULATED WITH SALMONELLA TYPHIMURIUM DT104 USING VARIOUS
ANTIMICROBIAL TREATMENTS.

 

 

TREATMENT TIME
30 sec 1 min 3 min 5 min 10 min

Sonication alone 0.42 0.39 0.41 0.58 0.39
Water control * 0.39 0.20 0.28 0.16 0.28
Clorox 200 ppm 1.45 1.69 1.66 1.16 1.33
Water control 0.39 0.20 0.28 0.16 0.28
Clorox 20,000 ppm 0.59 0.56 0.58 0.67 0.73
Water control 0.46 0.91 0.81 0.93 0.76
Clorox 200 ppm + sonication 1.47 1.45 1.24 1.85 1.61
Water control 0.39 0.20 0.28 0.16 0.28
Tsunami 80 ppm 1.68 2.98 2.98 2.98 2.98
Water control 0.39 0.20 0.28 0.16 0.28
Tsunami 800 ppm 1.42 1.46 1.08 1.03 1.73
Water control 0.46 0.91 0.81 0.93 0.76
Tsunami 80 ppm + sonication 2.49 1.47 0.76 1.85 1.86
Water control 0.39 0.20 0.28 0.16 0.28
Vegi-Clean 1% 1.48 1.47 1.47 1.36 0.98
Water control 0.39 0.20 0.28 0.16 0.28
Vegi-Clean 2% 1.55 1.00 2.55 2.61 2.35
Water control 0.46 0.91 0.81 0.93 0.76
Veg-Clean 1% + sonication 1.20 1.51 2.02 1.63 1.35
Water control 0.39 0.20 0.28 0.16 0.28
Copper ion 1 ppm 1.13 0.49 0.47 0.42 0.42
Water control 0.39 0.20 0.28 0.16 0.28
Copper ion + sonication 0.39 0.85 0.49 0.64 0.51
Water control 0.39 0.20 0.28 0.16 0.28

 

* All water control data in this table have to be compared to the treatment results reported on the row placed
above them

151

TABLE A.3.- REDUCTION OF MESOPHILIC AEROBIC BACTERIA (10810 CFU/g) ON ALFALFA
SPROUTS PREVIOUSLY INOCULATED ESCHERICHIA COLI 0157:H7 USING VARIOUS
ANTIMICROBIAL TREATMENTS.

 

 

TREATMENT TIME
30 sec 1 min 3 min 5 min 10 min

Clorox 1,000 ppm 3.57 4.24 4.78 5.49
Water control" 1.08 1.02 0.79
Clorox 2,000 ppm 3.67 5.02 5.77
Water control 1.08 1.02 0.79
Clorox 10,000 ppm 3.94 3.90 4.29 4.67 5.73
Water control 0.81 0.70 0.75 0.77 0.76
Clorox10,000 ppm+Cu ion 3.86 3.89 4.61 5.18 5.25
Water control 0.81 0.70 0.75 0.77 0.76
Vegi-Clean 5% 1.31 1.76 1.73 2.08 2.70
Water control 0.43 0.80 0.60 0.38
Vegi-Clean 5%+copper ion 1.40 1.76 1.80 1.88 2.36
Water control 0.43 0.80 0.60 0.38

 

"' All water control data in this table have to be compared to the treatment results reported on the row placed
above them

152

TABLE A.4.- REDUCTION OF MESOPHILIC AEROBIC BACTERIA (logIo CPU/g) ON ALFALFA
SPROUTS PREVIOUSLY INOCULATED WITH SALMONELLA TYPHIMURIUM DT104 USING
VARIOUS ANTIMICROBIAL TREATMENTS.

 

 

TREATMENT TIME
30 sec 1min 3 min 5 min 10min

Sonication alone 0.51 0.78 0.87
Water control * 0.61 0.41 0.78 0.70 1.07
Clorox 200 ppm 0.82 1.36 1.60 2.27 2.20
Water control 0.61 0.41 0.78 0.70 1.07
Clorox 20,000 ppm 3.80 3.38 2.90 3.10 5.34
Water control 0.38 0.57 0.47 0.43 0.42
Clorox 200 ppm + sonication 1.22 1.34 2.38 2.85 2.73
Water control 0.61 0.41 0.78 0.70 1.07
Tsunami 80 ppm 0.94 0.85 0.89 0.92 1.15
Water control 0.61 0.41 0.78 0.70 1.07
Tsunami 800 ppm 2.08 2.23 2.78 3.04 3.18
Water control 0.38 0.57 0.47 0.43 0.42
Tsunami 80 ppm + sonication 0.85 1.15 2.31 1.46 1.51
Water control 0.61 0.41 0.78 0.70 1.07
Vegi-Clean 1% 0.82 1.25 1.50 1.39 1.67
Water control 0.61 0.41 0.78 0.70 1.07
Vegi-Clean 2% 1.15 1.30 1.43 1.39 1.67
Water control 0.38 0.57 0.47 0.43 0.42
Veg-Clean 1% + sonication 0.70 1.30 1.51 1.57 2.58
Water control 0.61 0.41 0.78 0.70 1.07
Copper ion 1 ppm 0.73 0.33 0.48 0.42 0.82
Water control 0.61 0.41 0.78 0.70 1.07
Copper ion + sonication 0.22 0.34 0.45 0.77
Water control 0.61 0.41 0.78 0.70 1.07

 

* All water control data in this table have to be compared to the treatment results reported on the row placed
above them.

153

TABLE A.5.- REDUCTION OF MESOPHILIC AEROBIC BACTERIA (logIo CPU/g) ON ALFALFA
SPROUTS PREVIOUSLY INOCULATED WITH LISTERIA MONOCYTOGENES USING VARIOUS
ANTIMICROBIAL TREATMENTS.

 

 

TREATMENT TIME

30 sec 1min 3 min 5 min 10min
Clorox 20,000 ppm 2.46 3.47 3.59 4.59 5.53
Water control“ 0.64 0.45 0.49 0.53 0.40
Vegi-Clean 2% 0.97 1.18 1.60 1.85 1.90
Water control 0.64 0.45 0.49 0.53 0.40

 

* All water control data in this table have to be compared to the treatment results reported on the row placed
above them.

154

APPENDIX B

155

EFFICACY OF A F ATTY A CID-BASED s ANITIZER T O I NACTIVATE M ESOPHILIC A EROBIC
BACTERIA ON ALFALFA SEEDS ARTIFICIALLY CONTAMINATED ESCHERICHIA coLI
0157:H7, SALMoNELLA TYPHIMURIUM DT104 AND LISTERIA MONOCYTOGENES.

TABLE B.1.- INACTIVATION OF MESOPHILIC AEROBIC BACTERIA ON ALFALFA SEEDS
ARTIFICIALLY CONTAMINATED WITH E. COLI 0157:H7 USING 5x, 10x AND 15x
CONCENTRATIONS OF A PATTY ACID-BASED SANITIZER.

 

Mesophilic Aerobic Bacteria (log CFU/g)

 

 

Sanitizer Exposure Time After Treatment Reduction
Concentration (min)
0x 5 7.05 1 0.10a 0.12 1 0.10a
(Water control) 10 6.99 1 0.05al 0.18 1 0.05“1
5x 5 4.65 1 0.04b 2.52 1 0.04b
10 4.98 1 0.01b 2.19 1 0.01b
10x 5 < 1.30 1 0.00c > 5.88 1 0.00c
10 <1.76 1 0.47c > 5.421 047°
15x 5 < 1.30 1 0.00c > 5.88 1 0.00c
10 < 1.30 1 0.00c > 5.88 .1 0.0.0c

 

Inoculated seeds average: 7.18 i 0.04‘3 log CFU/g

Means 1: standard deviation (n = 3)
Means with different letters are significantly different (p < 0.05)

156

TABLE B.2.— INACTIVATION OF MESOPHILIC AEROBIC BACTERIA ON ALFALFA SEEDS
ARTIFICIALLY CONTAMINATED WITH E. COLI 0157:H7 USING 10x AND 15x
CONCENTRATIONS OF A FATTY ACID-BASED SANITIZER.

 

Mesophilic Aerobic Bacteria (log CPU/g)

 

 

Sanitizer Exposure Time Afier Treatment Reduction
Concentration (min)

0" 1 6.91 1 0.051“ 0.27 1 0.053
(Watercomml) 3 6.83 1 0.11a 0.34 1 0.113
5 6.90 1 0.08a 0.27 1 0.08al
10 6.97 1 0.083 0.20 1 0.08al
10x 1 b b

< 1.16 1 0.46 >6.021 0.46
< 0.70 1 0.00b >6.481 0.00b
< 0.70 1 0.00b >6.481 0.00b
‘0 < 1.16 1 0.46b >6.021 0.46b
15x 1 b b

< 0.70 1 0.00 >6.481 0.00
3 < 0.70 1 0.00b >6.481 0.00b
< 0.70 1 0.00b >6.481 000"
10 < 0.70 1 0.00b >6.481 0.00b

 

Inoculated seeds average: 7.18 i 0.08a log CFU/g

Means i standard deviation (n = 3)
Means with different letters are significantly different (p < 0.05)

157

TABLE B.3.— INACTIVATION OF MESOPHILIC AEROBIC BACTERIA ON ALFALFA SEEDS
ARTIFICIALLY CONTAMINATED WITH SALMONELLA TYPHIMURIUM DT 104 USING 10x AND

15X CONCENTRATIONS OF A FATTY ACID-BASED SANITIZER.

 

Mesophilic Aerobic Bacteria (log CFU/g)

 

 

Sanitizer Exposure Time After Treatment Reduction

Concentration (min)

0* 1 7.45 1 0.04a 0.08 1 004°

(Water 00111101) 3 7.41 1 0.07al 0.12 1 007°1

5 7.49 1 0.09a 0.04 1 009°

10* 1 4.91 1 026" 2.62 1 0.26b

3 2.95 1 033° 4.58 1 033°

5 2.18 1 0.04d 5.36 1 0.04d

15* 1 < 0.70 1 000° >683 1 000°

< 0.70 1 000° >6.83 1 000°

5 < 0.70 1 000° >683 1 000°

 

Inoculated seeds average: 7.53 i 0.02‘1 log CFU/g

Means 2t standard deviation (n = 3)
Means with different letters are significantly different (p < 0.05)

158

 

TABLE B.4.- INACTIVATION OF MESOPHILIC AEROBIC BACTERIA ON ALFALFA SEEDS
ARTIFICIALLY CONTAMINATED WITH LISTERIA MONOCYTOGENES USING 10X AND 15X
CONCENTRATIONS OF A FATTY ACID-BASED SANITIZER.

 

Mesophilic Aerobic Bacteria (log CFU/g)

 

 

Sanitizer Exposure Time After Treatment Reduction

Concentration (min)

0* 1 6.91 1 002° 0.65 1 002°

(W3ter 00111101) 3 6.94 1 009° 0.61 1 009°

7.00 1 003° 0.56 1 003°

10" 1 5.54 1 037° 2.01 1 037°

3 4.57 1 042° 2.99 1 042°

5 4.32 1 036° 3.23 1 036°

15* 1 < 0.70 1 0.00c > 6.86 1 000°

3 < 0.70 1 000° > 6.86 1 000°

< 0.70 1 000° > 6.86 1 000°

 

Inoculated seeds average: 7.56 i 0.09a log CFU/g

Means 1: standard deviation (n = 3)
Means with difi‘erent letters are significantly different (p < 0.05)

159

 

TABLE B.5.- INACTIVATION OF MESOPHILIC AEROBIC BACTERIA ON ALFALFA SEEDS
PREVIOUSLY INOCULATED WITH S. TYPHIMURIUM DT 104 USING PEROXYACID (PA),
CAPRIC/CAPRYLIC ACID (EMERY 658), LACTIC ACID (m), GLYCEROLMONOLAURATE (GM)
ALONE ORINCOMBINATION. -

 

Mesophilic Aerobic Bacteria (log CFU/g)

 

 

Sanitizer Exposure Time After Treatment Reduction
Treatment (min) (log CFU/g) (S) (log CFU/g)
Water control 3 757 i 0.28 abc 0.75 :1: 0.28
5 7.52 1 0.09 abc 0.80 1 0.09
A.- (ASEP 1+2) 3 0.70 1 0.00 h > 7.62 1 0.00
0.70 i 0.00 h > 7.62 :1: 0.00

B.- (ASEP 1) 3 6.52 1 0.41 W 1.80 1 0.41
5 6.07 1 0.19 “19 2.25 1 0.19

C.- (ASEP 2) 3 4.49 1 1.05 91 3.83 1 1.05
5 5.32 1 038 def 3.00 1 0.38

D.- (E658 + LA) 3 251 :1: 1.57 g 5.81 :1: 1.57
5 2.3611518" 5.961151

E.- (E658 + GM) 3 7.01 1 0.67 abcd 1.31 1 0.67
5 65110.41de 1.811041

F.- (GM + LA) 7.41 :1: 0.08 abc 0.91 :1: 0.08
7.42 1 0.08 °°° 0'90 i 0-03

G.- (E658) 3 739 1 0.13 abc 0.93 1 0.13
5 7.94 1 0.06 315° 0.38 1 0.06

H.- (LA) 73,4 1 0.23 abc 0.98 1 0.23
5 7.281011°°° 104350-11

I.- (GM) 3 7.89 1 013 ab 0.43 1 0.13
7.94 1 0.06 °° 0-33 i 0’06

 

Inoculated seeds average: 8.32 i 0.238 log CFU/g

Means i standard deviation (n = 3).
Means with different letters are significantly different ( p< 0.05)

 

TABLE B.6.- INACTIVATION OF MESOPHILIC AEROBIC BACTERIA ON ALFALFA SEEDS
PREVIOUSLY INOCULATED WITH E. coLI 0157:H7 USING PEROXYACID (PA),
CAPRIC/CAPRYLIC ACID (EMERY 658), LACTIC ACID (LA), GLYCEROLMONOLAURATE (GM)
ALONE OR IN COMBINATION.

 

Mesophilic Aerobic Bacteria (log CFU/ g)

 

 

Sanitizer Exposure Time After Treatment Reduction
Treatment (min)
Water control 3 7.77 i 0.36 ab 0.64 :1: 0.36
7.57 1 0.24 ab 0.84 1 0.24
A.- (ASEP 1+2) 3 0.70 1 0.00 f > 7.71 1 0.00
5 0.70 :1: 0.00 f > 7.71 :1: 0.00
B.- (ASEP 1) 3 6.30 1 007 be 1.61 1 0.07
5 6.28 i 054 bed 2.13 :1: 0.54
C.- (ASEP 2) 3 538 1 028 cd 2.53 1 0.28
5 3.61 21:08] e 4.80i0.81
D.- (E658 + LA) 3 5.50 1 0.25 “1 2.91 1 0.25
5 5.05 1 1.44 <16 33611.44

 

Inoculated seeds average: 8.41 i 0.12a log CFU/g

Means 1 standard deviation (n = 3).
Means with different letters are significantly different (p < 0.05)

161

TABLE B.7.- INACTIVATION OF MESOPHILIC AEROBIC BACTERIA ON ALFALFA SEEDS
PREVIOUSLY INOCULATED WITH LISTERIA MONOCYTOGENES USING PEROXYACID (PA),
CAPRIC/CAPRYLIC ACID (EMERY 658), LACTIC ACID (LA), GLYCEROL MONOLAURATE (GM)
ALONE OR IN COMBINATION.

 

Mesophilic Aerobic Bacteria (log CFU/g)

 

 

Sanitizer Exposure Time After Treatment Reduction

Treatment (min)

Water control 3 7.17 :1: 0263 0.31 d: 0.26
5 7.18 i 0.09a 0.30 :1: 0.09

A- (Asep 1+2) 3 0.70 3: 000° >6.79 i 0.00

C- (ASEP 2) 3 4_39 1 0851’ 3.09 1 0.85
5 3.76 i 0.22b 3.72 :1: 0.22

D- (E658 + LA) 3 3.95 1 0.5513 3.53 1 0.55
5 4.34 :1: 0.651) 3.15 i 0.65

 

Inoculated seeds average: 7.49 i 0.05a

Means :1: standard deviation (n = 3).
Means with different letters are significantly different (p < 0.05)

162

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