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CONTROL OF CLOSTRIDIUM BOTULINUM BY BACTERIOCINS AND CHARACTERIZATION OF NISIN ACTION DURING SPORE-TO-CELL TRANSFORMATION

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree

Doctor of Philosophy in the Graduate School of

The Ohio State University

By

Yoon-Kyimg Chimg, M. S.

*****

The Ohio State University 2oei

Dissertation Committee: Approved By

Dr. Ahmed E. Yousef, Advisor

Dr. Grady W. Chism m

Dr. David B. Min Food Science and Nutrition Dr. Polly D. Courtney UMI Number; 3031190

UMI'

UMI Microform 3031190 Copyright 2002 by Bell & Howell Information and Leaming Company. All rights reserved. This microform edition is protected against unauthorized copying under Titie 17, United States Code.

Bell & Howell Information and Leaming Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT

Bacteriocins from lactic acid bacteria (LAB) can serve as a natural preservative to control foodbome pathogens in minimally processed foods. For effective biocontrol, type of bacteriocin, level of application, and stage of application against target pathogen, such as C. botulinum in this study, are the some of the factors need to be optimized. The purposes of this study were (1) to determine the most effective bacteriocin against C. botulinum, (2) to verify the effect of bacteriocin in a food system,

(3) to monitor the germination of C. botulinum spores after heat treatment and in the presence of bacteriocin, and (4) to define the function of bacteriocin at different stages of spore-to-cell transformation

Clostridium botulinum was more effectively inactivated by the culture supernatant of a nisin-producing bacterium than that of pediocin P02 or lacidin A.

Combination of nisin and its-producer effectively delayed the growth of C. botidinum in mashed potatoes stored at 25°C for 5 days. In addition, commercial nisin (50-250 pg/g potatoes), was a very effective bactericidal agent against C. botulinum in roasted potatoes packaged under modified atmosphere (100 % N% or 20% CO 2 :80% Nz).

The effect of nisin and pH (2.8 and 3.4) on the activation and subsequent germination of C. botulinum spores was investigated. Spores of C. botulinum were not

ii inactivated by heat treatments ug to 92“C for 2 hours. Heat treatment at 85°C for 90 min

was selected as the optimal activation condition based on monitoring subsequent

germination. L-alanine alone or combined with L-cysteine was not sufficient to

germinate the spores of this strain. Additional germinant(s) in Tryptone-Peptone-

Glucose-Yeast extract (TPGY) broth were required for germination. Lower pH and

presence of nisin during heat activation increased the degree of phase darkening, i.e.,

germination. However, extended heating in the presence of nisin at pH 2.8 decreased

the spore culturability on recovery medium (TPGY agar).

The action of nisin against vegetative cells and spores of C. botulinum was

investigated. Nisin was added at various stages of spore-to-vegetative cell transition and

changes in sensitivity to the bacteriocin were observed. Nisin was bactericidal against

vegetative cells, but was sporostatic against spores of C. botulinum under the same

concentration. The degree of bactericidal and sporostatic action of nisin was dependent

on the concentration of nisin used. Under combined treatment of heat and nisin, the

bacteriocin enhanced spore activation, and subsequently triggered the germination.

Nisin delayed germination of activated spores, when it was added after heat activation.

In addition, nisin prevented the late event of germination stage (after loss of heat resistance and before phase darkening) when added after heat activation. Finally, nisin inhibited the outgrowth of germinated spores for 20 days when added after germination stage.

Findings firom this study indicate that LAB bacteriocins may be effective biocontrol agents against C. botulinum or other pathogenic/spoilage microorganisms in ready-to-eat refrigerated foods such as roasted potatoes. In addition, it is suggested that

iii the bacteriocin application time i& very important for the effective control of spores during the heating process of foods. It may be possible to apply nisin at the stage of processing that coincides with the most sensitive stage of spore transformation. Finally, the action of nisin is influenced by other factors such as pH and NaCl in food systems.

IV Dedicated to my mother and late father ACKNOWLEDGMENTS

I would like to express my sincere gratitude to Dr. Ahmed Yousef for his constant guidance, encouragement, advice, and patience throughout my study. I gratefully acknowledge that this fruition comes largely from his support. I greatly thank him for being a mentor, not just for serving as my advisor. I also would like to express thanks to Dr. Grady Chism, Dr. David Min, and Dr. Polly Courtney, my dissertation committee members, for their valuable advices, discussions and time.

I wish to deliver special thanks to Dr. Mohammed BChadre, Dr. Jin-Gab Kim, and Dr. Xia Liu for their helpful suggestions and discussions. Many thanks to our laboratory group. Dr. Robert Gilmore, Dr. Abdullatif Tay, Luis Rodriguez-Romo,

Hyun-Jung Chung, and Beatrice Lado, for their friendship and help in many ways. 1 wish to thank the Department of Food Science and Technology, Hillshire Farm and

Kahns Company (Cinciimati, Ohio), and The Center for Advanced Food Processing and

Packaging Studies (Raleigh, North Carolina) for supporting my research.

Most of all, I wish to share the joy of this accomplishment with my family. I want to give special thanks to my daughter, Mee-Ree, who was bom during my first year of PhT). study. She has shared all difSculties with me throughout the study. My deepest thank goes to my mother, who has not hesitated to bear all kinds of sacrifices for me; to whom I owe everything.

VI VITA

December 2, 1964...... Bom, Seoul, Korea

1987 ...... B. S., Seoul National University, Seoul, Korea

1987-198 8 ...... Graduate school, Seoul National University, Seoul, Korea

1988-199 1 ...... M. S., Iowa State University, Ames, Iowa

1992-1994...... M. S., University of Maryland, College Park, Maryland

1995-Present...... Graduate Research Associate, The Ohio State University, Columbus, Ohio

FIELDS OF STUDY

Major Field: Food Science and Nutrition

vu TABLE OF CONTENTS

Page

Abstract...... ü

Dedication ...... v

Acknowledgments ...... vi

Vita...... vii

List of Tables ...... x

List of Figures ...... xi

Introduction ...... 1

Chapters: l . Literature Review...... 4

Clostridium botulinum...... 4 Clostridium botulinum and botulism ...... 4 Spore structure and resistance ...... 5 Spore-to-cell transition ...... 6

Bacteriocins of lâche acid bacteria ...... Il Definition of bacteriocins ...... 11 Chemical composition and classification ...... 12 Properties of bacteriocins ...... 13 Inhibitory spectrum ...... 14 Mode of action ...... 14 Genetic aspects of bacteriocin production ...... 16

Application of bacteriocins in minimally processed foods ...... 16 Minimally processed firuits and vegetables ...... 16 Clostridium botulhrum in minimally processed foods ...... 17 Risk of Clostridium botulinum in potato products ...... 20 viii Application of LAB bacteriocins in biopreservation ...... 23 Factors affecting LAB bacteriocin action ...... 23 Storage stability of bacteriocin ...... 26

Inactivation of Clostridium botulinum by LAB bacteriocins ...... 27 Effects of LAB bacteriocins against Clostridium botulinum in media and foods...... 27 Activity of LAB bacteriocins against spores ...... 29

2. Inactivation of Clostridium botulinum Type A by bacteriocins of lactic acid bacteria in laboratory broth and solid food system ...... 44

Abstract...... 44 Introduction ...... 45 Materials and Methods ...... 47 Results and Discussion ...... 53 References...... 59

3. Culturability of Clostridium botulinum spores under different germination conditions, sublethal heat treatments, and in the presence of nisin ...... 75

Abstract...... 75 Introduction ...... 76 Materials and Methods ...... 78 Results and Discussion ...... 83 References...... 90

4. Action of pure and commercial nisin preparations on clostridium botulinum during spore-to-cell transformation ...... 100

Abstract...... 100 Introduction ...... 101 Materials and Methods ...... 103 Results and Discussion ...... 107 Conclusion ...... 112 References...... 114

List of References ...... 125

IX LIST OF TABLES

Table Page

1.1 Distinguishing characteristics of Clostridium botulinum serotypes ...... 32

1.2 Examples of foodbome botulism outbreaks ...... 33

1.3 Transformation of a dormant spore into a vegetative cell ...... 34

1.4 Germination conditions for Clostridium botulinum strains...... 35

1.5 The inhibitory spectrum of bacteriocins of potential significance in food ...... 36

2.1 Bacteriocin producing and indicator bacteria used in this study and media in which these bacteria were grown ...... 64

2.2 The antimicrobial activity of bacteriocins determined by agar-diffiision assay ...... 65

3.1 Media used in this study for germination of Clostridium botulinum spores ...... 80 LIST OF FIGURES

Figure Page

1.1 Diagram of typical spore structure ...... 41

1.2 Structure of nisin. Dha = Dehydroalanine, Dhb = Dehydrobutyrine, ABA = a-Amino butyric acid, Ala-S-Ala = Lanthionine, ABA-S-Ala = P-Methyl lanthionie . (Gross etal., 1971) ...... 42

1.3 Mechanism of action of nisin. (Montville et al., 1995) ...... 43

2.1 Comparison of activity of crude extracts of nisin and pediocin P02 preparation, measured as zone of inhibition, against different indicator microorganisms using agar difhision assay ...... 66

2.2 Activity of commercial nisin solution (2500 pg/ml), prepared by different methods, against Lactobacillus leichimannii ATCC 4797 ...... 67

2.3 Comparison of activity of commercial nisin (2.5 pg/ml) at different pHs against three indicator microorganisms. Diameter of zone of inhibition was measured for non-diluted samples ...... 68

2.4 Behavior of Clostridium botulinum ATCC 25763 in TPGY broth containing supernatant of bacteriocin producing cultures, nisin producing Lactococcus lactis ATCC 11454, pediocin P02 producing Pediococcus acidilactici P02, and lacidin A producing Lactobacillus acidophilus OSU133...... 69

2.5 Behavior of Clostridium botulinum ATCC 25763 in TPGY broth containing commercial nisin (2.5 pg/ml) ...... 70

2.6 Behavior of Clostridium botulinum ATCC 25763 in mashed potatoes stored at 25°C in the presence of nisin (0.125 pg/g potato) ...... 71

2.7 Behavior of Clostridium botulinum ATCC 25763 in autoclaved mashed potatoes in the presence of (a) pediocin P02 (Each data points are average values of 3 individual experiments) and (b) lacidin A ...... 72

XI 2.8 Behavior of Clostridium botulinum ATCC 25763 in nitrogen-packaged (100% Nz) roasted potato cubes in the presence of commercial nisin (50 pg/g potatoes). Each data points are average values of 3 individual experiments ...... 73

2.9 Behavior of Clostridium botulinum ATCC 25763 in modified-atmosphere- packaged (20% COz + 80% Nz) roasted potato cubes in the presence of commercial nisin (250 pg/g potatoes) ...... 74

3.1 Representative survivior curves for Clostridium botulinum ATCC 25763 spores suspended in distilled water ...... 94

3.2 Monitoring germination of C. botulinum spores. Spore suspension was heat activated at 80°C for 15 min, 85°C for 15 min, or 85“C for 90 min, incubated at 37°C in the presence of germinant. Samples were reheated at 80°C for 30 min and culturable spores were counted on TPGY agar. Non-reheated spores were also plated as a control treatment. Heat activation treatment: (a) at 80°C for 15 min; (b) at 85°C for 15 min; (c) at 85°C for 90 min ...... 95

3.3 Activation of C. botulinum spores by intermittent heating. Spore suspension was pre-heated at 80-85°C for 15-90 min and then reheated at 80“C for 30 min ...... 96

3.4 Monitoring germination of C. botulinum spores by phase change observation. Spore suspension was heat activated at 80, 85, or 90°C for 15-90 min, and incubated at 37®C in the germination medium. Samples were examined under phase-contrast microscope at intervals ...... 97

3.5 Culturability of C. botulinum spores after heating at two different pH values and in the presence or absence of nisin (250 pg/ml) ...... 98

3.6 Phase-change observation of C. botulinum spores before and after heat activation treatment (at 85°C for 90 min). Different activation conditions were compared under different pH and in the presence or absence of nisin (250 pg/ml) ...... 99

4.1 Monitoring germination by determining loss of heat resistance and observing phase-darkening of spores ...... 117

4.2 Behavior of vegetative cells and spores of Clostridium botulinum in TPGY broth at 37“C in the presence of nisin. (a) 25 pg/ml; (b) 2.5 pg/ml; (c) 0.25 pg/ml ...... 118

xu 4.3 Lag^time of gowth from spore inocula, produced by mcreasing msm concentrations in the growth media ...... 119

4.4 Monitoring germination of C. botulinum spores. Spore suspensions were heat activated at 85°C for 90 min in the presence of nisin (250 pg/ml). (a) activated at pH 2.8 in the presence of Nisaplin; (b) activated at pH 3.4 in the presence of pure nisin. Activated spores were transferred to the germination medium and incubated for germination. At intervals, phase- change of spores was observed. Each data points indicate the average values of at least two individual experiments ...... 120

4.5 Monitoring germination of C. botulinum spores. Spore suspensions were heat activated at 85°C for 90 min in the presence of nisin (250 jig/ml). (a) activated at pH 2.8 in the presence of Nisaplin; (b) activated at pH 3.4 in the presence of pure nisin. Activated spores were transferred to the germination medium and incubated for germination. At intervals, samples were heated at 80“C for 30 min, and enumerated on TPGY agar. Each data points indicate the average values of at least two individual experiments ...... 121

4.6 Monitoring germination of C. botulinum spores in the presence of nisin (250 pg/ml) by observing phase-contrast change after heat-activation at 85®C for 30 min. (a) at pH 2.8 in the presence of Nisaplin; (b) at pH 3.4 in the presence of pure nisin. Each data points indicate the average values of at least two individual experiments ...... 122

4.7 Monitoring germination of C. botulinum spores in the presence of nisin (250 pg/ml) by plate count method preceded by heat treatment (80°C, 30 min) after heat-activation at 85®C for 30 min. (a) at pH 2.8; (b) at pH 3.4. Each data points indicate the average values of at least two individual experiments 123

4.8 Effect of nisin (250 pg/ml) on outgrowth stage of C. botulinum spores when added after 3-hour germination incubation at 37“C ...... 124

xui INTRODUCTION

In spite of the introduction of modem technologies and safety concepts (e. g.

HACCP), the reported occurrence of food-home illnesses is not decreasing. On the

other hand, an increasing number of consumers prefer minimally processed foods,

which are prepared without chemical preservatives, and have ‘fresh-like’ or ‘mild’

sensory attributes. In fact, many of these ‘ready-to-eat’ and novel food types represent a

new category of risky products (Holzaphel et al., 1995). One of the critical concerns is

the potential for pathogenesis during temperature abuse, since the safety of these

products relies exclusively on refrigeration (Gombas, 1989). Clostridium botulinum is

one of the most hazardous pathogens in minimally processed foods. Therefore, this

study is based on the hypothesis that the application of bacteriocins from lactic acid bacteria can be used to control C. botulinum in minimally processed foods.

Bacteriocins from Generally Recognized as Safe (GRAS) lactic acid bacteria

(LAE) raised a great deal of interest as a novel approach to control food-home pathogens (Spelhaug and Harlander, 1989; Lewus et al., 1991; Okereke and Montville,

1991a & b; Stiles and Hastings, 1991; Hanlin et al., 1993). Because lactic acid bacteria naturally occur in a wide range of food products, including vegetables, meats, and cheeses, the use of bacteriocins produced by these microorganisms could serve as natural means of food preservation (Christensen and Hutkins, 1992). During

biopreservation of minimally processed foods, the bacteriocin-producing lactic acid

bacteria barely grow if the products are properly refrigerated. If the products are

temperature-abused, the bacteriocin-producing cultures grow and produce bacteriocins

to inhibit pathogenic and spoilage microorganisms (Hutton et al., 1991). However,

before biocontrol could be utilized as an additional barrier, it must be evaluated for its

effectiveness in preventing growth and toxin production by one of the most concerning

pathogens, C. botulinum, in minimally processed foods under temperature abuse. In

addition, this application needs to be optimized for effective control of the pathogen.

Therefore, the goal of this research was to produce safer foods by incorporating

bacteriocins, as natural preservatives, against C. botulinum as a target. Type of

bacteriocin, level of application, stage of application, and incorporation of the producer

are some of the factors studied to maximize the effectiveness of the proposed

biopreservation techniques. C. botulinum spores may exist in processed foods through

raw materials or by post-processing contamination of foods (Juneja and Marks, 1999).

Since inactivation of spores in foods is not always a practical approach, measures to

prevent outgrowth from spores are adequate for ensuring the safety of foods. Therefore,

understanding the different inactivation kinetics and mechanisms by bacteriocins against vegetative and spore forms of C. botidinum is important. Furthermore, it is

important to understand their mode of action on the spores and vegetative cells, especially on the processes involved in the spore-to-cell transition. These spore transformation stages would be ideal targets against which to apply specific inhibitors such as bacteriocins. In order to achieve these goals, following empirical objectives

2 were pursued: CO Screening bacterlQcin& and their producing cultures, for efhcaoy against C. botulinum and suitability for application in the food biopreservation. (2)

Testing a selected biopreservation system in cooked potatoes to inhibit the growth of C. botulinum. (3) Examining culturability of C. botulinum spores under different germination and heat treatment conditions to predict the ability of spores to survive the heating process and grow subsequently in foods. (4) Investigating the action of nisin against C. botulinum at various stages of spore germination and outgrowth. C H A PTER 1

LITERATURE REVIEW

Clostridium botulinum

Clostridium botulinum and foodbome botulism. Clostridium botulinum, is a

Gram-positive, sporeforming, rod-shaped bacterium that grows in the absence of

oxygen. These characteristics allow it to survive in foods that are incorrectly or

minimally processed and to grow in a vacuum-packed and modified atmosphere

environment. Food-home botulism is caused by the consumption of foods containing a

neurotoxin produced by C. botulinum. There are seven recognized serotypes of botulinal toxin (A, B, C, D, E, F, and G), based on the antigenic specificity of the toxin produced by each strain (Sperber, 1982). Even though these toxins are similar in action, there are some differences among the strains in characteristics such as proteolytic activity, growth temperature, spore heat resistance and host specificity (Lynt et al.,

1982). The type A, B, and E strains are associated with human botulism. C. botulinum type A is most common in land-based products, and it is a common contaminant on processing equipment. C. botulinum type E is most common in fish and fishery products, and it is of particular concern because it grows at temperature as law as 3&°F and. pioduces little evidence of spoilage. Table L I shows

some distinctive characteristics of C. botulinum serotypes

C. botulinum is found throughout the environment and has been isolated from

soil, water, vegetables, meats, dairy products, ocean sediments, the intestinal tracts of

fish, and the gills and viscera of crabs and other shellfish. The widespread of food-borne

botulism throughout the world is probably associated with the prevalence of spores in

the environment. Some examples of botulism outbreaks in commercially prepared foods

or home- or restaurant-prepared foods are presented in Table 1.2.

Spore structure and resistance. Spore structure is related to the resistance of

spores to stress factors. Spores are morphologically different from vegetative cells

(Figure 1.1). Many spore structures, including the exosporium and coats, have no

counterparts in the vegetative cell (Setlow and Johnson, 1997). The outermost spore

layer, the exosporium, varies significantly in size between species, and its various

components are not well characterized (Murrell, 1969). Underlying the exosporium are the spore coats, which are two layers, i.e., outer and inner spore coats. The spore coats protect the spore cortex from attack by lytic enzymes. The coats also may provide an initial barrier to chemicals such as oxidizing agents. However, spore coats play no significant role in maintenance of spore resistance to heat or radiation (Setlow and

Johnson, 1997). These outer layers, including inner- and outer coat and exosporium, contain mainly spore proteins, which are unique in terms of composition. These proteins contain nonpolar amino acids, glycine, lysine, aspartate, glutamate, and high amount of cysteine (Tipper and Gautier, 1972). It is hypothesized that a high degree of

5 hydrophobic interaction and disulfide bonds can be formed fiom the amino acid composition of the spore protein. Therefore, it is likely that a coat protein rich in cysteine, stabilized by S-S linkages, is responsible for maintaining the dormant state.

Reduction of these linkages, due to reducing agents, will change the tertiary structure of coat proteins and result in partial unfolding of the proteins. Underlying the inner spore coat (or outer membrane) is the cortex. This large peptidoglycan layer is structurally similar to cell wall peptidoglycan, but with several differences, including that it is loosely cross-linked and lacks amino acid cross-links between adjacent peptide chains

(Gould, 1999). Underlying the cortex is the germ cell wall, whose structure is similar to that in vegetative cell (Setlow and Johnson, 1997). The next structure, irmer membrane, is a strong permeability barrier to hydrophilic molecules and is compositionally similar to the vegetative cell membrane (Setlow and Johnson, 1997). Finally, the innermost region, the core, contains the DNA, RNA, ribosomes, and most enzymes, as well as dipicolinic acid (DPA) and divalent cations (Setlow and Johnson, 1997). Low water content in the core plays a major role in spore dormancy and in spore resistance to various agents (Setlow and Johnson, 1997).

Spore-to-cell transition. The transition of dormant bacterial spores to fully active vegetative forms can be divided into four sequential processes: activation, germination initiation, germination, and outgrowth. General characteristics during spore transformation are summarized in Table 1.3.

Activation. Activation of spores or the breaking of dormancy is commonly achieved by heating spores in aqueous suspension, a process first described by Evans

6 and Cuiran (1943). The temperature and duration of optimal heating of this effect vary widely among different species and even among different spore preparations of the same strain.

The effects o f heat activation: A substantial but sublethal heat treatment appears to render the spores less dormant, and in turn enhances germination process.

Consequently, heat activation is often a prerequisite for the enumeration of maximum populations of spores (Busta and Ordal, 1964). Relatively mild heating of mesophilic aerobic spores has been shown to increase the rate of subsequent germination (Evans and Curran, 1943). Curran and Evans (1945) reported that the germination process of spores from 12 thermotolerant and thermophilic aerobes was influenced by the preheating. In the absence of preheating treatment, a large proportion of the potentially viable spores did not germinate (Curran and Evans, 1945). Rowley and Feeherry (1970) indicated that the rate and extent of germination were also increased when spore suspension of C. botulinum 62A was heated at 80°C for 60 minutes. In addition,

Treadwell et al. (1958) also reported that activation at 75“C for 20 minutes enhanced germination of spores of C. botulinum 62A.

Factors influencing activation o f spores: Powell and Hunter (1955) reported that heat activation must be performed in the presence of water. The optimal temperature of heat activation vary widely among different species and even among different spore preparations of the same strain. Gibbs (1964) showed that maximum germination of C. bifermentans in CMB medium (2% acid-hydrolysed casein; 0.5%

KH2 PO 4 ; 10 ug/ml of biotin, nicotinamide, pyridoxal and calcium pantothenate; 1.6 mg/ml MgS 0 4 7 H2O; 80 ug/ml MnSO44H20; 80 ug/ml PeS 0 4 7 H2O) + yeast extract at

7 37°G was^ attained following » heat shoelefof KKminutes at 80-&5“G. Little germinatiort occurred when heated at 70°C or below, and temperature of 90°C and above prevented subsequent germination (Gibbs, 1964).

Heat activation is also pH dependent (Keynan et. al, 1964; Gibbs, 1967).

Keynan et al. (1964) indicated that the activation of Bacillus cereus strain T was inhibited by high pH (above 8.5). Below pH 4.5, the pH dependence of spontaneous activation parallels that of activation induced by reducing agents, whereas between pH

4.5 and 7.2, the activation was more pronounced in the presence of reducing agents

(Keynan et al., 1964). However, spores of Clostridium bifermentans were more activated at 37®G when spores were held at below pH 3.0 or above pH 10.0 (Gibbs,

1967). The same researcher indicated that activation occurs over a wide range of pH values, but that the rate of activation is greater at the extremes of the range. If activation is due to a reversible denaturation-like process of spore-coat protein, as suggested by

Keynan et al. (1964), then it is expected that the rate of activation would increase as extreme of pH value or temperature are approached. The heat activation phenomenon could be explained by assuming that heat or reducing agents change the tertiary structure of a protein responsible for the maintenance of the dormant state by reducing the disulfide linkages, which stabilize the protein in a specific configuration (Keynan et al., 1964).

In addition, the composition of the medium in which spores are suspended during heating influences the degree of activation. Gurran and Evans (1945) showed that suspending medium determined the degree of activation of Bacillus species, the following media could be arranged in order of decreasing efficiency upon heat

8 activatiolu glucose, lactose, peptone, skim milk, glucose nutrient agar, beef extract,

glucose nutrient broth, distilled water, and sodium chloride. In addition, Foerster (1985)

reported that activation in solutions of sodium nitrite greatly reduced the time required

for the activation of spores of Bacillus stearothermophilus NGBIOl at 30°C.

Furthermore, Montville (1981) indicated that the composition of the plating medium also affected the expression of heat activation. C. botulinum 62A and ATCC 25763 spores required heat activation for maximal colony formation when plated on reinforced clostridial agar but not when plated on botulinum assay medium. In addition, spores from C. botulinum strains B-aphis and 53B did not exhibit heat activation when plated on either medium. Rowley and Feeherry (1970) demonstrated that C. botulinum 62A spores exhibit heat activation in a chemically defined medium (8 mM L-cysteine, 11.9 mM sodium bicarbonate, 4.4 mM sodium thioglycolate; buffered with 100 mM TES, pH 7.0).

Germination. Foster and Johnson (1989) proposed that germination is defined as “ a series of degradative events triggered by specific germinants which leads to the loss of typical spore properties”. These changes can be conveniently divided into two categiries: (I) events including commitment, loss of heat resistance, and release of dipicolinic acid (DPA); and (2) events including loss of absorbance, cortex hydrolysis and the onset of spore metabolism which are initiated at a later stage (Foster and

Johnson, 1989).

Factors affecting germination: The optimum conditions for germination of spores of C. bifermentans suspended in phosphate buffer were incubation at 37®C within the range pH 6.0-7.8 following heat shock at 85®C for 10 minutes. Anaerobic

9 conditions were not necessary for germinatioiL The minimum requirement of compounds needed for the germination of spores of C. bifermentans was the presence of

L-alanine, L-phenylalanine and lactate (Gibbs, 1964). Uehara and Frank (1965) found that germination of spores of C. sporogenes PA 3679h was stimulated by thioglycollate

(0.1%) in an alanine + pyrophosphate system, in which the two latter components were essential. In addition, Rowley and Feehery (1970) showed that the addition of low concentrations of thioglycollate to cysteine and bicarbonate induced a most effective response for C. botulinum 62A, 90% in 60 min as compared with 6% in 120 min in its absence. On the other hand, Gibbs (1964) showed that germination of C. bifermentans could be markedly decreased by a low concentration of the agent (0.005%). Gibbs

(1964) clearly demonstrated that the effect was specifically associated with the germination stage and did not apply to outgrowth and vegetative growth; for these latter stages its presence was seemingly essential. Holland et al. (1969) explained some of the apparent inconsistencies in the effects of thioglycollate on the germination process, extending at times to contradictory responses even for the identical strain (C. botulinum

62A). Barker and Wolf (1971) concluded that the effect of thioglycollate on germination of clostridial spores is dependent on the medium. While no major effect of thioglycollate was observed when complex germination medium was used, thioglycollate was inhibitory or stimulatory when inadequate germination condition was used (Barker and Wolf, 1971). Germination conditions for some spores of C. botulinum strains are summarized in Table 1.4.

10 Bacteriocins o f lactic acid bacteria.

Lactic acid bacteria (LAB) have been responsible for the fermentative preservation of many foods. The study on bacteriocins in LAB has been reviewed by many researchers over the past few decades (Tagg et al., 1976; Piard and Desmazeaud,

1992; Klaenhammer, 1993; Nettles and Barefoot, 1993; Dodd and Gasson, 1994; Jack et al., 1995; Stiles, 1996).

Definition of bacteriocins. Bacteriocins are biologically-active ribosomally synthesized proteins with inhibitory properties against sensitive bacterial species but not to the producer microorganism (Klaenhammer, 1993; Nettles and Barefoot, 1993;

Montville and Bruno, 1994; Holzaphel et al., 1995). Tagg et al. (1976) proposed a more extensive definition stating that bacteriocins; (i) have a narrow inhibitory spectrum of activity; (ii) contain an essential, biologically active protein moiety; (iii) exhibit a bactericidal mode of action; (iv) are able to attach to specific cell receptors; (v) have plasmid-bome genetic determinants of production and host immunity; and (vi) produced by lethal biosynthesis. However, it has become clear that very few bacteriocins meet all six criteria of Tagg’s classical definition, as the heterogeneity of bacteriocins has been pointed out by other studies (Klaenhammer, 1993; Nettles and Barefoot, 1993; Jack et al., 1995). Bacteriocins, are a heterogeneous group of anti-bacterial proteins that vary in spectrum of activity, mode of action, molecular weight, genetic origin, and biochemical properties (Abee et al., 1995).

II Chemical—composition- and- classificatioii^Bacteriocins- are an extremeiy

heterogeneous group of substances. According to a classification by Klaenhammer

(1993), four distinct groups of lactic acid bacterial bacteriocins can be defined based on

their chemical compositions:

(I) Lantibiotics, small membrane-active peptides (<5K-Da) containing the

unusual amino acid lanthionine and dehydrated residues, e.g., nisin;

(II) Small (<10-kDa), relatively heat-stable, non-lanthionine-containing

membrane-active peptides, subdivided into (Ha) Z/j/erm-active pediocin-like

bacteriocins, e.g., pediocin PA-1 (Chikindas et al., 1993), (Ilb) poration

complexes requiring two different peptides for activity, e.g., lactacin F

(Abee et al., 1994a), and (lie) thiol-activated peptides requiring reduced

cysteine residues for activity, e.g., lactococcin B (Venema et al., 1993);

(III) Large (>30-kDa), heat labile proteins, e.g., helveticin J (Thompson et al.,

1996);

(TV) Complex bacteriocins that contain essential lipid or carbohydrate moieties in

addition to protein, e.g., plantaricin S (Jimenez-Diaz et al., 1993).

Although chemically diverse, the one unifying property is the presence of an

essential protein part (Tagg et al., 1976; Daw and Falkiner, 1996). LAB bacteriocins

are, in general, small cationic proteins containing 30 to 60 amino acids (Montville

and Bruno, 1994).

Nisin. Nisin was discovered by Rogers and Whittier in England in 1928 and characterized and named by Mattick and Hirsch in 1947 (Rogers and Whittier,

12 1928L Mattick and Hirsch» 1947), Lactococcus lactis subsp, lactis^ the producer

organism, belongs to the Group N Lancefield serotyping scheme, so nisin referred to as

‘Group N Inhibitory Substance’ and followed by the sufBx ‘-in’. The first commercial

preparation of nisin (Nisapiin) was made in 1953 by Aplin and Barrett Ltd., for the

purpose of preventing clostridial spoilage of processed cheese. In 1969, a joint

F AO/WHO expert committee on food additives recognized nisin as a safe and legal

biological food preservative (FAG/WHO, 1969). Then, nisin was awarded GRAS status

in the United States in 1988 to inhibit the clostridial spores in certain pasteurized cheese

products (Federal Register, 1988). Nisin is defined as a Group A lantibiotic, which

contains characteristic lanthionine rings with monosulfide bridges (Figure 1.2). All

Group A lantibiotics are cationic and amphiphilic polypeptides with elongated forms

and include nisin, subtilin, epidermin, gallidermin, PepS and lacticin 481 (De Vos et al.,

1995).

Properties of bacteriocins. LAB bacteriocins usually have high isoelectric

points and amphiphilic characteristics, i.e., the presence of regions within the molecule,

which are predominantly hydrophilic or hydrophobic (Montville and Bruno, 1994).

Most bacteriocins are susceptible to proteolytic enemies such as a-chymotrypsin,

trypsin, pronase, and pepsin (Piard and Desmazeaud, 1992; Stiles, 1996). The heat tolerance of class 1 and 11 bacteriocins is generally high, even though it may be

significantly reduced afier purification. This heat resistance also suggests that the activities of class 1 and n bacteriocins are based on molecular structures which are relatively small and uncomplicated, probably lacking a tertiary structure (Piard and

13 Desmazeaud, 1992). Most bacteriocins are hydrophobic^ hence they can. be bound by fats and phospholipids (Stiles, 1996). LAB bacteriocins are generally stable at acid or neutral pH, indicating that the substances are well adapted to the environment of bacteria producing them (Piard and Desmazeaud, 1992).

Inhibitory spectrum. Most bacteriocins of Gram-positive bacteria exhibit activity against a wide range of bacterial species, whereas most bacteriocins produced by Gram-negative bacteria act on very closely related species (Tagg et al., 1976). A number of Gram-positive pathogenic bacteria have been found sensitive to bacteriocins of certain LAB. For example, pediocin A produced from Pediococcus pentosaceus is active against pathogens such as Listeria monocytogenes. Staphylococcus aureus,

Clostridium perjringens, and Clostridium botulinum (Daeschel and Klaenhammer,

1985). However, Gram-negative bacteria such as salmonellae are not sensitive to LAB bacteriocins because their outer membrane protects them by excluding the bacteriocins

(Holzaphel et al., 1995). Table 1.5 shows the inhibitory spectrum of some bacteriocins, which have potential significance in foods.

Mode of action. For most bacteriocins, the antimicrobial effect seems to be bactericidal (Schillinger, 1990), with some exceptions, e.g., leuconocin S (Lewus et al.,

1992) being bacteriostatic. Several researchers observed a bactericidal action without reduction in optical density indicating that the lethal effect is not accompanied by a significant cell lysis (Barefoot and Klaenhammer, 1984; Bhunia et al., 1988). The target site of bacteriocin action is the cytoplasmic membrane of sensitive cells. Based on the

14 bactenocins’ amphiphilic chamctedstics^they may act by membrane permeabilizatioa mechanism. Bacteriocins may act by a poration complex in which bacteriocin monomers bind, insert and oligomerize in the cytoplasmic membrane to form a pore with a hydrophilic residues facing inward and the hydrophobic ones facing the hydrophobic regions of phospholipid molecules in the interior of the membrane (Figure

1.3: Montville et al., 1995).

The addition of these membrane-active peptides to vegetative cells causes a rapid and non-specific efflux of small molecular weight compounds (Montville et al.,

1995). Exposure of sensitive cells to nisin (10 pg/mL) causes efflux of pre-accumulated cations (Rb^ and amino acids from the cytoplasm of various Gram-positive bacteria

(Ruhr and Sahl, 1985). Nisin (1.5 pg/mL) also causes rapid leakage of intracellular ATP in L. monocytogenes cells (Winkowski et al., 1994).

The changes in membrane permeability induced by bacteriocins decrease or deplete the proton motive force (PMF) of sensitive cells. PMF is an electrochemical gradient composed of a membrane potential (Ay) and a pH gradient (ApH). These gradients serve as the driving force for many vital energy-dependent cellular processes.

Bacteriocins from four LAB genera act similarly; nisin (2.5 pg/mL), pediocin PA-1 (20 pg/mL), leuconocin S (29.1 pg/mL), and lactacin F (13.5 pg/mL) dissipate the PMF in sensitive cells (Bruno et al., 1992; Bruno and Montville, 1993; Okereke and Montville,

1992). In turn, dissipation of PMF may render the cell unable to protect its cytoplasm from the surrounding medium, arrest all energy-dependent cellular processes and lead to growth inhibition, if not death (Montville et al., 1995).

15 Genetic-aspects, of bacteriocin-production^Most-of the genetic determinants,

of LAB bacteriocins are plasmid-bome. For example, the genetic determinants for both

production of and immunity to lactococcin from L. lactics subsp. lactis ADRIA 8SL030

have been linked to lOkb region of piamid, pOS5 (Dufour et al., 1991). Several strains

of Pediococcus have been also found to produce bacteriocins as a consequence of

plamid-encoded genetic determinants (Daeschel and BClaenhammer, 1985; Ray et al.,

1989). However, another molecular analysis has established that the genes for nisin

biosynthesis are located on the chromosome (Dodd et al., 1990) and are carried by a

conjugative transposon (Horn etal., 1991). ^

In general, bacteriocins of LAB appear to be translated as prepeptides that are

subsequently modified to form the mature biologically active molecules. The

biosynthesis of nisin occurs in two steps: a precursor peptide (prenisin) is synthesized

ribosomally followed by post-translational enzymatic modifications converting the

inactive precursor into a biologically active peptide (De Vuyst and Vandamme, 1994).

The bacteriocin-associated genes of LAB appear to be arranged in multigene operon-

like structures with the first gene typically encoding the structural protein. Additional

gene products may be required for transcriptional regulation, post-translational

modifications, processing, translocation to the exterior of the cell, and producer strain

self-protection (Van de Meer et al., 1993; Venema et al., 1995).

Application of bacteriocins in minimally processed foods

Minimalty processed fruits and vegetables.Consumer’s demand for firesh convenience foods with minimal or no preservatives has led to a relatively new area of

16 food preservation» i.e.» minimally processed foods (King and Bolin, 1989). By

definition, minimal processing encompasses any procedure, short of any traditional

complete preservation procedures (heat sterilization, freezing etc.), that adds value

(King and Bolin, 1989). Forms of minimally processed fruits and vegetables vary

widely, depending on the unprocessed commodity and how it is normally consumed

(HuxsoU and Bolin, 1989). Minimally processed fruits and vegetables in the market

include packaged shredded lettuce and cabbage, cut fruits, peeled and sliced potatoes

(King and Bolin, 1989). Most of these products are usually trimmed, cut, washed, and

often sanitized, to supply consumers with ready-to-eat produce. Such products are

packaged in sealed pouches or in plastic trays sealed with polymeric films. Safety risks

associated with consumption of these products continue to be a concern (Hao et al.,

1998).

Clostridium botulinum in minimally processed foods. Novel food processing

techniques associated with minimally processed foods have created a new niche for the

growth of microorganisms. This niche has three important characteristics: (i) Many of

the foods are packaged under vacuum or an anaerobic atmosphere. This restricts the

growth of aerobic bacteria, but favors the growth of anaerobic bacteria; (ii) The foods

receive a mild heat treatment that should eliminate vegetative cells, but not bacterial

spores; (iii) The foods are stored at refrigeration temperature (Peck, 1997). This niche

favors colonization by microorganisms that produce heat-resistant spores and grow in the absence of oxygen at the refrigeration temperature. In particular, concern exists

17 about the potential for growth and toxin production by C. botulinum in the absence of competitive microorganisms (Notermans et al., 1990; Peck, 1997).

At 22 and 25°C, shredded cabbage packaged under modified atmosphere conditions (70% CO 2 and 30% N2) and inoculated with spores firom type A strains of C. botulinum became toxic after 4, 5, or 6 days (Solomon et al., 1990). In this study, an inoculum of approximately 10^ type A spores/g of cabbage produced toxin, while the cabbage was still organoleptically acceptable, as determined by appearance, odor, and texture (Solomon et al., 1990). Coleslaw prepared from shredded cabbage was implicated in four cases of botulism in Sarasota, Florida, in December 1987. It was suspected that the cabbage had been packaged in a modified atmosphere (Solomon et al., 1990). Packaged mushrooms (Agaricus bisporus) also became toxic after storage for

4 days at room temperature, but high numbers of spores were necessary (Sugiyama and

Yang, 1975). In contrast, spores of C. botulinum (types A, B, and E) did not produce toxin on fresh celery even after 8 weeks at 21°C under anaerobic conditions, presumably because of lack of nutrients or the presence of inhibitors (Johnson, 1979).

Tomatoes kept under modified atmosphere ( 1% O2, 20% CO2, balance N2) packaging at

23°C were toxic 26 days after inoculation with types A and B spores of C. botulinum

(Hotchkiss et al., 1992). Petran et al. (1995) investigated the effect of storage and packaging conditions for toxin production by C. botulinum in romaine lettuce and shredded cabbage. Toxin was produced in nonvented pouches of romaine lettuce and cabbage after 14 days and 7 days, respectively, at 21°C. In vented pouches, romaine samples at 21°C became toxic after 21 days. However, no toxin was detected with either storage method at 4.4 and 12.T*C (Petran et al., 1995).

18 Austin et ai. (1998) performed the challenge studies using a variety of&eshrCUt packaged salads and vegetables inoculated with both proteolytic and non-proteolytic C. botulinum strains. Botulinum toxin was produced by proteolytic types A and B on onion, butternut squash, rutabaga, salad, and stir-fiy vegetables at I5“C or higher. Non- proteolytic strains produced toxin on butternut squash and salad at temperatures as low as 5°C (Austin et al., 1998). The strict maintenance of low temperatures (< 5°C) is recommended in order to control the potential growth of C. botulinum on fresh-cut vegetables packaged in a modified atmosphere (Austin et al., 1998). The effect of temperature on the behavior of C. botulinum on mushrooms was also indicated in other studies (Sugiyama and Yang, 1975; Malizio and Johnson, 1991). In vacuum-packaged enoki mushrooms {Flammulina velutipes), or in Agaricus bisporus mushrooms, toxin was detected in packs inoculated with proteolytic spores of C. botulinum after storage at

15°C or higher but not at 4 or 6 °C (Sugiyama and Yang, 1975; Malizio and Johnson,

1991). Growth and toxin production by non-proteolytic C. botulinum strains occurred in cooked cauliflower, mushrooms, broccoli, asparagus, and kale at temperatures between

5 and 16“C (Carlin and Peck, 1996).

In October 1983, sautéed onions in “patty-melt” sandwiches were epidemiologically responsible for a large outbreak of botulism in Peoria, Illinois

(MacDonald et al., 1985). In addition, garlic in oil caused an outbreak of botulism in

Vancouver, British Columbia, Canada (Anonymous, 1985). Solomon and Kautter

(1986) indicated that, probably, margarine in sautéed onion patty-melt sandwiches provided an anaerobic condition. Both garlic and onions were thought to contain ingredients inhibitory to bacterial growth, however, when cooked in margarine or oil

19 they support the growth and toxin, production^by C^ botuUmm. (Solomoa and Kautter^

1986). Simpson et al. (1995) investigated the risk of survival of, and toxin production

by, proteolytic strains of C. botulinum spores in a sous-vide spaghetti and meat-sauce

product at mild temperature-abuse storage conditions. Toxin was detected in samples of

pH >5.5 after 14-21 days and in products of pH 5.25 after 35 days at 15°C (Simpson et

al., 1995).

Risk of Clostridium botulinum in potato products. Since vegetables such as

potatoes are often in contact with soil, which is regarded as a major habitat of

Clostridium spp., these foods can easily be contaminated with spores of C. botulinum.

Between 1899 and 1976, about 53% of the outbreaks of botulism were associated with

vegetables (Pierson and Reddy, 1988). Sugiyama et al. (1981) reported that C.

botulinum was isolated from each of three samples of unpeeled potatoes from the

supermarket in North America. Raw, cooked or baked potatoes can support the growth

and the toxin production by C. botulinum (Sugiyama et al., 1981; Notermans et al.,

1981; Notermans et al., 1985). Spores of both proteloytic and non-proteolytic C.

botulinum in vacuum-packaged potatoes survived the cooking process at 95°C

(Notermans et al., 1981). Lund et al. (1988) indicated that spores of C. botulinum in

pasteurized potatoes remained viable and resulted in growth and formation of toxin

within 5-9 days at 25°C. The heat resistance of Clostridium sporogenes PA 3679 in

mashed potato/alginate mixture in the presence of oil was tested by Cemy et al. (1990).

It was reported that C. sporogenes spores remained viable at 106°C and indicated that

20 lipids appeared to provide the protection for spores against the heat stress (Cemy et al.»

1990).

Sliced, fresh, raw potatoes inoculated with types A or B spores, packaged under

vacuum and held at 22°C became toxic in 3 days (Solomon et al., 1994). In their report,

untreated potatoes deteriorated before or at the same time that toxin was produced.

However, when such potatoes were treated with NaHSO], they remained

organoleptically acceptable for up to 6 days although toxic on day 4 (Solomon et al.,

1994). Solomon et al. (1998) conducted similar study with modified atmosphere

packaged (70% CO 2 and 30% N2) sliced raw potatoes and found that potatoes treated

with NaHSOa appeared acceptable for human consumption through day 7, even though

they became toxic in 4 days.

The ability of C. botulinum to grow and produce toxin is affected by barriers

incorporated within a food such as pH and competitive microflora, and storage

conditions such as temperature and modified atmosphere packaging. Cooked, vacuum-

packaged potato samples inoculated with spores of a mixture of type A and proteolytic

type B strains were studied by Dodds (1989). Toxin was produced at pH levels > 4.75 when the water activity was > 0.97, pH ^ 5.25 when the water activity was 0.965, and pH > 5.75 at water activity of 0.96, and no toxin was detected at water activity of 0.955

(Dodds, 1989). Baumgart (1987) showed that pasteurized potatoes, dipped in 0.4 and

0.5% acetic acid for 15 min, stored at 20°C, produced toxin after 21 days at pH 4.8.

Toxin was also formed after 14 days at 7®C in pasteurized potatoes with 0.4% acetic acid (at pH 5.2) in this study (Baumgart, 1987). C. botulinum toxin was detected in some inoculated, cooked, vacuum-packed potatoes stored at 15 or 22°C, but no toxin 21 was detected in samples stored at 5°C CTamminga et al.» 1980). In this study» the maximum storage life of 3 weeks in vacuum-packaged precooked potatoes is suggested even under refrigeration (Tamminga et al., 1980).

Dignan (1985) studied the sporicidal effects of modified atmosphere on C. botulinum spores in raw diced potatoes. The results indicated that neither SO 2 (0-100%) treatment nor packaging under modified atmosphere (100% CO 2) had any significant sporicidal effects and that toxigenesis could occur without usual signs of spoilage

(Dignan, 1985). However, in other study, the spores of C. botulinum in sliced raw potatoes were killed when exposed either to a mixture of ethylene oxide ( 10%) and CO2

(90%) or to pure SO 2 and SO2 (50%) air mixtures (Kaffezakis et al., 1969).

The risks of bacterial contamination and spoilage are increased when the vegetables are prepared for “ready-to-use” purposes (Giannuzzi and Zaritzky, 1993). A botulism hazard is created when foil-wrapped, baked potatoes are held at room temperature for several days. Miller (1984) indicated that the baked potatoes were found to be the cause of severe outbreaks of botulism. Potato caused hospitalization of a

37-year old man when the baked product, wrapped in foil, was kept at room temperature overnight, reheated and consumed (Miller, 1984). The potential is greater if such potatoes are not further heated before consumption as when used for potato salads

(Sugiyama et al., 1981). Brent et al. (1995) investigated an outbreak of botulism in

Colorado, USA, in 1992 and reported that potato salad, which had been temperature abused in the patient’s home, was the probable vehicle of infection.

22 Application of LAB bacteriocins in biopreservationvThere is interest in possible use of LAB as biocontrol agent to ensure safety of minimally processed, refrigerated foods, which are not acidified. Many refrigerated minimally processed foods have an intermediate redox potential (Eh), a high water activity (a*), a pH>4.6 and are free of chemical preservatives. If this type of refrigerated food is temperature- abused in distribution, by the retailer, or by the consumer, the only defense against botulinum toxigenesis is the presence of competitive microorganisms. If the growth of competitive microflora has been retarded by modified-atmosphere package, or eliminated by a mild heat treatment, then only the refrigeration barrier exists to prevent the germination and outgrowth of heat resistant spores of C. botulinum. Therefore, a second line of defense to guard against the consequences of temperature abuse is necessary for this type of food (Hutton et al., 1991). In the process of biopreservation for a refrigerated food, the lactic acid bacteria barely grow if the temperature is properly refngerated. If the product is temperature abused, the lactic acid bacteria grow and produce antimicrobials such as lactic acid and bacteriocins at a rate that is sufficient to inhibit pathogens such as C. botulinum (Hutton et al., 1991).

Factors affecting LAB bacteriocin action. In food matrices, the bacteriocin activity may be affected by (i) changes in solubility and charge of bacteriocins, (ii) binding of the bacteriocins to food components, (iii) inactivation by proteases, and (iv) changes in the cell membrane of the target organisms as a response to environmental factors (Ganzle et al., 1999). Recent reports emphasized the importance of interactions of bacteriocins with food components. The effectiveness of bacteriocins may depend on

23 the amount of bactedocia inactivated by the înteiactîoiL with food components

(Muriana, 1996). Jung et al. (1992) reported that nisin lost 8 8 % of its activity in the

presence of 13% fat. Their study suggested that nisin adsorbs to milk fat globules,

which render it unavailable for microbial inactivation. Dean and Zottola (1996) also

indicated that the effect of nisin on L. monocytogenes was decreased in the higher fat

ice cream. In addition, Degnan et al. (1993) found that up to 90% of pediocin activity

applied to food emulsion could be protected by liposomal encapsulation. The edect of

propionicin on survival of Lactobacillus delbrueckii was compared in MRS broth and

skim milk (Hsieh and Glatz, 1996). The residual viable population in broth stayed

constant up to 9 days of incubation, but the remaining cells in milk eventually grew.

Therefore, this study suggested that some ingredients present in milk might have

interacting effect either with the bacteriocin itself or with cells (Hsieh and Glatz, 1996).

Similar observations have been reported for the effects of camocin CP5 on L.

monocytogenes in skim milk (Mathieu et al., 1994). Rogers and Montville (1994)

showed the effect of food ingredients on nisin activity against C. botulinum. According to this study, albumen, carrageenan, and soluble starch reduced nisin inhibition, while xanthan and soy flour potentiated nisin activity against C. botulinum (Rogers and

Montville, 1994).

The efiBciency of nisin Z against L. monocytogenes was shown to be significantly reduced in the presence of divalent cations such as Mg^^ and Ca^\ which may interact with the negatively charged phospholipid headgroups present in the cytoplasmic membrane (Abee et al., 1994b). This can result in inhibition of electrostatic interactions between positive charges on the bacteriocins and the negatively charged

24 headgoups of phospholipid molecules^ and/or neutralization inducing a condensation of these lipids resulting in a more rigid membrane (Abee et al., 1994b). The temperature, phospholipid content, and pH affected growth inhibition of C. botulinum by nisin in a model food system (Roger and Montville, 1994). The action of nisin Z is also dependent on the temperature. The rate of nisin Z-induced K* efflux from L. monocytogenes grown at 30°C was shown to be severely reduced at decreased temperatures. The ordering of the lipid hydrocarbon chains, which occurs at lower temperatures resulting in a decrease in membrane fluidity, are probably responsible for the reduced nisin Z efficiency observed (Abee et al., 1994b).

Bacteriocins, including nisin, pediocin P02, brevicin 286, and piscicolin 126, were extracted from growth media by adsorption onto Micro-Cel (a food-grade diatomite calcium silicate anticaking agent) and subsequent desorption (Coventry et al.,

1996). All four bacteriocins were absorbed, with little activity detected in the supernatants and 100% activity recovered in the Micro-Cel. Absorption of bacteriocins onto Micro-Cel most probably involved both electrostatic and hydrophobic interactions.

The electrostatic interactions would be facilitated by the negatively charged diatomite particles over a wide range of pH (2.0 to 11.0) and by the positively charged bacteriocin molecules at a pH below their isoelectric points which fall within alkaline range (Ray &

Hoover, 1993). The hydrophobic interactions between the Micro-Cel surface and the bacteriocin molecules were influenced by the use of an appropriate surfactant (Coventry et al., 1996). Micro-Cel-adsorbed piscicolin was effectively desorbed by SDS but not by

Tween 80 or monoglycerides. This study suggested that the hydrophobic interactions between Micro-Cel particles and piscicolin molecules are more important than the

25 electrostatic interactions and that they were overcome more effectively by the use of strong surfactant such as SDS (Coventry et al., 1996).

Storage stability of bacteriocins. Bacteriocins for commercial application as food preservatives need to survive the long periods of time required for processing, distribution, and storage. Hsieh and Glatz (1996) reported long-term storage stability and effectiveness of propionicin PLG-I, a bacteriocin produced by Propionibacterium theonii, in a food model system. They found that bacteriocin activity increased by as much as 200% over the first ID days of storage in nonlyophilized samples stored at 25 or 4“C. Activity then decreased gradually for samples stored at 25°C while samples stored at 4°C retained high activity through 14 weeks of storage (Hsieh and Glatz,

1996). In this study, propionicin inhibited Lactobacillus delbrueckii, with the extent of killing dependent on concentration of bacteriocin present. With 100 AU/ml of propionicin, the original cell population was reduced by 98% in 6 hours. However, culture growth resumed after this period of inhibition and maximum count were reached in 48 hours.

Dean and Zottola (1996) monitored the stability of nisin throughout frozen storage of ice creams for 3 months. They reported that the nisin level in the 10% fat ice cream samples was lower throughout the storage when compared to the 3% fat ice cream samples. The antilisterial effect of Lactobacillus plantarum WHE92, pediocin

AcH producer, was investigated in a smear-surface soft cheese (Ennahar et al., 1998).

Pediocin AcH activity reached to the maximum at the end of exponential phase of L. plantarum during 4-5 day period. In addition, the L. monocytogenes count dropped to

26 10^ CEU/ml between. 3 and 4 days, of incubation, in. the presence of L, plantarum^ Le.^ during the period of intense production of pediocin AcH by this bacterium. However, no further reduction was observed, indicating that the amount of bacteriocin present in the culture broth was not sufficient for total elimination of L. monocytogenes. Also, pediocin AcH activity decreased gradually firom 8 days of incubation and disappeared at the end of 3-week period (Ennahar et al., 1998). This study suggested that the residual antibacterial activity would be too weak to avoid a further recontamination and re­ growth of L. monocytogenes.

Inactivation of Clostridium botulinum by LAB bacteriocins.

Effect of LAB bacteriocins against Clostridium botulinum in media and foods. Pediocin A produced by Pediococcus pentosaceus 43200 and 43201 inhibited both type A and B strains of C. botulinum (Daeschel and Klaenhammer, 1985; Okereke and Montville, 1991a & b). However, pediocin AcH produced by Pediococcus acidilactici had no activity against types A and B strains of C. botulinum (Bhunia et al.,

1988; Okereke and Montville, 1991a & b). No botulinum toxin was detected in chicken salad that was formulated with P. acidilactici at 10* CFU/g, whereas toxin was detected in chicken salad without P. acidilactici and stored at 35, 30 and 15°C for 7, 14 and 28 days, respectively (Hutton et al., 1991).

The sensitivities of proteolytic and nonproteolytic C. botulinum strains to nisin and other bacteriocins were investigated by Montville et al. (1992). Different nisin sensitivities among vegetative cells and their spores from 18 C. botulinum strains were indicated, but these differences were not biotype-associated (Montville et al., 1992).

27 Higher level of nisin (100 and 250. ppmX were, required to prevent outgrowth, of botulinal spores in cheese spreads with 57% moisture level, whereas 12.5 ppm nisin was able to prevent outgrowth and toxin production in a cheese spread of 52% moisture

(Somers and Taylor, 1987). Nisin is more effective as an antibotulinal agent in process cheese spreads than in meat emulsions or bacon in other studies. For example, 550 ppm of nisin in combination with 60 ppm of nitrite failed to prevent outgrowth of C. botulinum spores in pork slurries adjusted to pH 5.8 (Rayman et al., 1983). Addition of nisin at levels up to 500 ppm allowed only 1-week extension of the shelf life of chicken frankfurter emulsions challenged with spores of C. botulinum and incubated at 27°C

(Taylor et al., 1985). In addition, nisin alone had no antibotulinal effectiveness in bacon

(Taylor and Sommers, 1985). Similarly, 500-2000 lU/ml of nisin inhibits by 50% botulinal spore outgrowth in Tryptone-Peptone-Yeast extract-Glucose (TPYG) broth, but levels up to 10,000 lU/ml are ineffective in cooked meat medium (Scott and Taylor,

1981a & b). This limited effectiveness of nisin in meat emulsions is likely due to its binding to meat particles (Taylor et al., 1985). In bacon, Nisapiin is relatively insoluble in the brine, and distribution of nisin is likely the limiting factor (Taylor and Sommers,

1985). Furthermore, 10 lU/ml nisin inhibits Bacillus licheniformis spores in basal medium, but 100 lU/ml is needed in the presence of 1% salt (Bell and De Lacy, 1985).

This may be caused by salt interference with nisin absorption to the spore. In contrast, nisin is relatively easy to incorporate in process cheese spreads, and it seems to be reasonably stable in cheese emulsions (Somers and Taylor, 1987). In these studies, nisin clearly serves as one part of a muliple barrier inhibition system.

28 Activity of LAB hacteriociii& a^iinst spores^ Mechanistic infonnation. about the action of bacteriocin against spores is not clearly understood, although it is known to be different from that against vegetative cell. Most of the studies are published in

1950-60’s and deal primarily with nisin. The bacteriocins produced by Pediococcus pentosaceus and Lactobacillus plantarum are also active against botulinal spores

(Okereke and Montville, 1991 a & b), but nothing about their mechanism is known.

Nisin is normally sporostatic, but its sporicidal effects have been reported depending on both the strain and severity of any heat treatment (De Vuyst and

Vandamne, 1994). In general, spores are more sensitive than cells, and the sensitivity is increased in acidic substrates, if the spores have suffered injury (e. g. heat treatment).

Lewis et al. (1954) reported that complementary action of heat and nisin caused a 55% decrease in D-value of C. sporogenes PA 3679. Other studies confirmed preservative effectiveness of nisin combined with mild heat against C. sporogenes PA 3679, C. thermosaccharolyticum. Bacillus coagulans, and B. stearothermophilus (O’Brien et al.,

1956; Campbell et al., 1960). Similarly, Campbell and Sniff (1959) indicated that the number of B. coagulans spores was greatly reduced in the presence of nisin, when the spores were heated at 107.2‘*C. They suggested that nisin was strongly bound by the spores and was not removed by the dilution procedures employed. If nisin were bound to the spores, it would be carried over into the subculture medium and would be available to inhibit germination or outgrowth of the survivors (Campbell and Sniff,

1959). Campbell and O’Brien (1955) also demonstrated that 0.03 ppm nisin in carry­ over was sufficient to inhibit the development of severely heated spores of Clostridum and Bacillus sp. Campbell and Snifi" (1959) further indicated that nisin was firmly

29 bound by the spores and inhibited spore recovery by preventing, spore germination, or outgrowth, because survivor curves in the absence and presence of nisin were superimposable when nisin was inactivated by trypsin after heating. In addition, the spores germinated at a slower rate in the presence of nisin (125 pg/ml) than the absence of nisin, however, outgrowth was inhibited by nisin until nisin was inactivated by the addtion of trypsin (Campbell and Sniff, 1959).

Hirsch and Grinsted (1959) indicated that nisin was actively sporicidal in the case of Clostridium butyricum, but the spore coimt of C. sporogenes and C. bifermentans did not change. Therefore, they suggested that nisin acts, according to the nature of test organism and the numbers present, either sporicidally or as an inhibitor of germination (Hirsch and Grinsted, 1959). Mikolajcik et al. (1965) also reported that nisin had a sporicidal effect against B. licheniformis-A with the degree of destruction being proportional to the nisin concentration and the length of exposure time in the buffer system. Similarly, dramatic increase in nisin sensitivity was noted in spores of

Bacillus and Clostridum sp. as the severity of heating increased (Denny et al., 1961;

Heinemann et al., 1965).

Several studies indicated that nisin could function as an effective inhibitor of B. cereus and C. botulinum spore outgrowth by inactivating the membrane sulfhydryl groups in newly germinated spores (Scott and Taylor, 1981; Somers and Taylor, 1981;

Morris et al., 1984; Liu and Hansen, 1990). It has been reported that the activity of nisin is directly related to the presence of dehydroalanine residues, which can act as acceptors for electrophilic agents and readily react with sulfhydryl groups (Gross and Morrell,

1967; Liu and Hansen, 1990). The sulfhydryl groups are involved in many enzymatic

30 reactions, of the. cell ancL therefor&serve as. potential sites of inhibitioa by sulfhydryl agents (Rose et al., 1999). Gross and Morrell (1967) suggested that metabolically important compounds, such as glutathione or coenzyme A, which are sulfhydryl- containing enzymes, may be intercepted by nisin.

Temperature of heat-shocking, length of the heating period, pH, and spore load were all shown to be important variables that had an impact on the effectiveness of nisin in preventing the outgrowth of C. botulinum spores (Scott and Taylor, 1981). Nisin was more effective in preventing outgrowth at pH 6 than in pH 7 and 8 . C. botulinum spores become less likely to germinate and outgrow as the pH decreases and nisin may magnify this effect. For example, the stability of nisin in solution is dependent on the pH of the solution with nisin being more stable at acid pHs (Lipinska, 1977). Heat also has a profound impact on the effectiveness of nisin. Nisin was more effective in inhibiting spore outgrowth following heating at the higher heat-shocking temperatures.

In addition, heating at a specified high heat-shocking temperature for progressively longer periods of time showed that longer heating periods decreased required inhibitory concentration of nisin even further (Scott and Taylor, 1981).

31 Serotype Charcteristics Proteolytic Psychrophilic Heat resistant Human pathogen A, some B & F + - + +

C,D - - +/- -

E, G, some B & F - + - +

Table 1.1: Distingushing characteristics of Clostridium botulinum serotypes (Sperber, 1982)

32 Food product Year Location Toxin No. of No. o f Reference type cases death Duck paste 1922 UK A 8 8 Brett, 1999 Minced meat pie 1935 UKB 1 1 Brett, 1999 Pickled fish 1955 UK A 6 0 Brett, 1999 Potato salad 1969 USA A 6 0 CDC, 1969; Ryan & Cerington, 1971. Canned jalapeno peppers 1977 USAB 59 0 Tenanova etal., 1978 Potato salad 1978 USAA 34 2 CDC, 1978 Potato salad 1978 USAA 8 0 Seals etal., 1981 Canned Alaskan salmon 1978 UK E 4 2 Ball etal., 1979. Kapchunka (salt-cured, uneviscerated whitefish) 1981 USAB 1 0 Johnson, 2000 Beef pot pie 1982 USAA 1 0 Johnson, 2000 Bologna sausage 1982 Madagascar E 60 30 Vicens et al., 1985 Sauteed onion 1983 USA A 28 1 CDC, 1984 Karahi-renkon (deep-fried, mustard-stuffed, lotus root) 1984 Japan A 36 II Otoftiji et al., 1987 Baked potatoes 1984 USAA I 0 MacDonald et al., 1986 Chopped garlic-in-oil 1985 Canada B 36 0 HWC, 1986 Bottled peanuts 1986 Taiwan A II 0 HWC, 1987 Mushrooms (lightly salted water in bottle) 1987 Canada A 9 2 Chou et al., 1988 Rice and vegetables, shelf-stable airlinp meal 1987 UK w A 1 0 Colebatch et al., 1989 w Kapchunka 1987 USA/Israel E 8 2 Johnson, 2000 Chopped garlic-in-oil 1989 USA A 3 0 Food chemical news, 1989 Hazelnut yogurt 1989 UKB 27 I Critchley et al., 1989. Faseikh (salted fish) 1991 Egypt E 92 20 Weber etal., 1993 Cheese sauce 1993 USA A 5 1 Johnson, 2000 Skordalia (Greek salad with baked potato) 1994 USAA 30 0 Johnson, 2000 Clam chowder soup (vacuum-packed in a plastic) 1994 USA A 2 0 Anonymous, 1994 Marscapone cheese 1997 Italy A 8 1 Aureli et al., 2000 Muktuk (fermented blubber, skin, and meat from a white whale) 1997 Canada E 4 1 HPB, 1997 Pickled eggs (hard-boiled eggs with beets, hot peppers & vinegar) 1997 USAB 1 0 CDC. 2000 Matambre (Argentine meat roll) 1998 Argentina A 9 0 V illaretal., 1999 Peyote tea (dried, alkaline-ground peyote in water) 1998 USAB 3 0 Hashimoto and Parko, 1998 Green olives (in salty water in bottle) 1998 Japan B 5 0 The Daily Yomiuri, 1998 Home-preserved mushrooms bottled in oil 1998 Italy B 2 0 Brusin and Salmaso, 1998 Vegetable soup 1998 Italy AII Brusin and Salmaso, 1998 Fermented salmon roe 2001 Canada - 4 0 Infectious Diseases News Brief, 20QI Fermented beaver tail 2001 USAE 14 0 CDC, 2001

Table 1.2: Examples of food-bome botulism outbreaks Step Requirement Physical & Structural Comments Changes Activation • Heat • Loss of protein • Reversible • Chemicals that mimic from spore coats • Conditioning of heat treatment (e.g. reducing • Expose binding spores to increase rate agents) sites (receptors) for & extent of • High Pressure germinants germination Processing • Needed for quantification Initiation • Germ inants: • Spores remain • “Commitment” Amino acids (e.g. L-alanine) refractile or “Triggering” Sugars (e.g. glucose) • Cortex • Takes minutes Salts (e.g. Ca-dipicolinic hydrolyzing enzymes acid) are activated Enzymes (e.g. lysozyme) • Interaction of germinants with receptors Germination • Water • Loss of heat • Easily measured • Optimum temperature resistance microscopically • Excrete Ca-DPA • Stainable • Loss of retractility • Degradation of protein, RNA, lipids • Decrease in CD • Start of swelling Outgrowth • Water • Emergence of • End of • Nutrients vegetative cell germination

Table 1.3: Transformation of a dormant spore into a vegetative cell

34 Microorganism Activation Germination Medium pH Reference

C. botulimtm 75*C BHI+1% YE+HCOj' 99% in 4h Treadwell et 62A 15-20 min CAA+1% YE+HCOj- 89% in 4h al., 1958 CAA+HCO3 "36%in4h L-ala+L-phe+L-arg+HCOs' 12% in 4h

C. botulinum 80"C L-cysteine + Sodium bicarbonate + 7.0 Rowley & 62A 60 min Sodium thioglycolate in TES buffer- Feeherry, 93% in 2h 1970

C. botulinum 80“C L-alanine or L-cysteine + Sodium 7.0 Montville et B-aphis & 60 min bicarbonate + Sodium thioglycolate in al., 1985 Ba410 TES buffer

C. botulinum 80“C L-alanine + Sodium bicarbonate + 7.0 Billon et al., 62A 60 min Sodium thioglycolate in potassium 1997 phosphate buffer

C. botulinum 65''C L-alanine + DT^-Iactic acid + glucose 7.0 Evans et al., NCIB4270 15 min + NaHCOs + sodium thioglycolate in 1997 (non-proteolytic, potassium phosphate buffer psychotolerant) C. botulinum 80"C L-alanine in phosphate-citrate buffer 7.0 Chaibi et al., 62A 15 min 1996 & 1997

C. botulinum 80”C L-alanine or L-cysteine in phosphate- 7.0 Blocher & 62A 15 min citrate buffer or 2 -(N-morpholino)- Busta, 1985 ethanesulfonate buffer

C. botulinum 80“C L-alanine + L-cysteine in 2-(N- 6 .0 Mazzotta& 169B 10 min morpholino)-ethanesulfonate buffer Montville, (proteolytic) 1999

Table 1.4: Germination conditions for Clostridivm botulirmm strains

35 Baclcriocin Producer Inblbilory Spectrum Reference '

Palbogens Non-patbogeiu Acidocin B Lactobacillus acidophilus M4é Bacillus cereus Clostridium sporogenes Brink et a!., 1994 Listeria titoitocyiogenes Enterococcusfaecalls AcidocinJll32 Lactobacillus acidophilus Lactobacillus easel, Lfermentum, L planlarum Tahara et al., 199^ JCMII32 BbcI829 Staphylococcus aureus Staphylococcus aureus RN4220 Streptococcus suis Crupper and ^ KSII829 Corynebacterlum psudotuberculosis, C. Corynebacterlum renale, landolo, 1996 diphtherlae Haemophilus parasuls Bordetella pertussis. B. bronchlseptica Moraxella bovis Pasteurella multoclda Bacillin Ills Bacillus subtllls OS Y111S Listeria monocytogenes Bacillus megaterlum Ku. 1993 Enterococcus faecalls Bievicin 286 Lactobacillus brevis VB286 Listeria monocytogenes Lactobacillus sp, L curvatus Coventry el al., ' Enterococcus faecalls Listeria Innocua, L. ivanovil, L seeligerl 1996a Streptococcus thermophilus Enterococcus faecium Camobacterium sp. Collcin Escherichia coll ‘ Escherichia coll 0IS7 H7 Murinda et al., ' 1996 CurvBticIn FS47 Lactobacillus curvatus FS47 Listeria monocytogenes Lactobacillus sp. Carver and ' Pedlococcus sp. Muriana, 1994 Enterococcus sp. Bacillus sp. Dlacelln B LactococtMS lactls subgp. lactls Clostridium perfrtngens Clostridium butyricum, C. tyrobutyricum, C. All et al., 1995 " bv. DIacetylaclls UL730 Listeria monocytogenes sporogenes Staphylococcus aureus Lactobacillus cremoris, L. plantarum, L diacetylactis Listeria Ivanovli. L Innocua

Enterocin P Enterococcusfaeclum p 13 Staphylococcia aureus Lactobacillus cuvatus, L fermentum, L reuterl CIntasetal., 1997 Listeria monocytogenes Pedlococcus pentosaceus Clostridium perfrtngens, C. botulinum Staphylococcus camosus Enterococcus faecalls Listeria innocua Clostridium sporogenes, C. tyrobutyricum Propionibactertum sp. (Continued)

Table 1.5: The inhibitory spectrum of bacteriocins of potential significance in food Table 1,5: Continued Enterocin 4 Enterococcus faecalis (NIA 4 Listeria monocytogenes Clostridium tyrobutyricum Joostenetal., 199^ Staphylococcus aureus Lactobacillus buchnerl, L brevis Salmonella cholerasuis Enterococcusfaecltun Listeria Innoctta Fcrmenticin B Lactobacillus fermentim Lactobacillus plantarum, L pentosus, L brevis, VanL and Lee, 199) delbrueckll subsp.bulgarlcta, L helvetlcus Micrococcus luteus Jensenlin G Proplontbaclerium Jehfenii Lactobacillus bulgarlcus, L delbrueckll subsp. Grinstead and ' PI26 lactls, L helvetlcus, L lactls Barefoot, 1992 Propionibactertum acldlproplonlcl Lacidin A Lactobacillus acidophilus Staphylococcus aureus Lactobacillus delbrueckll subsp./aclB ATCC Liao et al., 1994 ' OSUI33 Enterococcus faecalis 4797, L acldophlllus NCK89 Leuconostoc mesenteroldes Lactacin B Lactobacillus acidophilus N2 Lactobacillus bulgarlcus, L helvetlcus, Barefoot and Lactococciu lactls, Lactobacllha lelchlmannll Klaenhammer, ATCC 4797 1984 LaclacinF Lactobacillus johnsonti LactobtKlllus debruecki, L Abee et al„ 1994a’ VPI11088 helvetlcus,Enterococcus faecalls Lacllcln3l47 Lactococcus lactls subip. lactls Bacillus cerues. Acetobacter acetl McAulifle et al., ^ DPC3147 Listeria monocytogenes Bacillus subtllls 1998; Staphylococcus aureus Lactobacillus acidophilus, L fermentlcum, L. Rysn et al.,1996; Streptococcus agalactlae, curvatus, L bulgarlcus, L helvetlcus, L easel, L Ryan et al., 1998 S. dysgalactiae kefir, L lelchlmannll, L reuterl, L sake. Lactococcus lactls subsp. cremoris Listeria Innocua Pedlococcus pentrlceans, P. pentosaceus Streptococcus thermophilus, S. uberl

Lactococcin R Lactococcus lactls subfp. L. monocytogenes Scott A Listeria Innocua, L Ivanovil, L murrayl, L. gntyl, Yildirim and ^ cremoris R Bacillus cerues L seeligerl, L tvelshlmerl Johnson, 1998 Staphylococcus aureus Enterococau faeclum Clostridium perjringens Bacillus subtllls, B. megaterlum Enterococcusfaecalls Lactobacllltis virldans, L acldophlllus, L. Streptococcus agalactlae plantarum, L easel Leuconostoc oenos, L. mensenteroldes Pedlococcus acldllactlcl, P. dextrlnlcus, P parvulus, P. urtnaegul Streptococcus thermophilus Mtcrococcus roseus, M. luteus Staphylococcus lentus, S. hycus Clostridium sporogenes (Continued) Table 1,5: Continued Lcuconocin S Leuconostoc paramesenteroides Listeria monocytogenes Lewis cl al., 1991' OX Yersinia enterocolltica Okcrekc and Clostridium botulinum Montville, 1991

LinocinMlS Brevlbaclerlum linens M18 Listeria monocytogenes Valdes-stauberani) .Schctcr, 1996 NIsin A Lactococcus lactls subip. lactls Bacillus cereus Listeria innocua, L ivanovil, L seeligerl, L Spelhaug and ' ATCC 11454 Clostridium perjringens uelshlmerl, L murrayl, L welshmerl Harlandcr, 1989 JS2I Listeria monocytogenes MicrococcusJIavus, M. luteus DeVos el al., 1993 NIZ0R5 Staphylococcus aureus Streptococcus thermophilus Meghtousetal., INRA 1-6 Aeromonas hydrophlla Clostridium tyrobutyricum 1999 NP40 Escherichia coll 0I57 H7 Lactococcus lactls subsp. cremoris, L NZI yibrlo cholerae, K. parahaemolytlcus diacetylactis NCD02III.NCD0S1 Enterococcusfaecalls Lactobacillus plantarum ILCI3 Pedlococcus acldllactlcl

NislnZ Lactococcus lactls subip. lactls Bacillus cereus MicrococcusJIavus DeVos et al., 1993 NIZ022I86.NIZ0N9 Listeria monocytogenes Streptococcus thermophilus Abee et al., 1994b ATCC 7962 Clostridium tyrobutyricum NCD02118.NCD02597. Lactococcus lactls subsp. cremoris NCDO209I NCK400 UN80 ILCll, ILC19, ILCI26, 1LCSL5, ILCSL20,10| Pediocin A Pedlococcus pentosaceus Lactobacillus acidophilus, L. bulgarlcus, L Piva and Headon, ' FBD6I Jermemtum, L helvetlcus, L sake 1994 Proplonlbacterlum acldlproplonlcl Lactococcus cremoris Streptococcus thermophilus Staphylococcia camosus Clostridium sporogenes, C. tyrobutlrlcum Lactococcus lactls

Pediocin AcH Pedlococcus acidilactiçl H Aeromonas hydrophllla Lactobacillus lelchlmannll, L plantarum, !.. Bhunia et al., 1988 Bacillus cereus virldecens Clostridium perjringens Leuconostoc mesenteroldes Listeria monocytogenes Pseudomonas putlda Staphylococcus aureus (Continued) Table 1.5: Continued Pediocin AcH Laclobaclllus plantarim WHE Listeria monocytogenes Listeria Innocua, L seelegerl Ennahai et al., 92 Bacillus cereus Enterococau faeclum 1996 Lactobacillus curvatus Pedlococcus pentosaceus Mtcrococcus sedentarius Staphylococcus xylosus Pediocin PA-1 Pediococctu acldllactlcl Listeria monocytogenes Pedlococcus pentosaceus, P. acldllactlcl, P. Gonzalez and PACl.O cerevlslae Kunka, 1987 Lactobacillus plantarum, L. easel, L. bifermentans Pucci etal., 1988 Leuconostoc mesenteroldes, L dextranlcum Henderson et al., Enterococau sp 1992

Pediocin Si-1 Pedlococcus acldllactlcl Si-1 Clostridium perjringens Lactobacillus plantarum, L lelchlmannll, L Schvedetal., 199) Listeria monocytogenes brevis

Pediocin L50 Pedlococcus acldllactlcl Clostridium perfrtngens, C. botulinum Lactobacillus sp. Cintas et al., I99f Listeria monocytogenes Pedlococcus pentosaceus Stapbylococcus aureus Leuconostoc cremoris Enterococatsfaecalls Lactococcus lactls Staphylococcus camosus Clostrlditun sporogenes, C. tyrobutyricum

Pediocin PC Pedlococcus acldllactlcl PÙ Clostridium perfrtngens Listeria innocua, L ivanovli, L seeligerl, L iager and Listeria monocytogenes welshimeri Harlander, 1992 Leuconostoc mesenteroldes

Piscicocins VI Camobacterium pisclaola VI Listeria monocytogenes Camobacterium divergens Bhugaloo-vial et ' Enterococcusfaecalls Lactobacillus curvatus, L sake, L plantarum al., 1996 Leuconostoc mensenteroldes Pedlococcus acldllactici. Listeria innocua Plantaricin D Lactobacillus plantarum BFE Listeria monocytogenes Lactobacillus sake Franz etal., 1998 ' 905 Piantaricin S Lactobacillus plantanim Enterococcus faecalls Clostridium tyrobutyricum iimenez-Diaz et LPCOlO Proplonlbacterim sp. al., 1993 Lactococcus lactls subsp. cremoris, L. lacils subsp. lactls Leuconostoc mesenteroldes, L. paramesenteroides Lactobacillus delbrueckll, Lfermentum, L. helvetlcus, L sake, L. curvatus Mtcrococcus sp., Pedlococcus pentosaceus Streptococcus thermophilus (Continued) Table 1.5: Continued Plantaricin S Lactobacillus plantanitn Enterococcusfaecalls Clostridium tyrobutyricum Jimcnez-Diaz et ' LPCOlO Proplonlbacterim sp. al.. 1993 Lactococcus lactls subsp. cremoris, L lactls subsp. lactls Leuconostoc mesenteroldes, L paramesenteroides Lactobacillus ilelbrueckli, Lfermentum, L. helvetlcus, L sake, L curvatus MIcrococcus sp., Pedlococcus pentosaceus Streptococcus thermophilus Plantaricin SA6 Lactobacillus plantanm SA6 Lactobacillus brevis, L buchnerl Rckhifctal.. 199^ Leuconostoc paramesenteroides Listeria grayl Propionicin Pl.Q-1 Proplonibacteriutn thofitil Listeria monocytogenes Psudomonas fluorescence Lyon ct al., 1993 ' yibrlo parahaemolyticus Corynebacterlum sp. Yersinia enterocolltica Salivacin 140 Lactobacillus salh/arlus subsp. Listeria monocytogenes Lactobacillus easel, L celloblosus, L delbrueckll Ariharactal., '' SalIcInlusTlAO Staphylococcus aureus subsp. delbrueckll, L delbrueckll subsp. lactls, L 1996 Yersinia enterocolltica helvetlcus, L planlarum, Actinomyces viscosus Staphylococcus mutons, Proplonlbacterlum acnes Themtophilln 13 Streptococcus thermopfiilus Listeria monocytogenes S9 Streptococcus thermophilus Marcisct ct al.. ' Bacillus cereus C 14 Enterococcusfaeclum 1997 Clostrliilum botulinum 100003 Lactococcus cremoris Lactobacillus acidophilus, L helvetlcus, L fermentum, Leuconostoc cremoris, L mesenteroldes, Bffldobacterlum bffldum Listeria Innocua, Bacillus subtllls Clostridium tyrobutyricum Staphyrococcus camosus, MIcrococcus varions Tticnnophilln 347 Streptococcus thermopfiilus 347 Listeria monocytogenes Lactococcus lactls subsp. lactls, L lactls subsp. Villanictal., 1995 Enterococcus faecalls cremoris, L lactls subsp. diacetylactis Streptococcus thermophilus Enterococau faeclum, E. durons, E. casseliflavus

Variacin MIcrococcus varions Listeria monocytogenes Lactobacillus helvetlcus, L. bulgarlcus, L lactls, Pridmore ct al.. Clostridium botulinum L delbrueckll, L acidophilus, L plantarum, L. 1996 Staphylococcus aureus sake, L curvatus, Leuconostoc mesenteroldes Bacillus cereus Streptococcus thermophilus Enterococcus faecalls Lactococcus lactls, Enterococcus faeclum Listeria Innocua, Clostridium sp. Staphylococcus camosus, S. saprophytlcus Bacillus subiilis, B. pumills Core

Inner membrane

Germ cell wall~ Peptidoglycan Cortex ----- layers Outer membrane —, Protein layers w/ Coat layers high S-S bonds

Exosporlum

Figure 1.1: Diagram of typical spore structure. Structure is not drawn to actual scale

41 ^Dha^ ^Ala-Leu lie Leu . Sv Gly Met I I / \ + I I hfeN-lle-Dhb-Ala Ala-ABA Ala-Lys-ABA Gly \ s / \ / \ / Pro -Gly ® “Ala Asn I _ Met + / I His—Ala ABA - Lys+ + + / \ / HOOC- Lys -Dha- Val-Hls-lle-Ser-Ala ABA-Ala 34 ^ s ^

Figure 1.2: Structure of nisin. Dha = Dehydroaianine, Dhb = Dehydrobutyrine, ABA = a-Amino butyric acid, Ala-S-Ala = Lanthionine, ABA-S-Ala = P-Methyl lanthionie (Gross et ai-, 1971)

42 tnisin

membrane

Figure 1.3: Mechanism of action of nisin. The cytoplasmic membrane of bacterial cell is the primary target for nisin activity. Three steps of nisin mode of action on membranes are generally accepted; (1) binding, (2) insertion, and (3) pore formation. Adapted from Montville et al., 1995.

43 C H A PT E R 2

IN A CTIV A TIO N O F CLOSTRIDIUM BOTULINUM A BY

BACTERIOCINS OF LACTIC ACH) BACTERIA IN LABORATORY BROTH

AND SOLID FOOD SYSTEM

ABSTRACT

Bacteriocins from three lactic acid bacteria (LAB), Lactococcus lactis ssp. lactis

ATCC 11454, Pedlococcus acldllactici P02, and Lactobacillus acidophilus OSU133 were screened for their inhibitory activity against Clostridium botulirmm Type A as a target microorganism. EfScacy of bacteriocins against C. botulirmm was tested by agar diffusion assay and by monitoring growth, of bottdiman in the presence of bacteriocin in both Tryptone-Peptone-GIucose-Yeast extract (TPGY) broth and model solid food systems. Feasibility of biopreservation method was also investigated by a challenge study using nisin, which was added to modifred-atmosphere packaged roasted potatoes that was inoculated with C. botulinum.

44 Culture supernatant of a nisia-producing^ bacterium, was more active against C botulinum than that of pediocin P02 or lacidin A producer. The preparation method and pH of nisin solution was important to measure the bacteriocin activity effectively.

Lactococcus lactis culture supernatant inhibited the growth of C. botulinum in mashed potatoes stored at 25®C for 5 days. In addition, commercial nisin (50-250 pg/g potatoes) prevented the growth of C. botulinum for 5 days in modified-atmosphere (100 % Ni or

20% CO2: 80% N 2) packaged roasted potatoes stored at 25°C.

INTRODUCTION

Some foods receive heat treatments to increase their shelf life and are then refrigerated to ensure their safety. Such products have been referred to in published papers as minimally processed refrigerated foods (Kvenberg, 1990), new generation refrigerated foods (Rhodehamel, 1992), extended shelf life refrigerated foods (Conner et al., 1989), or refrigerated pasteurized foods of extended durability (REPFEDS)

(Notermans et al., 1990; Limd and Notermans, 1993). Presence of Clostridium botuliman in such products is a potential health hazard, since the thermal treatment applied, often referred to as pasteurization, is not sufficient to inactivate spores of the pathogen. In addition, botulism risk increases in these products, when such foods are packaged under vacuum or modified atmosphere (Conner et al., 1989). Therefore, many such foods require refrigeration to avoid microbial spoilage. However, temperature abuse can readily generate a hazard and the safety of these foods cannot be guaranteed

45 with. reMgeratioii alone. To ensure the safe^ of minimally processed foodSv especially

those in the low acid category, a second antimicrobial barrier in addition to low

temperature must be included (King and Bolin, 1989; Skinner et al., 1999). Biological

barriers are tested in the form o f ‘protective cultures’ or their metabolites, notably

bacteriocins (Holzaphel et al., 1995).

Discussions on the risks associated with refrigerated foods have mainly focused

on nonproteolytic types of C. botulinum because of their ability to form toxin at

refrigeration temperatures (Conner et al., 1989; Notermans et al., 1990; Doyle, 1991;

Rhodehamel, 1992; Lund and Notermans, 1993; Peck, 1997). Nevertheless, outbreaks

of botulism have been associated with refrigerated commercial clam chowder and bean

dip that were attributed to toxin production by proteolytic types of C. botulinum in

which improper storage temperature was implicated (Trevejo, 1995). In addition,

outbreaks of type A botulism also involved individuals who had eaten potato salad in

restaurant (Seals et al., 1981). The USDA approved a biopreservation method in 1986

for use in bacon. It has been evaluated for effectiveness in delaying spoilage in many

commodities including beef and poultry meats (Gombas, 1989), chicken salad (Hutton

et al., 1991), a model gravy system (Crandall and Montville, 1993), and “sous-vide”

salmon and rice (Simpson et al., 1993). However, few attempts have been made about

the inactivation of C. botulinum by biopreservation in cooked potatoes, even though

several botulism outbreaks are linked to the consumption of this product (Ryan and

Cerington, 1971; CDC, 1978; Seals et al., 1981: MacDonald et al., 1984). Thus, cooked potatoes were chosen as a model food system in this study.

46 Biopreservation may consist of Q) adding bacterial strains that grow rapidly and produce antagonistic substances (e.g., bacteriocins), (2) adding purified antagonistic substances, (3) adding the fermentation liquor or concentrate fiom an antagonistic organism, or (4) adding mesophilic LAB as a fail-safe protection against temperature abuse (Stiles, 1996). In the process of biopreservation for minimally processed foods, the bacteriocin-producing lactic acid bacteria barely grow if the products are properly refiigerated. If the products are temperature-abused, the bacteriocin-producing cultures grow and produce bacteriocins to inhibit pathogenic and spoilage microorganisms

(Hutton et al., 1991). Before biocontrol could be utilized as an additional barrier, it must be evaluated for its effectiveness in preventing growth and toxin production by pathogens such as C. botulirmm in minimally processed foods under temperature abuse.

Therefore, the goal of this research is to investigate the feasibility of using biopreservation method to inhibit C. botulirmm in minimally processed foods. For this purpose, three bacteriocins of lactic acid bacteria are evaluated for their effectiveness against C. botulinum in laboratory growth broth media and a food system.

MATERIALS AND METHODS

Bacterial strains and media

Three bacteriocin-producing strains, Lactococcus lactis ssp. lactis ATCC 11454,

Pedlococcus acidilactici P02, and Lactobacillus acidophilus OSU133 and target or indicator microorganisms, Clostridium botulinum Type A ATCC 25763, Lactobacillus

47 hichimanmi ATCC 4797^ ClosteidhatL sporogeaes. OSUS92^ and Ustepia monocytogenes ScottA were used in this study (Table 2.1). L. lactis ssp. lactis ATCC

11454, a nisin-producing strain, and C. sporogenes OSU392 were obtained from the culture collection of The Ohio State University - Department of Microbiology

(Columbus, OH). Pediococcus acidilactici P02, a pediocin P02 producer, L. acidophilus OSU133, a lacidin A producer, L. leichimannii ATCC 4797, and L. monocytogenes Scott A were obtained from the culture collection of the food safety laboratories. Department of Food Science and Technology, The Ohio State University

(Columbus, OH). Clostridium botulinum Type A ATCC 25763 was obtained from the

American Type Culture Collection (Bethesda, MD). Cultures were kept frozen at -80“C in appropriate media containing 20% (v/v) sterile 80%-glycerol (Sigma Chemical Co.,

St. Louis, MO) until use. All cultures were grown at 37°C, except L. lactis ssp. lactis

ATCC 11454, which is grown at 3 1°C for 24 h, and transferred at least twice before use.

TPGY broth consisted of (per liter preparation): tryptone (Difco), 50 g; peptone (Difco),

5g; yeast extract (Difco), 20 g; dextrose (Difco), 4 g; L-cysteine hydrochloride (Sigma),

1 g. All agar media were prepared by adding 15 g agar (Difco) per liter of broth. Soft agar media were prepared to contain 7.5 g/L agar.

Bacteriocin preparation

Nisin and pediocin P02 were prepared in two forms, (a) bacteriocin-producing culture supernatant fluid and (b) dried, crude extracted bacteriocin. For culture supernatant fluid, bacteriocin-producing culture was grown for 18 h and centrifuged at

12,000 rpm for 10 min to remove the cells. This cell-free supernatant from bacteriocin

48 producer was. directly tested for the. activity of bacteriocia. Aitemadveiy, supernatant fluid was boiled for 10 tnin to ensure that bacteriocin-producing cells are removed before use in the study.

The complete procedures for crude bacteriocin preparation are included in

Burianek and Yousef (2000). Concisely, overnight (18 h) culture of bacteriocin- producing cells was pelleted in a centrifuge and the bacteriocin-containing supernatant fluid was collected. Chloroform was added to the supernatant fluid and mixed vigorously, the mixture was centrifuged, and the sediments at the interface between aqueous and chloroform layers were collected. The interface material was resuspended in Tris buffer and centrifuged again to remove the remaining chloroform and medium.

The pellet was dried in aluminum pan in a chemical hood overnight and kept firozen until use. Dried pellets were rehydrated with sterile distilled water before use as a source of bacteriocin.

Commercial nisin solution

Commercial nisin (Nisaplin) was purchased from Sigma Chemical Co. (St.

Louis, MO). Stock solution was prepared in 0.02N HCl solution to contain 10® lU/ml nisin (Scott and Taylor, 1981). The pH of the stock solution was adjusted to 2 with HCl solution and the mixture was autoclaved at 121°C for 10 min. Alternatively, stock solution was fliter-sterilized using 0.45 pm membrane filter (Acrodisc; Gelman

Sciences, Ann Arbor, MI) and the filtrate was used for the nisin assay. For another preparation method of clean nisin solution, stock solution of nisin was centrifuged at

12,000 rpm for 10 min, and the supernatant fluid was used. Bacteriocin activity in these

49 sterile nisin. solutions, prepared by three methods, was. tested against JL leichimannii

ATCC 4797. In addition, pH of the nisin stock solution was adjusted to 5.2, 3.6, or 2.8

with HCl, and tested for the activity.

Comparison of antimicrobial activity of bacteriocins

Commercial and laboratory produced nisin and preparations of pediocin P02

were tested against C. botulinum, C. sporogenes, and selected sensitive indicators.

Bacteriocin activity was analyzed using agar diffusion assay (Barefoot and

Klaenhammer, 1983). Ten microliters of 18 h indicator culture was mixed with 7 ml

soft agar. Inoculated soft agar was overlaid onto solidified agar plate. Two-fold serial

dilutions of bacteriocin preparations were made using sterile water. Portions (5 pi) of

original or diluted samples were spotted onto the seeded soft overlay agar plate. Assay

plates were incubated at 37°C for 24 h. Plates seeded with anaerobic indicators, C.

botulinum and C. sporogenes, were incubated under anaerobic conditions. After

incubation, the plates were examined for the presence of a clear zone of inhibition.

Diameter of inhibition area produced by non-diluted sample was measured as an index of activity. Additionally, dilutions that produced growth inhibition were recorded.

Bacteriocin activity was defined as the reciprocal of the highest dilution exhibiting complete inhibition of the indicator lawn, expressed in arbitrary units (AU) per milliliter. Alternatively, zone o f inhibition resulting finm spotting bacteriocin preparations on indicator’s lawn was used as an indication of bacteriocin activity.

50 Inactivation of Cbotulinum by bacteriocin in liquid medium

Behavior of C. botulinum in the presence of bacteriocins in liquid media was examined. Overnight (18 h) culture of C. botulinum was diluted with 0.1% peptone water to contain 10^-10“* CFU/ml, and 1 ml of diluted culture was inoculated into TPGY broth tubes. One milliliter of bacteriocin-producing culture supernatant or commercial nisin solution was added to the inoculated broth tubes. Treated broth tubes were incubated at 37®C in an anaerobic chamber containing an atmosphere of 5% Hz, 10%

COz, and 85% Nz (Forma Scientific Inc., Marietta, OH). Samples were taken at appropriate intervals, diluted, and spread-plated (0.1 ml) onto TPGY agar plate. Plates were incubated at 3T"C for 48 h in the anaerobic chamber.

Inactivation of C.botulinum by bacteriocin in cooked potatoes

Russet potatoes were purchased from local grocery and were cleaned with running water. The cleaned potatoes were cooked in a steamer until they were soft, then peeled, diced by knife, and mashed by stomaching (Lab-Blender 400, Cooke Laboratory products, Alexandria, VA) for 2 min. Portions of the mashed potatoes (20 g) were weighed in 250 ml beaker, autoclaved at 15-20 psi for 15 min, and stored in the anaerobic chamber overnight before use. Stock solution (10^ lU/ml) of commercial nisin was adjusted to 3200 AU/ml to simulate bacteriocin activity in nisin-producing L. lactis culture supernatant (3200 AU/ml against C. botulinum). The conversion between the two units (AU and lU) of bacteriocin activity was calculated using L. leichimannii

ATCC 4797 as a indicator microorganism. For example, 10^ lU/ml of commercial nisin

51 produced the activity of 3 2 x LQ® AU/ml against L, leidàmanmi ATCC 4797^

Therefore, 1 lU is approximately equivalent to 32 AU against this indicator microorganism. Pediocin P02 and lacidin A supernatant fluids were prepared as indicated in previous section of bacteriocin preparation. One milliliter of diluted C. botulinum culture (18 h) was added into the mashed potato samples, mixed thoroughly to distribute the culture inoculum evenly in the sample. One milliliter of each bacteriocin preparation was added to the inoculated potato samples and the samples were mixed.

Treated potato samples were stored at room temperature (25°C) in the anaerobic chamber. At appropriate intervals, potato samples (20 g) were diluted with 80 ml of sterile 0.1% peptone water and stirred with magnetic stirrer for 2 min. Diluted samples

(0.1 ml) were then spread-plated onto TPGY agar. The plates were incubated at 37®C for 48 h in the anaerobic chamber and the colonies formed on agar were enumerated.

Challenge study using modified-atmosphere-packaged roasted potatoes inoculated with C. botulinum

Frozen roasted potatoes (Hillshire Farm and Kahns, Cincinnati, OH) were thawed and 50 ± 0.1 g was weighed into sterile WHIRL-?AK bags (Nasco

International, Modesto, CA). These bags were packaged (Sipromac vacuum packaging machine, St. Germain, Quebec, Canada) with two different gas atmospheres; (i) modified atmosphere (20% CO 2 + 80% N 2), (ii) Nitrogen (100%). One ml of diluted C. botulinum culture and bacteriocin preparations were added through a rubber septum

(Alltech Inc., Deerfield, IL) that was attached outside of the package. The total volume 52 o f the. additives in diffeient treatments wa& at^uste&even^ with 0.1%. peptone water.

The concentrations of added nisin in modified atmosphere packaged and nitrogen

packaged roasted potatoes were 250 and 50 pg/g, respectively. Treated potato sample

packages were stored at room temperature (25°C).

Potato samples were taken at appropriate intervals and stomached for 3 min with

200 ml of sterile 0.1% peptone water. Diluted samples (0.1 ml) were then spread-plated

onto appropriate agar media, and incubated at 37°C for 48 h. Total Plate Count (TPC)

was obtained using Plate Count Agar (PCA, Difco) and the viable count of C. botulinum

was carried on Clostrisel Agar (Difco) containing sodium thioglycollate and sodium

formaldehyde sulfoxylate as reducing agents, and neomycin sulfate and sodium azide as

selective agents.

RESULTS AND DISCUSSION

Comparison of antimicrobial activity of bacteriocins

Nisin, pediocin P02, and lacidin A, in the form of culture supernatants or as crude preparations, were tested for inhibitory effect against C. botulinum and other indicator microorganisms using agar diffusion assay (Table 2.2). The spot-on-the-lawn method was selected for detecting bacteriocins among agar diffusion assays, since it was shown to be more reproducible, rapid and easy to score than fiip-plate or well- diffusion assay (Spelhaug and Harlander, 1989; Lewus and Montville, 1991).

Lactococcus lactis culture supernatant was more effective than that of P. acidilactici

53 £02 or L. acidophilus, iir inhibiting, the. vegetative cells.o£ botulinunu AntibotulinaL

activity of nisin was demonstrated in other studies (Okereke and Montville, 1991a & b).

While cell free supernatant fluid from P. acidilactici £02 showed a weak inhibitory

activity against C. botulinum, crude extract of pediocin £02 showed stronger activity.

Pediococcus acidilactici was shown to be effective against C. botulinum spores (Hutton

et al., 1991), but another strain of the same bacterium was not effective (Okereke and

Montville, 1991a). Lacidin A, produced by L. acidophilus OSU133, showed inhibitory

activity against pathogens such as Staphylococcus aureus, and other lactic acid bacteria

strains (Liao et al., 1994). However, inhibitory effect of lacidin A against C. botulinum

was not previously reported.

Results in Table 2.2 show that the inhibition area diameter is proportionally

related to the bacteriocin activity (expressed in arbitrary unit/ml). Therefore, either of

these two parameters can be used for the indication of bacteriocin activity. However,

measurement of activity in AU provides only discontinuous values, since serial dilution

of bacteriocin solution is involved. All of the indicator strains tested were inhibited by

the crude extracts of nisin and pediocin £02, but their sensitivities varied greatly (Table

2.2 & Figure 2.1). Clostridium botulimtm strain used in this study was slightly more sensitive to nisin preparation than was L. monocytogenes Scott A, and less sensitive than was L. leichimannii ATCC 4797. On the other hand, C. sporogenes, which is commonly used as a surrogate strain for C. botulinum, was much less sensitive to pediocin £02 preparation than was L. monocytogenes Scott A. Earlier reports agree with our results, which showed that pediocin was a more efflcient inhibitor of L. monocytogenes than nisin (Motlagh et al., 1991; Meghrous et al., 1999). Meghrous et al.

54 Cl999) concluded that there are large differences in the effectiveness of pediocin and

nisin on the vegetative cells and spores of Gram-positive bacteria. The relationship

between the diameter of inhibition area and the reciprocal of dilution factor in

logarithmic scale was linear for the indicator strains tested (Figure 2.1). However, the

slopes of these linear best-fit lines were slightly different among the indicators. This

was related to the several factors affecting diffusion of bacteriocins, including thickness

and composition of agar. Outward diffusion of bacteriocin is more rapid in a thin layer

agar, so the area of inhibition is larger on thin agar plates. In addition, composition of

agar medium influences the size of the inhibition area. Most agars carry an overall

negative charge, due to acid and sulfate groups on the polysaccharides. Therefore,

cationic bacteriocin molecule, such as nisin may be electrostatically bound to acid or

sulfate groups on agar, and consequently the diffusion rate will be reduced (Barry,

1986). Furthermore, certain ingredients in agar medium such as Tween 80 increase the

diffusion rate of bacteriocin (Nissen-Meyer et al., 1992). Therefore, standardization of

the agar diffusion assay method is important for the accurate measurement of

bacteriocin activity.

Currently, nisin is the only bacteriocin that is commonly used as a purified

substance for food preservation. Crude pediocin has been shown to be a good preservative in food system (Yousef et al., 1991). During purification of bacteriocin, or processing and storage of foods, bacteriocins are commonly exposed to various conditions. Therefore, it is essential to determine their stability against pH, temperature, filtration, and lyophilisation. This study simply compared the different methods to obtain the sterile nisin solution (Figure 2.2). Autoclaving at 121“C for 10 min under pH

55 2.& did not decrease, the nishk activity^ Filtration, and centrifugation^ however, greatly decreased the nisin activity. Nisin binding to the filtration unit was avoided by using low protein binding membrane. Nisin may have remained bound to the particulates that were included in Nisaplin as a carrier material. Rammelsberg and Radier (1990) also reported that nisin could be heated to 121“C for 30 min at pH values between pH 2.0 and 4.0 without loss of activity. In terms of pH stability, nisin was stable between pH

2.0 to 5.0, and solubility, consequently nisin activity, decreased at higher pH values

(Liu and Hansen, 1990). Nisin activity was compared at three pH values, 2.8, 3.6, or

5.2, in Figure 2.3. Activity measurement (diameter of zone of inhibition) was not greatly different among the three pH values. However, slightly smaller inhibition zone was formed at pH 5.2 than at pH 3.6 and 2.8, when C. sporogenes was used as an indicator microorganism (Figure 2.3a).

Inactivation of C. botulinum by bacteriocin in liquid medium

To determine the antimicrobial properties of a substance, endpoint or descriptive tests may be used. In descriptive tests, periodic sampling is carried out to determine changes in viable cell numbers over time in the presence of antimicrobial substances

(Davidson and Parish, 1989). Therefore, as a next screening test, behavior of C. botulirmm in TPGY broth containing bacteriocin-producing culture supernatant was examined for 8 h (Figure 2.4). Little difference in survivor finction was observed between the samples with and without bacteriocin-producing culture supernatant. This indicates that low concentration (320 AU/ml, i.e., diluted to 1/10 of initial concentration, 3200 AU/ml against C. botulinum fiom agar diffusion assay) of

56 bacteriocins. in- culture supernatant have no- discernable eifect ou C. botulinum^ iu nutrient-rich broth such as TPGY.

In food products, processors are interested in using the minimal antimicrobial concentration needed to cause inhibition of potential spoilage microorganisms. Even extension of the lag phase of a microorganism can be important under abusive conditions (Davidson and Parish, 1989). Therefore, it is important to examine the growth of microorganism in the presence o f antimicrobials over time. Nisin is the only bacteriocin for which a standard solution of known activity is available. Therefore, growth of C. botulinum in TPGY broth in the presence of nisin was monitored for 50 h

(Figure 2.5). Nisin (2.5 pg/ml) killed C. botulinum cells incidentally, and small percentage of survived population remained static for 16 h, and then began to grow.

Inactivation ofC. botulinum by bacteriocin in cooked potatoes

While in-vitro tests can give good information on antimicrobial performance, they cannot duplicate all the variability, which might exist in real food systems

(Davidson and Parish, 1989). Some outbreaks of botulism in US have been related to the consumption of potato products such as potato salad and baked potatoes, when these products were temperature abused (Ryan and Cerington, 1971; CDC, 1978; Seals et al.,

1981: MacDonald et al., 1984). Potatoes can harbor C. botulinum readily, since C. botulinum is a soil microorganism. Therefore, potatoes were chosen as a model food system. Behavior of C. botulinum in nisin-containing mashed potatoes was examined

(Figure 2.6). Low concentration of commercial nisin (0.125 pg/g of potatoes) initially decreased the count of C. botulinum for 2 days, and then cells began to grow.

57 Noticeably^ supernatant Êom nisin-pmducing A lactis. culture, inhibited the growth, for 5

days at 25°C. If low numbers of L. lactis cells were present in supernatant, these cells

grew and exerted the inhibitory effect. This is desirable when minimally processed

foods, which rely on refrigeration for storage, are temperature abused. However, no

further test to confirm the presence of L lactis cells in supernatant (e.g., plate count of

L. lactis cells) was performed in this study. The mere presence of bacteriocin producing

cell is effective to control the growth of pathogenic bacteria, as long as the lactic culture

strain do not affect the quality of food adversely (Ray, 1992). To separate the effect of

bacteriocin-producing cells from the bacteriocin itself, supernatant fluids of P.

acidilactici and L. acidophilus were boiled for 10 min, and growth of C. botulinum in

mashed potatoes was observed in the presence of pediocin P02 and lacidin A (Figure

2.7). Pediocin P02 and lacidin A were not very effective, compared to nisin.

Challenge study using modified-atmosphere-packaged roasted potatoes inoculated with C. botulinum

Nisin was selected for next application study. Behavior of C. botulinum cells in modified-atmosphere-packaged (MAP) roasted potato cubes in the presence of nisin was investigated (Figure 2.8 & Figure 2.9). Both 100% Nz and 20% COz + 80% Nz - packaged potatoes supported the growth of C. botulinum. The count reached to maximum population in 5 days at 25°C. In 100% Nz-pack, nisin (50 pg/g potatoes) decreased the count of C. botulinum incidentally, and small population of survivors began to grow in 3 days (Figure 2.8). Higher concentration of nisin (250 pg/g potatoes), used in 20% COz + 80% Nz-pack, prevented the growth of C. botulinum for 5 days at

58 25“C. The ability of C0% to inhibit competing spoilage microorganisms while

stimulating germinaiton of C. botulinum spores was reported in other studies

(Foegeding and Busta, 1983; Farber, 1991) and this adds more concerns on the

microbiological safety of MAP products. Studies indicated that raw, cooked, or baked

potatoes supported the growth and the toxin production by C. botulinum (Sugiyama et

al., 1981; Notermans et al., 1981; Notermans et al., 1985). Spores of both proteloytic

and non-proteolytic C. botulinum in vacuum-packaged potatoes survived the cooking

process and resulted in growth and formation of toxin within 5-9 days at 25"C

(Notermans et al., 1981; Lund et al., 1988). In addition, sliced, fresh, raw potatoes

inoculated with types A or B spores, packaged under vacuum and held at 22“C became

toxic in 3 days (Solomon et al., 1994). All these studies demonstrated that potatoes,

regardless of raw or cooked, or vacuum-packed or packaged under modified atmosphere, are probable causes of botulism. As far as we have investigated, currently, no bacteriocin has been tested in potato products. Results in this study clearly show that growth of C. botulinum can be prevented in the presence of nisin, otherwise C. botulinum readily grow in potato samples and may produce toxin.

REFERENCES

Barefoot, S. F. and Klaenhammer, T. R. 1983. Detection and activity of lactacin B, a bacteriocin produced by Lactobacillus acidophilus. Appl. Environ. Microbiol. 45:1808- 1815.

59 Bany, A. L. L9S6. Procedure, for testing antimicrobial agents, uragar media: Theoretical considerations. In Lorian p. I

Burianek, L. L. and Yousef, A. E. 2000. Solvent extraction of bacteriocin from liquid culture. Lett. Appl. Microbiol. 31:193-197.

Centers for Disease Control. 1978. Morbidity Mortality Weekly Report. 27:138

Conner, D. E., Scott, V. N., Bernard, D. T., and Kautter, D. A. 1989. Potential Clostridium botulimm hazards associated with extended shelf life refrigerated foods: A review. J. Food Safety. 10:131-153.

Crandall, A. D. and Montville, T. J. 1993. Inhibition of Clostridium botulinum growth and toxigenesis in a model gravy system by coinoculation with bacteriocin-producing lactic acid bacteria. J. Food Prot. 56:485-488,492.

Davidson, P. M. and Parish, M. E. 1989. Methods for testing the efficacy of food antimicrobials. Food Technol. 43(1):148-155.

Doyle, M. P. 1991. Evaluating the potential risk from extended-shelf-life refrigerated foods by Clostridium botulinum inoculation studies. Food Technol. 45(4):154-156.

Farber, J. M. 1991. Microbiological aspects of modified-atmosphere packaging technology - a review. J. Food Prot. 54:58-70.

Foegeding,P. M. and Busta, F. F. 1983. Effect of carbon dioxide, nitrogen, and hydrogen gases on germination of Clostridium botulinum spores. J. Food Prot. 46:987

Gombas, D. E. 1989. Biological competition as a preserving mechanism. J. Food Safety. 10:107-117.

Holzaphel, W. H., Geisen, R., and Schillinger, U. 1995. Biological preservation of foods with reference to protective cultures, bacteriocins and food-grade enzymes. Int. J. Food Microbiol. 24:343-362.

Hutton, M. T., Chehak, P. A., and Hanlin, J. H. 1991. Inhibition of botulinum toxin production by Pediococcus acidilactici in temperature abused refrigerated foods. J. Food Safety. 11:255-267.

King, A. D. and Bolin, H. R. 1989. Physiological and microbiological storage stability of minimally processed fruits and vegetables. Food Technol. 43(2):132-135.

Kvenberg, J. E. 1990. Microbiological criteria and regulatory aspects of minimally processed refrigerated foods. J. Food Prot 53:1990.

60 Lewus^ C. and. Montville^ Detection, of bacteriocins. produced by lactic acid bacteria. J. Microbiol. Methods 13:145-150.

Liao, C.-C., Yousef, A. E., Chism, G. W., and Richter, E. R. 1994. Inhibition of Staphylococcus auretts in buffer, culture media and foods by lacidin A, a bacteriocin produced by Lactobacillus acidophilus OSU133. J. Food Safety 14:87-101.

Liu, W. and Hansen, J. N. 1990. Some chemical and physical properties of nisin, a small protein antibiotic produced by Lactococcus lactis. Appl. Environ. Microbiol. 56:2551-2558.

Lund, B. M., Graham, A. P., and George, S. M. 1988. Growth and formation of toxin by Clostridium botulinum in peeled, inoculated, vacuum-packed potatoes after a double pasteurization and storage at 25°C. J. Appl. Bacteriol. 64:241-246.

Lund, B. M. and Notermans, S. H. W. 1993. Potential hazards associated with REPFEDS. In Clostridium botulinum. Ecology and Control in Foods. A. W. Hauschild and K. L. Dodds (Ed.). Pp. 279-303. Marcel Dekker, Inc., New York, NY.

MacDonald, K. L., Cohen, M. L., and Blake, P. A. 1986. The changing epidemiology of adult botulism in the United States. Amer. J. Epidemiol. 124:794-799.

Meghrous, J., Lacroix, C., and Simard, R. E. 1999. The effects on vegetative cells and spores of three bacteriocins from lactic acid bacteria. Food Microbiol. 16:105-114.

Motlagh, A. M., Johnson, M. C., and Ray, B. 1991. Viability loss of foodbome pathogens by starter culture metabolites. J. Food Prot. 54:873-878, 884.

Nissen-Meyer, J., Holo, H., Havarstein, L. S., Sletten, K., and Nes, I. F. 1992. A novel lactococcal bacteriocin whose activity depends on the complementary action of two peptides. J. Bacteriol. 174:5686-5692.

Notermans, S., Dufrenne, J., and Keiybets, M. J. H. 1981. Vacuum-packed cooked potatoes: Toxin production by Clostridium, botulinum. and shelf life. J. Food ProL 44:572-575.

Notermans, S., Dufrenne, J., and Keiybets, M. J. H. 1985. Use of preservatives to delay toxin formation by Clostridium botulinum (Type B, strain okra) in vacuum-packed, cooked potatoes. J. Food Prot 48:851-855.

Notermans, S., Dufrenne, J., and Lund, B. M. 1990. Botulism risk of refrigerated, processed foods of extended durability. J. Food Prot 53:1020-1024.

Okereke, A. and Montville, T. J. 1991a. Bacteriocin inhibition of Clostridium botulimm spores by lactic acid bacteria. J. Food Prot 54:349-353.

61 Okereke, A. and Montville, T. J. 1991b. Bacteriocin-mediated inhibition of Clostridium botulinum spores by lactic acid bacteria at refrigeration and abuse temperatures. Appl. Environ. Microbiol. 57:3423-3428.

Parente, E., Brienza, C., Moles, M., and Ricciardi, A. 1995. A comparison of methods for the measurement of bacteriocin activity. J. Microbiol. Methods 22:95-108.

Peck, M. W. 1997. Clostridium botulinum and the safety of refrigerated processed foods of extended durability. Trends Food Sci. Technol. 8:186-192.

Rammelsberg, M. and Radier, F. 1990. Antibacterial polypeptides of Lactobacillus species. J. Appl. Bacteriol. 69:177-184.

Rhodehamel, E. J. 1992. FDA’s concerns with sous vide processing. Food Technol. 46(12):73-76.

Ray, B. 1992. Bacteriocins of starter culture bacteria as food biopreservatives: an overview. In Food Biopreservatives of Microbial Origin. B. Ray and M.A. Daeschel (Ed.). Pp. 177-205. CRC press.

Ryan, D. W. and Cerington, M. 1971. Human type A botulism. JAMA. 216:513-514

Seals, J. E., Snyder, J. D., Edell, T. A., Hatheway, C. L., Johnson, C. J., Swanson, R. C., and Hughes, J. M. 1981. Restaurant-associated Type A botulism: Transmission by potato salad. Amer. J. Epidemiol. 113:436-444.

Skinner, G. E., Solomon, H. M., and Fingerhut, G. A. 1999. Prevention of Clostridium botulinum type A, proteolytic type B and E toxin formation in refrigerated pea soup by Lactobacillus plantarum ATCC 8014. J. Food Sci. 64:724-727.

Solomon, H. M., Rhodehamel, E. J., and Kautter, D. A. 1994. Growth and toxin production by Clostridium botulinum in sliced raw potatoes under vacuum with and without sulfite. J. Food Prot. 57:878-881.

Speihaug, S. R. and Harlander, S. K. 1989. Inhibition of foodbome bacterial pathogens by bacteriocins from Lactococcus lactis and Pediococcus pentosaceus. 52:856-862.

Stiles, M. E. 1996. Biopreservation by lactic acid bacteria. Antonie van Leeuwenhoek. 70:331-345.

Sugiyama, H., Woodbum, M., Yang, K. H., and Movroydis, C. 1981. Production of botulinum toxin in inoculated pack studies of foil-wrapped baked potatoes. J. Food Prot. 44:896-898.

62 Trevejo, R. T. 1995. Foodbome outbreaks in California. Dairy» Food EnviroiL Sanit. 15:611-615.

Yousef, A. E., Luchansky, J. B., Degnan, A. J., and Doyle, M. P. 1991. Behavior of Listeria monocytogenes in wiener exudates in the presence of Pediococcus acidilactici AcH or pediocin AcH during storage at 4 or 25°C. Appl. Environ. Microbiol. 57:1461- 1467.

63 Bacteriocin Producer Indicator

Bacterium Medium Bacterium Medium

Nisin Lactococcus lactis MRS Lactobacillus leichimarmii ATCC 4797 MRS ATCC 11454 Clostridium sporogenes OSU392 TPGY

Clostridium botulinum ATCC 25763 TPGY

Listeria monocytogenes Scott A TB;TA

Pediocin P02 Pediococcus acidilactici MRS Clostridium sporogenes OSU392 TPGY P02 Clostridium botulinum ATCC 25763 TPGY

Listeria monocytogenes Scott A TB;TA

Lacidin A Lactobacillus acidophilus MRS Lactobacillus leichimannii ATCC 4797 MRS OSU133 Clostridium sporogenes OSU392 TPGY

Clostridium botulinum ATCC 25763 TPGY

'Broth media were used to grow cultures and produce bacteriocins, regular agar for enumeration and soft agar (contains 0.75% agar) for overlaid media, which are seeded with bacteriocin indicators. All media were obtained from Difco (Ann Arbor, MI). MRS, deMan, Rogosa and Sharpe broth or agar, TPGY, Tryptone, Peptone, Glucose, Yeast extract broth or agar; TB, Tryptose broth; TA, Tryptose agar.

Table 2.1. Bacteriocin producing and indicator bacteria used in this study and media’ in which these bacteria were grown

64 Bacteriocin or Producer Sensitive Indicator Zone of Bacteriocin Inhibition* Activity (AU/ml)" Lactococcus lactis ATCC Clostridium botulinum ATCC 25763 + 3200 11454 Clostridium sporogenes OSU392 ++ 3200 Culture supernatant fluid Listeria monocytogenes Scott A + 3200 Lactobacillus leichimannii ATCC 4797 ++ 12800

Pediococcus acidilactici Clostridium botulinum ATCC 25763 + 800 P02 Clostridium sporogenes OSU392 - 0 Culture supernatant fluid Listeria monocytogenes Scott A ++ 6400 Lactobacillus leichimannii ATCC 4797 0

Lactobacillus acidophilus Clostridium botulinum ATCC 25763 + 800 OSU133 Clostridium sporogenes OSU392 - 0 Culture supernatant fluid Listeria monocytogenes Scott A N/A' N/A Lactobacillus leichimannii ATCC 4797 -H- 12800

Commercial nisin Clostridium botulinum ATCC 25763 ++ 102400 (10* lU/ml) Clostridium sporogenes 0SU392 ++ 3200 Listeria monocytogenes Scott A + 3200 Lactobacillus leichimannii ATCC 4797 +++ 3276800

Nisin preparation Clostridium botulinum ATCC 25763 ++ 102400 (Crude extracts) Clostridium sporogenes OSU392 ++ 3200 Listeria monocytogenes Scott A + 51200 Lactobacillus leichimannii ATCC 4797 +++ >819200

Pediocin P02 preparation Clostridium botulinum ATCC 25763 + 25600 (Crude extracts) Clostridium sporogenes OSU392 ++ 3200 Listeria monocytogenes Scott A +++ >819200 Lactobacillus leichimannii ATCC 4797 ++ 3200

diameter of inhibition zone at non-diluted concentration (2*^ < 10 mm; (++■) diameter of inhibition zone 10 to 20 mm; (+++) diameter of inhibition zone > 20 mm. '* A lf* Arbitrary unit, calculated as 200-x 1 /thu last dilution which shows inhibitioiT, corresponding to the sensitive indicators ' N/A, Not applicable; Previous study showed no activity (Liao et al., 1994)

Table 2.2: The antimicrobial activity of bacteriocins determined by agar-difhision assay

65 2 0 .

E & c so j= ç 30 *s 2 5 . O) s 2 0 . 15: î i 1 0 . Q

0 1 2 3 4 5 6 7 LOG (Reciprocal of dilution factor)

Figure 2.1 : Comparison of activity of crude extracts of nisin and pediocin P02 preparation, measured as zone of inhibition, against different indicator microorganisms using agar difiusion assay, (a) Nisin preparation, (b) Pediocin P02 preparation. (■ ) Clostridiim botulinum ATCC 25763; (A) Clostridium sporogenes OSU392; (^) Listeria monocytogenes Scott A; (#) Lactobacillus leichimarmii ATCC 4797.

66 3500000 3 2 7 6 8 0 0

3000000

2500000

2000000

;> 1500000

1000000

500000 102400 102400

Control Autoclave Filtration Centrifugation Preparation method

Figure 1 2 \Activity of commercial nisin solution (2500 ng/ml), prepared by different methods, against ùictobacillus leichimcamii ATCC 4797

67 .2 10

o 5. @)

N î

Q 20.

PH

Figure 2.3: Comparison of activity of commercial nisin (2.5 pg/ml) at different pHs against three indicator microorganisms. Diameter of zone of inhibition was measured for non-diluted samples, (a) Clostridiim sporogenes OSU392; (b) Listeria monocytogenes Scott A; (c) Lactobacillus leichimannii ATCC 4797.

68 10000

1000

o z z c 100 o

IW I co3

0 2 4 6 8 10 HousatST®

Figure 2.4: Behavior of Clostridium botulinum ATCC 25762 în TPGY broth containing supernatant of bacteriocin producing cultures, nisin producing Lactococcus lactis ATCC 11454, pediocin P02 producing Pediococcus acidilactici P02, and lacidin A producing Lactobacillus acidophilus OSU133. Data points shown are average values of 2 individual experiments. (■ ) C. botulinum', (#) C.botulinum + L. lactis supernatant; (♦) C. botulinum + P. acidilactici supernatant; (A) C. botulinum + L. acidophilus supernatant

69 1000000

100000 -

10000 -

z 1000 - z c o 100 -

I 10 - control - o —nisin I3 CO

0.01 -

0.001 0 10 20 30 40 50 6 0 Heurs at 37°C

Figure 2.5: Behavior of Clostridium botulinum ATCC 25763 in TPGY broth containing commercial nisin (2.5 ^ig/mi).

70 1000000

100000 -

o 10000 ■ z z 1000 - c o "S «5 10 0 - u . I 1 0 - 3 co

0.1

0.01 0 1 2 3 54 6

Days at 25°C

Figure 2.6: Behavior of Clostridium botulinum ATCC 25763 in mashed potatoes stored at 25°C in the presence of nisin (0.125 pg/g potato). ( ■ ) C. botulinum only; (#) C. botulinum + commercial nisin; ( A ) C. botulinum + L. lactis supernatant. Each data points represent the average values of 3-5 experiments.

71 100000

10000

1000

100

100. e 3 CO 10.

0.1

0.01 0 1 2 3 4 5 6 Days at 25°C

Figure 2.7: Behavior of Clostridiian botulinum ATCC 25763 in autoclaved mashed potatoes in the presence of (a) pediocin P02 (Each data points are average values of 3 individual experiments) and (t) lacidin A. (□ ) C. botulinum only; (#) C. botulinum + boiled supernatant from P. acidilactici P02; (O) C. botulinum + boiled supernatant from L. acidophilus OSU133.

72 10000000

1000000 -

100000 -

■ ± 10000 - z 1000 - § ts loo­

1 0 - £ w 0.1

0.01

0.001 0 1 2 3 4 5 6

Days at 25°C

Figure 2.8: Behavior of Clostridium botulinum ATCC 25763 in nitrogen-packaged ( 100% Na> roasted potato cubes in the presence of commercial nisin (50 pg/g potatoes). Each data points are average values of 3 individual experiments. (■) C. botulinum', (#) C. botulinum + nisin

73 1000000

100000 10000 o z 1000 z c 100 o u m

0.1 03 0.01

0.001

0.0001 1 20 3 4 5 6 Days at 25°C

Figure 2.9; Behavior of Clostridiim botulinum ATCC 25763 in modified-atmosphere- packaged ( 2 0 % CO2 + 80% N 2) roasted potato cubes in the presence of commercial nisin (250 pg/g potatoes). (■ ) C. botulinum; (#) C.botulinum + nisin

74 CHAPTERS

CULTURABILITY OF CLOSTRIDIUM BOTULINUM UNDER

DIFFERENT GERMINATION CONDITIONS, SUBLETHAL HEAT

TREATMENTS, AND IN THE PRESENCE OF NISIN

ABSTRACT

Thermal resistance and heat activation characteristics of Clostridium botulinum

ATCC 25763 spores were evaluated. The effect of nisin and pH on the activation and subsequent germination was also investigated. Spores of C. botulinum were not inactivated by heat treatments up to 92“C for 2 h. Heat treatment at 85°C for 90 min was selected, as the optimal activation condition based on monitoring subsequent germination. L-alanine alone or in combination with L-cysteine was not sufficient to germinate the spores of this strain. Tryptone-Peptone-Glucose-Yeast extract (TPGY) broth supplemented with L-alanine was used as a suitable germination medium.

Decreasing pH of activation suspension increased the degree of phase darkening, i.e..

75 germinatioa. la adëitioi^ presCTee-of^nisin-daring- aetivatiorr inereased the degree of

phase darkening. The majority of spore populations were dormant under pH 2.8 and

these populations required heat activation to increase the culturability on TPGY agar

medium. However, extended heating in the presence of nisin at pH 2.8 decreased the

spore count. Whereas, heat activation was less necessary at pH 3.4, compared with that

at pH 2.8.

INTRODUCTION

Thermal processing is still the most common method to preserve the food

products, despite the emergence of novel non-thermal techniques. Although thermal

processes are designed to kill bacterial pathogens, in general, the spores are resistant to

mild heat treatments such as pasteurization. In some cases, spores are activated by such

heating (Laurent et al., 1999). Effects of heat treatment on spores may be categorized as

three ways. First, sublethal heat may increase the culturable population, i.e., enables

spores to form colonies on agar medium; without heat, these spores would not have

formed colonies (Roberts, 1970). Secondly, heat treatments that do not result in complete inactivation of bacterial spores, still may damage them enough to prevent growth under the conditions that prevail after heating (Murrell, 1964). Lastly, severe heat treatments inactivate the spore population and leave only few surviving spores

(Roberts, 1970).

Activation describes changes in spores, which has been subjected to various

76 pretreatments. that alter the rate and. extent, of germination. (Lefehvre and Leblanc^

1988). Activation is most commonly achieved by sublethal heat treatment, although several other treatments, such as extremes of pH and reducing agents, have been used

(Gibbs, 1967; Keynan et al., 1969; Steinbuch, 1976; EI-Mabsout and Stevenson, 1979).

Germination of some spores following activation is rapid when suitable germinants are present. Heat activation results in an increased extent of germination as well as simplification of the requirements for the initiation of germination. For example,

Yousten (1975) reported that heat-activated B. cereus spores germinated in the presence of L-alanine or inosine, but only a combination of these two germinants were effective without heat activation. However, when the heat treatment is not carefully controlled, low spore counts will be obtained due to inadequate heat activation or excessive spore destruction (El-Mabsout and Stevenson, 1979). It is therefore important to be able to predict the ability of the spores to survive heat treatment and the effect of environmental conditions on their subsequent growth in foods (Laurent et al., 1999). Shacter and

Hashimoto (1975) indicated that germination response was changed by the sublethal heat injury of spores receiving extended heat-activation treatment. Such sublethal heat injury appears to be created by the combined effect of high activation temperature, duration of heat treatment, and pH and salt concentration of the medium in which spores was suspended during heat activation and germination. It has been substantiated that the importance of the heat treatment in the processing is to injure or sensitize the bacterial spores, thus making them less tolerant of antimicrobial agents. Therefore, spore injury during heat processing is important in that it amplifies the effect of heat. In this way, enhancement of injury may well contribute to the development of improved

77 preservatioii techniques in the future.

One possible method for enhancing the efifectiveness of thermal processes while using less severe heat treatments is through the addition of bacteriocin such as nisin to alter the thermal resistance of bacterial spores, in such a way that survivors of nisin treatment will be more susceptible to thermal processes. Actually, if foods are thermally processed with nisin, residual nisin most likely remain after heating, and it enhances microbiological safety of foods (Wandling et al., 1999). Therefore, this study was conducted to investigate the optimal heat activation conditions of C. botulinum spores.

In addition, the effect of nisin and pH during heat activation of spores was examined.

MATERIALS AND METHODS

Bacterial strains and preparation of spore suspensions

Proteolytic C. botulinum Type A ATCC 25763 was obtained from the American

Type Culture Collection (Bethesda, MD). Culture was grown in Tryptone-Peptone-

Glucose-Yeast extract (TPGY) broth and kept frozen at -80°C with 20% (v/v) sterile

80%-glycerol (Sigma Chemical Co., St. Louis, MO) added to the broth. Spores of C. botulinum were formed on the surface of TPGY agar plate. Overnight culture was spread onto TPGY agar and incubated at 37°C in the anaerobic chamber for 7-10 days until more than 90% of population were sporulated. Spores were harvested by adding 10 ml of sterile cold distilled water, releasing the colonies containing spores from the surface of the agar with the use of a sterile disposable inoculating loop. Spores were

78 collected by centrifugation at 6000 min at 4°C and the pellet was washed in sterile cold distilled water, and centrifuged again at 12,000 rpm for 10 min at 4°C. The washing procedure was repeated six times. Spore suspensions were then enzymatically cleaned by the methods of Billon et al. (1997). Lysozyme solution (200 pg/ml of lysozyme in 0.05 mol/L potassium phosphate buffer, pH 8.1) was added to the spore pellets and incubated at 45®C for 30 min. Then, trypsin solution (100 pg/ml of trypsin in

0.05 mol/L potassium phosphate buffer, pH 8.1) were added and incubated at 45°C for 2 more hours. Spore crops, then, were washed ten times in sterile distilled water by centrifugation at 12,000 rpm for 10 min at 4“C. The final pellets were resuspended in sterile cold distilled water and kept refrigerated. Microscopic observations were made to confirm the purity of spore suspensions.

Germination media

Various germination buffers were tested to determine which germinants were needed for C. botulinum ATCC 25763. Two germination buffers were used: citrate- phosphate buffer at pH 7 or 2-(/V-morpholino)-ethanesulfonate (MES) buffer at pH 6 .

Germinant such as L-alanine (100 mM or 200 mM) and/or L-cysteine (50 mM) was included in germination buffer. In addition, TPGY broth containing 200 mM L-alanine was tested as a germination medium. Germination media are listed in Table 3.1. Spore suspension (containing approximately 10^ spores/ml in distilled water) was dispensed in each 200-pl thin wall microtest tubes (Biorad Laboratories, Hercules, CA) and submerged and heated at 80“C for 15 min (Ababouch and Busta, 1986; Chaibi et al.,

1996 & 1997) in a circulating water bath (Haake, Germany). Activated spores were

79 traosfeired into^ the anaerobie chamber, added into- the diiferent germination buffers,

and incubated at 37°C for germination. Samples of spore buffer mixture are taken at

intervals, and phase-change of the spores in germination medium was examined by the

phase-contrast microscope (Bausch and Lomb, Rochester, NY). The numbers of phase-

bright (ungerminated) spores and phase-dark (germinated) spores were counted.

Germination pH Concentration of germinant Reference medium Citrate- 7.0 100 mM L-alanine Chaibi et al., 1996 phosphate 200 mM L-alanine &1997 buffer 100 mM L-alanine + 50 mM L-cysteine Blocher & Busta, 1985 MES buffer* 6.1 100 mM L-alanine + 50 mM L-cysteine Mazzotta & Montville, 1999 TPGY broth 6.8 200 mM L-alanine

Table 3.1 : Media used in this study for germination of Clostridium botulinum spores

Measuring degree of germination

Spore germination was measured by determining loss of heat resistance and observing phase darkening of spores. For monitoring germination, spore suspensions were heat activated at various temperatures, and then transferred to the germination buffer inside the anaerobic chamber. Spores in germination buffer were incubated at

37°C, and aliquots were withdrawn at predetermined intervals. Aliquots were spread-

80 plated iiv TPGY agar without reheating^ a t heated at 8Q°C for IQ min. and surviving,

spores were enumerated using TPGY agar medium. Samples of the incubated mixture

were also examined by the phase-contrast microscope (Bausch and Lomb, Rochester,

NY) at a magnification of I,OOOX. The numbers of phase-bright (ungerminated) spores

and phase-dark (germinated) spores were counted. Between 100 and 200 spores were

observed for each sample. All media and reagents were prepared at least 24 h before use

and kept at 22-25“C in the anaerobic chamber (Mazzotta and Montville, 1999).

Optimal heat activation treatment

The heat resistance was assayed at various temperatures ranging from 80 to

lOO^C to investigate the lethal and sublethal temperatures for C. botulinum ATCC

25763 spores. Aliquots of 200-pl (containing approximately lO’ spores/ml in distilled water) were dispensed in each 200-pl thin wall microtest tubes (Biorad Laboratories,

Hercules, CA) and submerged and heated in a circulating water bath (Haake, Germany).

Each sample tubes were withdrawn at predetermined intervals, kept in ice, and then transferred into the anaerobic chamber. Spore suspensions were serially diluted in 0.1% peptone water and were enumerated in TPGY agar after incubation at 37°C for 24-48 h under anaerobic conditions.

Heat treatments at selected sublethal temperatures were compared for their ability to enhance spore germination. Variable heating periods, i.e., 15,30,45, 60, or 90 min, at three different temperatures, i.e., 80, 85, or 90°C, were selected and germination was monitored by the methods described earlier.

81 Spore germination in different nisin preparations

Nisaplin (10® lU of nisin/g), a commercial nisin concentrate composed of 2.5%

nisin in denatured milk solids and sodium chloride, or pure nisin was obtained from

Alpine and Barrette Ltd. (Trowbridge, England). Stock solutions of nisin containing 10®

lU/ml in 0.02N HCl was prepared as described by Scott and Taylor (1981). The pH was

adjusted to 2-3 with HCl solution and the mixture was autoclaved (10 min at 121“C)

and stored at 4°C. Activity of nisin levels in these autoclaved stock solutions (prepared

as 10® lU/ml) were measured in Arbitrary Units (AU)/ml with the spot-on-the-lawn

assay employing Lactobacillus leichimannii ATCC 4797 as indicator microorganism.

The conversion between the two units (AU and lU) of bacteriocin activity was

calculated as follows: 10® lU/ml of commercial nisin produced the activity of 3.2 x 10®

AU/ml against L. leichimannii ATCC 4797. Therefore, 1 lU is approximately

equivalent to 32 AU against this indicator microorganism.

Different nisin preparation (Nisaplin or pure nisin) was added to the spore

suspension to achieve a concentration of 250 pg/ml. The addition of nisin preparation

lowered the pH of the activation medium (i.e., mixture of nisin stock solution and

spores in distilled water), since the pHs of stock solutions of nisin preparations were pH

2.0 and pH 2.5, for Nisaplin and pure nisin, respectively. Thus, resulting pH values of activation medium were 2.8 and 3.4, for Nisaplin and pure nisin-containing solution, respectively. The pH value of the spore suspension without nisin preparation was 4.9.

Peptone water adjusted pH to 2.8 and 3.4 was used as control, to differentiate the effect of pH and nisin. Different heat activation treatments, in the absence or presence of

82 nisin, under two pHs (2.8 and 3.4), were applied as follows: (i) no heat; (ii) heating at

85°C for 30 min; (iii) heating at 85°C for 90 min; (iv) heating at 85°C for 90 min, followed by 5 min of cooling, then reheating at 80°C for 30 min, and enumerated cultivable spores on TPGY agar. Phase change of spores was also examined before and after heat activation treatment in the presence of nisin.

RESULTS AND DISCUSSION

Germination media

In preliminary experiments, different media were compared for the germination capabilities of C. botulinum spores (Table 3.1). Examination of phase change of spores showed that germination occurred in TPGY broth, but not in other buffer media in the presence of germinant (Data are not shown). After heat activation at 80“C for 15 min, spores in the buffer media containing L-alanine and/or L-cysteine remained phase- bright (ungerminated) after incubated at 37°C overnight (Data are not shown).

Therefore, L-alanine alone or in combination with L-cysteine was not sufBcient to induce spore germination. It is apparent that ingredient(s) in TPGY broth aided germination, but this was not investigated in this study.

Generally, germination requirement in Clostridium species is complex, requiring a combination of several ingredients, compared to Bacillus species, where a single compound, e.g. L-alanine, often initiates germination. Amino acids alone did not stimulate the germination of spores of C. bifermentans, and the minimum requirement

83 of compounds needed foF the~ gecminatioo was the presence of L-alanine L-

phenylalanine and lactate (Gibbs, 1964). In addition, an unknown factor present in

yeast extract was suggested for enhanced germination (Gibbs, 1964). Treadwell et al.

(1958) also indicated that yeast extract and sodium bicarbonate were necessary for rapid

germination of C. botulinum 62A spores. Therefore, it is also probable that some

ingredient(s) in yeast extract, included in TPGY agar, was essential for the germination

of C. botulinum spores used in our study. Presence of sodium bicarbonate was also

important to enhance the germination of C. botulinum 62A spores (Rowley and

Feeherry, 1970). Uehara and Frank (1965) found that germination of spores of C.

sporogenes PA 3679h was stimulated by thioglycollate (0.1%) in L-alanine +

pyrophosphate system, in which the two latter components were essential. In conclusion

Grom early studies, L-alanine was essential for the germination of clostridial spores,

however, additional germinants were also required for enhanced germination.

Therefore, TPGY broth supplemented with L-alanine (200 mM) was selected as the germination medium for the strain used in this study.

Optimal heat activation treatment

To determine optimal activation treatment, spores of C. botulinum were heated at 80“C to 100°C for up to 90 min. Culturable spore population of this strain did not decrease by heat treatments up to 92°C for 90 min heating period; actually, culturability increased with extended heating (Figure 3.1). This indicates that considerable activation occurred and inactivation was not followed during 90 min heating at 80-92°C. Different sublethal heating treatments, 80“C for 15 min, 85“C for 15 min, and 85°C for 90 min,

84 were, con^iared for optimal activation, and subsequent germination. Loss of beat resistance was not observed during 3 h period of germination incubation at 37°C after heat activation at 80“C for 15 min, and the counts of heat-treated samples were even higher than control samples (Figure 3.2a). These results suggest that heat activation process at 80°C for 15 min did not fully activate the spores, so the spores did not germinate readily, and further activation, which occurred during reheating (80°C for 30 min), increased the count. Generally, heat activation treatment at 80°C for 10-15 min is used for the proteolytic clostridial spores in most other studies (Blocher and Busta,

1985; Okereke and Montville, 1991; Chaibi et al., 1996; Mazzotta and Montville,

1999). However, this treatment was not sufficient to activate the spores of this strain.

Therefore, higher degree of heat treatment was tested. Heat treatments for 15 and 90 min at 85°C increased heat sensitivity of spores, spore counts decreased as the germination proceeded (Figures 3.2b & 3.2c). Germination occurred more readily after heating for 90 min at 85°C than for 15 min heating. In addition, the effect of intermittent heating on the culturability of spore was examined after these three heat activation treatments (Figure 3.3). Culturability was increased after all three heat activation (pre­ heating) treatments, compared to non-heated spores. Reheating for 30 min at 80°C further increased the culturability of spore samples, which were activated at 80°C for 15 min and 85®C for 15 min. The effect of double heating was greater with lower degree of pre-heating treatment (80°C for 15 min), than 85°C for 15 min. This suggested that double-stage, intermittent heating with mild temperature was very effective to increase the culturability (i.e., germinated population on the agar medium) of spores. The spore count after double-heating (pre-heated at 80°C for 15 min, and reheated at 80°C for 30

85 min), was comparable to the count obtained after single-heating at 85°C for 90 min. Cho

et ai. (1999) also found that double-stage heating at lethal temperature inactivated B.

subtilis spores more effectively as compared to the single-stage heating, indicating

intermittent heating effectively increased the degree of activation. On the other hand,

reheating did not increase the culturability of spores, which was heat activated at 85°C

for 90 min (Figure 3.3). Therefore, treatment at 85°C for 90 min was sufficient to

activate the spore populations fully. In other words, the best activation was achieved

with single heating treatment at this condition.

Phase contrast microscopy observation also showed similar results. Heating at

85“C for 90 min produced the greatest degree of phase change of spores, compared to

the other heat activation treatments (Figure 3.4). At 80“C, even for extended period (90

min) of heating (single-stage heating), did not activate most of the spore population

(Figure 3.4a). Thus, less than 20% of spores were phase-darken after 2 h germination

incubation. On the other hand, at 85°C and 90°C, the degree of germination increased in

proportion to the heating time at each temperature (Figure 3.4b & 3.4c). However, in

general, germination occurred more readily in samples treated at 85°C than 90°C.

Therefore, activation treatment at 85*’C for 90 min was chosen for C. botulinum strain

used in this study. The temperature and duration of optimal heating for activation of

spores vary widely among different species and even among different spore preparations of the same strain (Keynan et al., 1964). Spores of C. bifermentans

germinated readily after activated at 80-90“C (optimal at 85°C), and heating at temperatures < 70°C or > 90°C, prevented subsequent germination (Gibbs, 1964).

86 Spore germination in different nisin preparations

Culturability of control samples (no nisin added) during activation at pH values

2.8 and 3.4 was compared. Lower pH (2.8) suppressed the activation by heat (Figure

3.5). In other words, heat activation treatment was necessary to increase the spore culturability under lower pH condition. In addition, at pH 2.8, culturability was increased in proportion to the heating duration. At pH 2.8, similar spore counts on agar plates, as the counts obtained from control samples under pH 3.4, could be obtained only after double-stage, intermittent heating (85®C for 90 min + 80°C for 30 min). On the other hand, there was no measurable increase in spore culturability after beat treatments at pH 3.4. Thus, the majority of spore populations (more than 90 % of maximum spore count) were dormant under lower pH (pH 2.8) and these populations required heat activation treatment to germinate on the nutrient medium.

On the other hand, presence of nisin decreased the heat activation requirement for the spores under low pH. In other words, short time, single-stage heating in the presence of nisin resulted in the similar degree of culturability as in spores treated with double-stage heating in the absence of nisin. However, extended heating at pH 2.8 in the presence of nisin decreased spore culturability. Steinbuch (1976) also found that heat activation in acidic condition resulted in destruction of spores during the heat treatment. Activation of B. stearothermophilus spores in a suspension of pH 3.5 for 20 min at 100°C caused no destruction, however, intensification of heat activation to 15 min at 105°C or 10 min at 110“C, decreased spore counts by 1.5 and 3 logs, respectively

(Steinbuch, 1976). Beard et al. (1999) reported that D-values of B. licheniformis spores

87 were loweE th& presence^ of nisin-ancfthis^ effect of nisiiw was-furthec enhanced under

lower pH.

Several studies indicated the effect of subtilin, a bacteriocin produced by

Bacillus subtilis, on the heat treatment of spores. Subtilin is a 32-amino acid peptide,

which is structurally similar to nisin, including same 5 lanthionine rings with

dehydroalanine residue in position 5 (Liu and Hansen, 1993). It also seems likely that

the mechanism of antimicrobial action of subtilin is fundamentally the same as that of

nisin (Hansen, 1994). Subtilin decreased the spore counts for some Bacillus and

Clostridium strains during heat treatment (Michener, 1953 & 1955). In 9 Bacillus

strains and 2 Clostridium strains, heat treatment (10 min at 85°C) in the presence of

subtilin reduced the spore count by 25% to 99.5% as compared with the absence of

subtilin (Michener, 1953). LeBlanc et al. (1953) also reported that heat resistance, in the

presence of subtilin (14 ppm), of spores of C. sporogenes PA 3679 and C. botulinum

were only 47% and 63%, respectively, of the resistance in its absence. In addition, the

lethal effect of heat and subtilin on spores was also shown in studies in which subtilin was added after heat activation treatment (Lewis et al., 1954; Michener, 1955).

High acidity during heat activation treatment increased the degree of phase darkening of spores, and nisin enhanced it synergistically (Figure 3.6). Different pH of the heating suspension during activation at 85°C for 90 min affected phase-change of spores (Figure 3.6a). Initially, % phase-dark spores in three different pH suspensions before heat activation were between 5-7%. After heat activation, lower pH (pH 2.8) enhanced phase darkening of spores, compared to higher pH values (pH 3.4 and 4.9).

Heat activation in suspension of pH 2.8 induced phase darkening to 17-18 % of spore

88 population (Figure 3.6a). Under similar pH values^ degree of phase darkening was greater in the presence of nisin than it’s absence (Figure 3.6b & 3.6c). In addition, % phase-dark spores were higher (40%) when spores were activated at low pH (pH 2.8) in the presence of nisin, than in suspension at pH 3.4 (15%) in the presence of nisin

(Figure 3.6b & 3.6c). Keynan et ai. (1964) also indicated that incubation of B. cereus spores in the presence of reducing agents such as mercaptoethanol or thioglycollate or under low pH (less than 4.5) resulted in a partial activation of spores. Even though the incubation with reducing agents or under low pH did not replace the heat activation completely, it increased the germination rate considerably compared to unheated samples (Keynan et al., 1964). Therefore, nisin and/or acidic environment activated spores, and furthermore some populations were germinated, by observing phase- darkening, without the addition of germinants.

It has been reported that nisin activity is directly related to the presence of dehydroalanine residues, which can readily react with sulfhydryl groups (Gross and

Morrell, 1971; Liu and Hansen, 1993). Spore coats mainly consist of spore proteins, which contains high amount of cysteine (Tipper and Gauthier, 1972), and it is likely that a coat protein rich in cysteine, stabilized by S-S linkages, is responsible for maintaining the dormant state (Keynan et al., 1964). Therefore, the effect of nisin and pH on the activation of spores could be related to the reduction of disulfide bonds in spore protein.

Hashimoto and Conti (1971) observed the comb-like stmctures in the spore coat in heat- activated B. cereus spores. They also indicated that these comb-like structures might represent cysteine-rich structural protein present in the spore coat and the rapid disappearance of this subcoat space occurred concomitantly with the loss of heat

89 resistance. Furthermore, heat activation (70“C for 20 min) resulted in alteration in structural proteins and enzymes found in B. cereus spore coats (Srivastava and Fitz-

James, 1981). They also observed the changes, such as increased glycosylation of coat proteins, alteration in polypeptide pattern on sodium dodecyl sulfate - polyacylamide gels, and an increase in free SH groups of proteins (Srivastava and Fitz-James, 1981).

All of these studies suggested that spore coat or spore coat proteins are the possible targets for activation of germination by reducing agent, heat or pH.

REFERENCES

Ababouch, L. and Busta, F. F. 1986. A modified Lee tube technique for the cultivation and enumeration of anaerobes. Int. J. Food Microbiol. 3:211-216.

Beard, B. M., Sheldon, B. W., and Foegeding, P. M. 1999. Thermal resistance of bacterial spores in milk-based beverages supplemented with nisin. J. Food Prot. 62:484- 491.

Billon, C. M. P., McKirgan, C. J., McClure, P. J., and Adair, C. 1997. The effect of temperature on the germination of single spores of Clostridium botulinum 62A. J. Appl. Microbiol. 82:48-56.

Blocher, J. C. and Busta, F. F. 1985. Multiple modes of inhibition of spore germination and outgrowth by reduced pH and sorbafe. J. Appl. BacterioL 59:469-478.

Chaibi, A., Ababouch, L. H., and Busta, F. F. 1996. Inhibition by monoglycerides of L- alanine-triggered Bacillus cereus and Clostridium botulinum spore germination and outgrowth. J. Food Prot. 59:832-837.

Chaibi, A., Ababouch, L. H., Belasri, K., Boucetta, S., and Busta, F. F. 1997. Inhibition of germination and vegetative growth of Bacillus cereus T and Clostridium botulinum 62A spores by essential oils. Food Microbiol. 14:161-174.

90 Cho» H.-Y., Yousef» A. E.» and Sastry» S. K. 1999. Kinetics of inactivation ofBacillus subtilis spores by continuous or intermittent ohmic and conventional heating. Biotechnol. Bioengineer. 62:368-372.

El-Mabsout, Y. E. and Stevenson, K, E. 1979. Activation of Bacillus stearothermophilus spores at low pH. J. Food Sci. 44:705-709.

Gibbs, P. A. 1964. Factors affecting the germination of spores of Clostridium bifermentans. J. Gen. Microbiol. 37:41-48.

Gibbs, P. A. 1967. The activation of spores of Clostridium bifermentans. J. Gen. Microbiol. 46:285-291.

Gross, E. and Morell, J. L. 1971. The presence of dehydroalanine in the antibiotic nisin and its relationship to activity. J. Amer. Chem. Soc. 93:4634-4635.

Hansen, J. N. 1994. Nisin as a model food preservative. Grit. Rev. Food Sci. Nutr. 34:69-93.

Hashimoto, T. and Conti, S. F. 1971. Ultrastructurai changes associated with activation and germination of Bacillus cereus T spores. J. Bacteriol. 105:361-368.

Hatheway, C. L. 1992. Chapter 1. Clostridium botulinum and other clostridia that produce botulinum neurotoxin. In Clostridium botulinum: ecology and control in foods. A. H. W. Hauschild and K. L. Dodds (Ed.). Marcel Dekker, Inc., New York.

Keynan, A., Evenchik, Z., Halvorson, H. 0., and Hastings, J. W. 1964. Activation of bacterial endospores. J. Bacteriol. 88:313-318.

Keynan, A. and Evenchik, Z. 1969. Activation. In The Bacterial Spore. G. W. Gould and A. Hurst (Ed.). Academic Press, Inc. London, pp. 359-395.

Laurent, Y., Arino, S., and Rosso, L. 1999. A quantitative approach for studying the effect of heat treatment conditions on resistance and recovery of Bacillus cereus spores. Int. J. Food Microbiol. 48:149-157.

LeBlanc, F. R., Delvin, K. A., and Stumbo, C. R. 1953. Antibiotics in food preservation. I. The influence of subtilin on the thermal resistance of spores of Clostridium botulinum and the Putrefactive Anaerobe 3679. Food Technol. 7:181-184.

Lefebvre, G. M. and Leblanc, R. 1988. Chapter 8 : The kinetics of change in bacterial spore germination. In Physiological Models in Microbiology. Vol. H. M. J. Bazin and J. I. Prosser (Ed.). CRC Press, Inc., Boca Raton, FL.

Lewis, J. C., Michener, H. D., Stumbo, C. R., and Titus, D. S. 1954. Additives accelerating death of spores by moist heat Agric. Food Chem. 2:298-302. 91 Liu, W. and Hansen, J. N. 1993. The antimicrobial effect of a structural variant of subtilin against outgrowing Bacillus cereus T spores and vegetative cells occurs by different mechanisms. Appl. Environ. Microbiol. 59:648-651.

Mazzotta, A. S. and Montville, T. J. 1999. Characterization of fatty acid composition, spore germination, and thermal resistance in a nisin-resistant mutant of Clostridium botulinum 169B and in the wild-type strain. Appl. Environ. Microbiol. 65:659-664.

Michener, H. D. 1953. Effect of subtilin on ungerminated bacterial spores. Proc. Bacteriol. p. 29.

Michener, H. D. 1955. The action of subtilin on heated bacterial spores. J. Bacteriol. 70:192-200.

Murrell, W. G. 1964. Heat resistance of microorganisms. Australasian J. Pharmacy 45:840

Okereke, A. and Montville, T. J. 1991. Bacteriocin-mediated inhibition of Clostridium botulinum spores by lactic acid bacteria at refrigeration and abuse temperatures. Appl. Environ. Microbiol. 57:3423-3428.

Roberts, T. A. 1970. Recovering spores damaged by heat, ionizing radiations or ethylene oxide. J. Appl. Microbiol. 33:74-94.

Rowley, D. B. and Feeherry, P. 1970. Conditions affecting germination of Clostridium botulinum 62A spores in a chemically defined medium. J. Bacteriol. 104:1151-1157.

Scott, V. N. and Taylor, S. L. 1981. Effect of nisin on the outgrowth of Clostridium botulinum spores. J. Food Sci. 46:117-120.

Shacter, S. M. and Hashimoto, T. 1975. Bimodal kinetics of germination of Bacillus cereus T spores. In Spores VI. P. Gerhardt, R. N., Costilow, and H. L. Sadoff (Ed.). American. Society for Microbiolo^. Washington. D. C.

Srivastava, O. P. and Fitz-James, P. C. 1981. Alteration by heat activation of enzymes localized in spore coats of Bacillus cereus. Can J. Microbiol. 27:408-416.

Steinbuch, E. 1976. The acid sensitization of heat resistant bacterial spores. In Spore Research. Vol. H. A. N. Barker, J. Wolf, D.J. Ellar, G.J. Dring, and G.W. Gould (Ed.)J*p.451-468. Academic Press.

Tipper, D. J. and Gauthier, J. J. 1972. Structure of the bacterial endospore. In Spores V. H. O. Halvorson, R. Hansen, and L. L. Campnell (Ed.). Pp. 3-12. American Society for Microbiology, Washington D C.

92 Treadwell, P. E., Jann, G. J., and Salle, A. J. 1958. Studies on factors affecting the rapid germinations of spores of Clostridium botulinum. Bacteriol. J. 76:549-556.

Uehara, M. and Frank, H. A. 1967. Sequence of events during germination of putrefactive anaerobe 3679 spores. J. Bacteriol. 94:506-511.

Wandling, L. R., Sheldon, B. W., and Foegeding, P. M. 1999. Nisin in milk sensitizes Bacillus spores to heat and prevents recovery of survivors. J. Food Prot. 62:492-498.

Yousten, A. A. 1975. Germination of Bacillus cereus endospores: a proposed role for heat shock and nucleosides. Can J. Microbiol. 21:1192-1197

93 100

1 0 -

o z z c o ■■s m I 3 0.01 - co

0.001 - A\

0.0001 0 20 40 60 80 100 Heating time (Minutes)

Figure 3.tri spores suspended in distilled water. (#) 80°C; (| I ) 85°C; (A) 92“C; (Q) 94.5°C; (□ ) 97.5”C; (A) 100°C.

94 !■—------— - -r (a) 8.5 &ig ------XK ------1

6.5 . 9 (b) i & CO O 7.5 o

6.5 9 (c) 8.5

7.5

6.5

Incubation hours at 37°C

Figure 3.2: Monitoring germination of C. botulinum spores. Spore suspension was heat activated at 80°C for 15 min, 85°C for 15 min, or 85°C for 90 min, incubated at 37°C in the presence of germinant. Samples were taken at intervals, reheated at 80“C for 30 min and culturable spores were counted on TPGY agar. Non-reheated spores were also plated as a control treatment Heat activation treatment: (a) at 80®C for 15 min; (b) at 85“C for 15 min; (c) at 85"C for 90 min. (O) no heating (control); ( g ) reheating at 80°C for 30 min during sampling.

95 8.5 -

_ 8 I I qL 7.5 CO O O 7 -

6.5

No heat 80C 15 min 85C15mln 85C90min Pre-heating treatments

Figure 3.3: Activation of C. botulinum spores by intermittent heating. Spore suspension was pre-heated at 80-85“C for 15-90 min and then reheated at 80°C for 30 min. (□)no pre-heating; (0 ) pre-heated only; (■ ) pre-heated and reheated. Bars denoted by different letters indicate significant (a = 0.05) differences as measured by ANOVA.

96 120 J (a) at80°C Î (b)at85“C (c)at90“C 100 i <1 ♦ I _ ♦ ♦ 80.

1 60- ■q. S •s 40. ♦ i t

1 20. ♦ 1 5 min ♦ 1 5 min ♦ 1 5 min ■45 min ■ 4 5 min I ■ 3 0 min • 9 0 min • 9 0 min • 6 0 min Z 0 . 1------1-----—I----r ------T" ' I 50 100 150 200 250 0 50 100 150 200 0 50 100 150 200 Time (Minutes)

Figure 3.4: Monitoring germination of C. botulinum spores by phase change observation. Spore suspension was heat activated at 80, 85, or 90“C for 15-90 min, and incubated at 37“C in the germination medium. Samples were examined under phase-contrast microscope at intervals. 0} 6 0.8.5 -

control nisin

Figure S.5: Culturability ot' C botulinum spores after heating at twodiff«ent pH values and in the presence or absence of nisin (250 pg/mi). ( □ ) no heat; (B ) heated at 85"C for 30 min; O ) heated at 85“C for 90 min; (■ ) heated at 85®C for 90 min + reheated at 80“C 30 min.

98 20 (a) ^ 15

10 - 5 - ri^rl pH 4.9 pH 3.4 pH 2.8

2 25 (b) pH 3.4 I 20 I 15

I 10 - CD 5 - 0 ÉÉ control nisin

50 (0) pH 2.8 40 30 H 20 10 -j 0 -I control nisin

Figure 3.6: Phase-change observation of C. botulinum spores before and after heat activation treatment (at 85“C for 90 min). Different activation conditions were compared under different pH and in the presence or absence of nisin (250 (ig/ml). Before activation (□); After activation (■).

99 CHAPTER 4

ACTION OF PURE AND COMMERCIAL NISIN PREPARATIONS ON

CLOSTRIDIUM BOTULINUM li\JÏC£SG SPORE-TO-CELL

TRANSFORMATION

ABSTRACT

It has been proposed that the mode of action of nisin against vegetative cells and spores of Clostridium botulinum is different. However, clear explanation is not available. Therefore, nisin action against vegetative cells and spores of C. botulinum was investigated in this study. Nisin was added at various stages of spore-to-vegetative cell transition and changes to sensitivity to the bacteriocin were observed. EMfferent nisin preparation (Nisaplin or pure nisin) was compared for their activity against different stages of spore transformation of C botulinum ATCC 25763. Germination was measured by determining loss of heat resistance and observing phase darkening of spores under phase-contrast microscope.

lOO Nisin. acted bactericidally against vegetative cells^ but acted sporostatically against spores of C. botulinum under the same concentration. This bactericidal and sporostatic action of nisin was dependent on the concentration of nisin used. Presence of nisin during spore activation by heat increased subsequent phase darkening and germination rates. However, nisin inhibited the germination and the outgrowth, when it was added after heat activation stage. Findings from this study suggest that the time of addition of nisin is very important for the effective control of spores during the heating process of foods. In addition, it may be possible to apply nisin at the stage of processing that coincide with the most sensitive stage of spore transformation.

INTRODUCTION

It is not always practical to eliminate C. botulinum spores during food processing, since harsh treatments may reduce the organoleptic and nutritional quality of foods. Furthermore, the presence of spores in the food does not lead to the toxin production unless a proper environment for germination and growth is provided (Kim and Foegeding, 1993). Therefore, the important consideration for the safety of foods is whether C. botulinum spores can germinate, grow and produce toxin. Suitable targets for control of C. botulinum toxin production in a food product might be found in the processes involved in the spore-to-cell transition, since this must take place for toxin production to ultimately occur. This transition, from activation and germination through outgrowth to cellular multiplication, would be an ideal target against which to design

101 specific inhibitors that m i^t serve as alternatives to conventional antibotulinal agents,

such as nitrite, that are commonly used in foods (Widdus and Busta, 1982).

Nisin is an antimicrobial peptide, which has been used to preserve foods, such as

pasteurized cheese spreads, nonstandardized salad dressings, and low cholesterol liquid

eggs in the U. S. (Dept. Health and Human Services 1994, 1995), since it was

recognized as a safe and legal biological food additive in 1969 (FAO/WHO 1969).

Some studies indicated that nisin acts either sporicidally or as an inhibitor of

germination or outgrowth (Campbell and Sniff, 1959; Hirsch and Grinsted, 1959;

Mikolajcik et al., 1965; Attwell et al., 1972; Scott and Taylor, 1981; Somers and Taylor,

1981; Morris et al., 1984; Liu and Hansen, 1990). These studies show that mode of

action of nisin varies with the test organism and the numbers present. Recently,

Mazzotta and Montville (1999) reported that nisin reduced the thermal resistance and

increased the germination rate of C. botulinum spores. Therefore, effect of nisin on germination of C. botulinum spores is currently controversial. In addition, the stage of transformation that is targeted by nisin and its mechanism of action are not known.

Therefore, this study was performed to compare the effect of nisin against C. botulinum at different stages in spore-to-cell transition and to determine the most susceptible stage of the life cycle of C. botulinum to this additive. Detecting and monitoring germination by conventional techniques (e.g., loss of heat resistance measurements) are tedious and time-consuming tasks. This direct approach, however, produces unambiguous results and thus was used in this study.

102 MATERIALS AND METHODS

Bacterial strains and preparation of spore suspensions

Proteolytic C. botulinum Type A ATCC 25763 was obtained from the American

Type Culture Collection (Bethesda, MD). Culture was grown in Tryptone-Peptone-

Glucose-Yeast extract (TPGY) broth and kept frozen at -80°C with 20% (v/v) sterile

80%-glycerol (Sigma Chemical Co., St. Louis, MO) added to the broth. TPGY broth consists of 50g tryptone (Difco), 5g peptone (Difco), 4g dextrose (Difco), 20g yeast extract (Difco), and I g L-cysteine hydrochloride (Sigma) per liter preparation (Scott and Taylor, 1981). Spores of C. botulinum were formed by growing the bacterium on

TPGY agar plate. An overnight culture was spread onto TPGY agar and incubated anaerobically at 37®C until more than 90% of population were sporulated, which took 7 to 10 days. Spores were harvested by adding 10 ml of sterile cold distilled water, releasing the colonies containing spores from the surface of the agar with the use of a sterile disposable inoculating loop. Suspensions were centrifuged at 6000 rpm for 10 min at 4°C and the resulting pellet was washed in sterile cold distilled water, and centrifuged again at 12,000 rpm for 10 min at 4°C. The washing procedure was repeated six times. Spore suspensions were then enzymatically cleaned by the methods of Billon et al. (1997). Lysozyme solution (200 ug/ml of lysozyme in 0.05 mol/L potassium phosphate bufrer, pH 8.1) was added to the spore pellets and incubated at 45®C for 30 min. Then, trypsin solution (100 ug/ml of trypsin in 0.05 mol/L potassium phosphate buffer, pH 8.1) were added and incubated at 45®C for 2 more hours. Spore crops, then, were washed ten times in sterile distilled water by centrifugation at 1 2,000 rpm for 10

103 min at 4°C. The final pellets, werejssuspendediit stenle. cold distilled water and kept at

4“C. Microscopic observations were made to confirm the purity of spore suspensions.

No vegetative cells were observed and spores were all refiractile under phase-contrast

microscope.

Preparation of nisin

Nisaplin (10® lU of nisin/g), a commercial nisin concentrate composed of 2.5%

nisin in denatured milk solids and sodium chloride, or pure nisin was obtained from

Aplin and Barrette Ltd. (Trowbridge, England). Stock solutions of nisin containing 10®

lU/ml in 0.02N HCl solution was prepared as recommended by Scott and Taylor

(1981). The pH was adjusted to 2-3 with HCl solution and the mixture was autoclaved

(10 min at 121°C) and stored at 4°C. Activity of nisin levels in these autoclaved stock

solutions (prepared as 10® lU/ml) were measured in Arbitrary Units (AU)/ml with the

spot-on-the-Iawn assay employing Lactobacillus leichimannii ATCC 4797 as indicator

microorganism. The conversion between the two units (AU and lU) of bacteriocin

activity was calculated as follows: 10® lU/ml of commercial nisin produced the activity

of 3.2 X 10® AU/ml against L. leichimannii ATCC 4797. Therefore, 1 lU is

approximately equivalent to 32 AU against this indicator microorganism.

Comparison of sensitivity of vegetative and spore formsbotulinum of C. to nisin

Sensitivity of vegetative and spore forms of C. botulinum to nisin in TPGY broth medium was examined. Overnight (18 h) culture of vegetative cells was diluted to appropriate concentrations in 0.1% peptone water. Spore suspensions were also diluted

104 to suitable concentrations in 0^1% peptone water. The diluted suspensions were heatr shocked at 80°C for 10 min. The heat-shocked suspensions were inoculated into TPGY broth containing nisin. The final vegetative cell and spore levels in the inoculated broth were approximately 10^ to 10^ CFU/ml TPGY broth. Duplicate TPGY tubes were inoculated for each set of conditions. Tubes without nisin served as controls. The nisin levels used were 10, 100, and 1000 lU/ml (0.25,2.5, and 25-pg/ml, respectively). Plate counts were obtained after incubated at 37®C for predetermined periods.

In all of the experiments, C. botulinum manipulation and incubation were done in an anaerobic chamber containing an atmosphere of 5% Hz, 10% COz, and 85% Nz

(Forma Scientific Inc., Marietta, OH). All media and reagents were prepared at least 24 h before use and kept at 22-25®C in the anaerobic chamber (Mazzotta and Montville,

1999).

Effect of nisin on spore-to-vegetative cell transition

Effect of nisin on activation stage.Optimal heat activation (85°C for 90 min) condition for this C. botulinum strain was chosen from previous experiments (Chapter

3). Nisaplin or pure nisin was added to the spore suspension to achieve a concentration of 250 pg/ml. The addition of nisin preparation to the spore suspension lowered the pH of the activation medium (i.e., mixture of nisin stock solution and spore in distilled water). The pH values of activation medium were 2.8 and 3.4, for Nisaplin and pure nisin-containing solution, respectively. Peptone water adjusted pH to 2.8 and 3.4 was used as control, to differentiate the effect of pH and nisin. The pH value of the spore suspension without nisin preparation was 4.9. Phase change of spores was examined

105 before and after heat activation, treatment in. the presence of nisin. hr addition^ spore counts were obtained at 0,30, and 90 min during heat activation treatment.

Effect of nisin on germination stage.Germination was monitored in the presence of nisin when it was added before or after heat activation treatment (i.e., pre­ activation or post-activation treatment, respectively). For pre-activation treatment, 250 pg/ml nisin (Nisaplin or pure nisin) was added to the spore suspension [nisin solution : spore suspension =1:9 (vol/vol)] and the mixture was heated at 85°C for 90 min. After heating, the heated mixture was transferred to the germination medium [heated mixture

: germination medium =1:9 (vol/vol)] and incubated at 37°C. TPGY broth containing

200 mM L-alanine was selected as a germination medium based on the previous experiments (Chapter 3). For post-activation treatment, spore suspension was heated at

85°C for 90 min, and heated spores were transferred to the germination medium containing 250 pg/ml nisin (Nisaplin or pure nisin). The volume ratio of transferred heated spore suspension in germination medium was 1:9 (vol/vol).

Monitoring spore germination.Spore germination was measured by determining loss of heat resistance (i.e., decrease in plate count upon heating) and observing the increase in phase darkening of spores. The general scheme of monitoring germination is shown in Figure 4.1. At predetermined intervals during germination incubation, samples were withdrawn and examined under phase contrast microscope

(Bausch and Lomb, Rochester, NY). Between 100 and 200 spores were counted for each sample, and the numbers of phase-bright (ungerminated) spores and phase-dark

(germinated) spores were recorded. Samples of the incubated mixture were also enumerated using TPGY agar medium without heat treatment or plated on TPGY agar

106 after heat treatment (80°C for 30 min).

Effect of nisin on the outgrowth stage.Spore suspension was heat-activated at

85°C for 90 min, transferred to the germination medium (TPGY broth containing 200 mM L-alanine) and incubated anaerobiaclly at 37“C. After 3-h germination incubation, nisin (250 pg/ml) was added to the germination medium. At appropriate intervals, samples were withdrawn and phase-bright/phase-dark spores were counted under a phase-contrast microscope (Bausch and Lomb, Rochester, NY).

RESULTS AND DISCUSSION

Comparison of sensitivity of vegetative cells and sporesbotulinum of C. to nisin

Sensitivities of spores and vegetative cells of C. botulinum were compared in

TPGY broth using three different concentrations of commercial nisin (Figure 4.2).

Population of C. botulinum vegetative cells decreased > 2 logs in mixtures containing

25 pg/ml nisin. The bacterium remained undetectable for 36 h incubation. Spore outgrowth was prevented for about 15 h by the same concentration of nisin, and then they grew and reached to 10* CFU/ml at 28-h period (Figure 4.2a). When 2.5 pg/ml nisin was used, population of vegetative cells decreased > 2.5 logs and remained undetectable (< 10 CFU/ml) for 15 h. A similar concentration of nisin prevented spore outgrowth for 9 h, and then the count increased to 10^ CFU/ml after 30 h incubation

(Figure 4.2b). When nisin concentration was lowered to 0.25 pg/ml, vegetative cells

107 wei& inhibited foc 2 L and then^staited ta paw ancLreached ta CFU/ml. However^ spores were not affected with the same concentration of nisin (Figure 4.2c).

This direct comparison between C. botulinum vegetative cells and spores in sensitivity to nisin illustrates the mode of action of nisin against this pathogen. Nisin acted bactericidally against vegetative cells, but acted sporostatically against spores of

C. botulinum under the same concentration (Figure 4.2). Sensitivity of vegetative cells and spores to nisin varies between genera and even between strains of the same species

(Delves-Broughton et al., 1996), but direct comparisons between vegetative and spore forms were not tested earlier. The bactericidal and sporostatic action of nisin was dependent on the concentration of nisin used. The lag in germination of C. botulinum spores was related proportionally with the concentration of nisin used (Figure 4.3). It has been known that nisin works in a concentration-dependent fashion both in terms of amount of nisin applied and the number of vegetative cells or spores that needs to be inhibited (Delves-Broughton et al., 1996). In addition, the lag period of vegetative cells was much longer than the lag time of powth from spore inocula with the same concentration of nisin. Therefore, vegetative cells were more sensitive to nisin than were the spores of C. botulinum.

Effect of nisin on different stages of spore-to-vegetative ceil transition

This experiment was done to determine the stages of spore-to-cell transition that are affected most by nisin Therefore, nisin was added at different stages of spore transformation and its action on the transforming spores was examined.

Nisin was added to the dormant spore suspension (pre-activation treatment),

108 spores were heat activated at 85“C for 90 and then subsequent germination was

monitored by observing phase contrast change (Figure 4.4) and by determining loss of

heat resistance at 80“C for 30 min (Figure 4.5). Presence of nisin during heat activation

of spores enhanced phase-change after activation treatment and subsequent germination

incubation as measured microscopically (Figure 4.4). The effect of nisin was greater when the pH of the heat-activated spore suspension was lower (pH 2.8 vs. pH 3.4).

However, pH effect during germination was not of concern, because the germination buffer containing activated spores was neutral (pH 6.8 ). In Figure 4.4, regarding the germination incubation period only, the number of phase-dark spores increased by 2 0 -

30% after the initial phase change during heat activation, regardless of presence of nisin in the germination medium. Therefore, germination was dependent on the activation condition (Figure 4.4). Testing for the loss of heat resistance showed that presence of nisin shortened the heating time required for spore activation (Figure 4.5). Maximum culturability was observed when spores were heated, in the presence of nisin, for 30 min at 85°C. However, after extended heating during activation (90 min), the spore count decreased. This observation may be resulted ftom sensitization of nisin-treated spores to heat. Therefore, sublethal heating intended to only activate spores became lethal when applied for an extended period. Alternatively, extended heating with nisin only activated spores rapidly, it also led to spore germination during the extended heating step. Considering the phase-change observation, as a parallel experiment, the latter explanation is supported. In other words, nisin enhanced activation and the subsequent germination, resulted in decreased spore count as the germination proceeded. Studies indicated that germination, due to the changes in the cortex structure, could be induced

109 by drastic means such as surface active agents (Rode and Foster» I960)» or heating

(Himnell and Ordall, 1961), in addition to the use of compounds that promote physiological germination such as L-alanine.

To investigate the effect of nisin on the germination stage per se, nisin was added into the germination medium after heat activation treatment. Nisin delayed phase darkening of spores and Nisaplin ftirther prevented germination than pure nisin did

(Figure 4.6). Therefore, opposite effect of nisin on the phase-change of spores was observed depending on the time of nisin addition. When added before heat activation, nisin enhanced phase darkening (germination) (Figure 4.4). On the other hand, nisin delayed phase darkening when it was added after heat activation (Figure 4.6). Phase darkening of spores is thought to correspond with an increase in spore hydration after peptidoglycan layers in cortex have been ruptured (Ross and Billing, 1957; Gould and

Hitchins, 1963).

No decrease in spore counts was observed in 2-h germination period in the presence of Nisaplin (Figure 4.7a). However, spore count during germination incubation in the presence of pure nisin decreased slightly faster than that in the control sample (Figure 4.7b). This may indicate that NaCl in Nisaplin prevents germination, because commercial nisin contains 77.5% NaCl as a carrier material (Aplin & Barrette

Ltd.). The amount of nisin solution in the germination medium was 10% (vol/vol), so the amount of NaCl in germination medium was 7.75%. Therefore, when nisin was added after heat activation stage, the discrepancy between the plate counting results from the experiments using different nisin preparation (Nisaplin and pure nisin), was probably attributed to the presence of NaCl. Warth and Strominger (1972) proposed the

110 contractile cortex, theory explaining the inhibition. o£ germination in. the presence of ionic solute, such as NaCl. Spore peptidoglycan is lightly cross-linked and carries a net negative charge resulting from free carboxyl groups. If these were neutralized by the cations, cortex might be expected to contract and the permeability of spore might be changed as well (Lewis et al., 1960). These events could interfere with the germination process.

In Figure 4.7b, pure nisin decreased the heat resistance slightly faster than the control, when it was added after heat activation. Apparently, this was not consistent with the result obtained from phase contrast observation, which indicated slower phase darkening in the presence of nisin than its absence (Figure 4.6b). However, the difference between the absence and presence of nisin may not be considerable. On the other hand, it may possibly suggest that nisin inhibited the step between the stage of loss of heat resistance and the stage of phase darkening during the transition of spores.

Hitchins et al. (1963) indicated that nisin prevented post-germination swelling and subsequent spore outgrowth. Several studies supported that measurable events during spore germination do not occur simultaneously, but rather constitute a time-ordered sequence (Mayall and Robinow, 1957; Levinson and Hyatt, 1966; Uehara and Frank,

1967; Dring and Gould, 1971; Hsieh and Vary, 1975). The observable changes during germination of B. megaterium QM B1551 spores occurred in the following sequence: loss of resistance to heat, release of dipicolinic acid (DPA), onset of stainability, darkening of individual spores under phase contrast microscope and decrease in turbidity of spore suspension (Levinson and Hyatt, 1966). Uehara and Frank (1967) also indicated that specific changes occurred in two distinct regions of spores, i.e., one

111 located a tthe^outer layer (spore-coats).and the second atthe^core. membrane. During, the:, initial stage of germination, DPA is released, and the cortex disintegrates rapidly until it is completely destroyed (Mayall and Robinow, 1957). In addition, researchers agreed that heat resistance is lost early during germination, even before all DPA has been released (Levinson and Hyatt, 1966; Uehara and Frank, 1967; Dring and Gould, 1971).

To examine the effect of nisin on the outgrowth stage of C. botulinum spores, nisin was added into the germination medium after 3-h germination incubation period

(usually, when 50-75% of spores have germinated). Outgrowth occurred in control sample, and some vegetative cells were observed in 4-h germination period. However, nisin prevented the outgrowth of germinated spores, and no vegetative cells were observed for 20 days from phase-contrast microscopic observation (Figure 4.8). This clearly indicates that nisin prevents the outgrowth of germinated spores of C. botulinum.

Nisin effectively inhibits B. cereus and C. botulinum spore outgrowth by inactivating the membrane sulfhydryl groups in newly germinated spores (Scott and Taylor, 1981;

Somers and Taylor, 1981; Morris et al., 1984; Liu and Hansen, 1990). It has been reported that the activity of nisin is directly related to the presence of dehydroalanine residues, which can act as acceptors for electrophilic reagents and readily react with sulfhydryl groups (Gross and Morrell, 1971; Liu and Hansen, 1993).

CONCLUSION

The mechanism of action of nisin against spores is not clear and research in this area has been inconclusive. Early studies suggested that nisin acts either sporicidally or

112 as an inWbitor of genninatipn or joutgowA (Cam Sniffy 1959 l Hirsck and

Grinsted, 1959; Mikolajcik et ai., 1965; Attwell et al., 1972). Even though the main purpose of this study was to investigate the action of nisin during spore transformation, the other factors, such as pH or NaCl, were also considered due to the characteristics of commercial nisin preparation. Therefore, our results show the compounding effects of three factors (e.g. pH, NaCl, and nisin). Two major effects of nisin preparations were observed during the spore transition. First, nisin activated spores during heat treatment and the effect of nisin on the activation was greater under low pH conditions. Secondly, nisin prevented the outgrowth of germinated spores. On the other hand, the effect of nisin on the germination stage of spores, was slightly different depending on the time of addition. When nisin was added before heat activation, germination proceeded slightly faster in the presence of nisin. Addition of nisin after heat activation decreased phase- darkening. Therefore, under combined treatment of heat and nisin, nisin activates spores, triggers initiation of germination, and enhances the subsequent germination.

Nisin, on the other hand, delays germination in the absence of heat treatment. In addition, nisin probably prevents the late event of germination stage (after loss of heat resistance and before phase darkening, i.e., pre-emergent swelling) in the absence of simultaneous heat treatment

It is suggested that the adding time of nisin is very important for the effective control of spores during the heating process of foods. The action of nisin, combined effect of other factors such as pH and NaCl in food systems, should also be considered during the process. In addition, it may be possible to ^ p ly nisin at the stage of processing that coincide with the most sensitive stage of spore transformation.

113 REFERENCES

Attwell, R. W., Cross, T., and Gould, G. W. 1972. Germination of Thermoactimyces vulgaris endospores: microscopic and optical density studies showing the influences of germinants, heat treatment, stain differences and antibiotics. J. Gen. Microbiol. 73:471- 481.

Billon, C. M. P., McKirgan, C. J., McClure, P. J., and Adair, C. 1997. The effect of temperature on the germination of single spores of Clostridium botulinum 62A. J. Appl. Microbiol. 82:48-56.

Campbell, L. L. and Sniff, E. E. 1959. Effect of subtilin and nisin on the spores of Bacillus coagulons. J. Bacteriol. 77:766-770.

Delves-Broughton, J., Blackburn, P., Evans, R. J., and Hugenholtz, J. 1996. Applications of the bacteriocin, nisin. Antonie van Leeuwenhoek 69:193-202.

Department of Health and Human Services. 1994. Aplin & Barrett Ltd.; filling of petition for affirmation of GRAS status. Federal Register. 59FR:12582-12583.

Department of Health and Human Services. 1995. Aplin & Barrett Ltd.; filling of petition for affirmation of GRAS status. Federal Register. 60:64167.

Dring, G. J. and Gould, G. W. 1971. Sequence of events during rapid germination of spores of Bacillus cereus. J. Gen. Microbiol. 65:101-104.

F AO/WHO Expert Committee on Food Additives. 1969. Specification for identity and purity of some antibiotics. 12*** Report. WHO Technical Report Series, No. 430.

Gould, G. W. and Hitchins, A. D. 1963. Sensitization of bacterial spores to lysozyme and to hydrogen peroxide with agents which rupture disulfide bonds. J. Gen. Microbiol. 33:413-423.

Gross, E. and Morell, J. L. 1971. The presence of dehydroalanine in the antibiotic nisin and its relationship to activity. J. Amer. Chem. Soc. 93:4634-4635.

Hirsch, A. and Grinsted, E. 1959. Methods for the growth and enumeration of anaerobic spore-formers from cheese, with observations on the effect of nisin. J. Dairy Research 21 :101- 110.

Hitchins, A. D., Gould, G. W., and Hurst, A. 1963. The swelling of bacterial spores during germination and outgrowth. J. Gen Microbiol. 30:445-453.

114 Hsieh^L>iL and, Yaiy^ L97S^EeptidogIycaa hydrolysis, during initiation, of spore germination in Bacillus megaterium. In Spores VI. P. Gerhardt, R. N., Costilow, and H. L. Sadoff (Ed.). American Society for Microbiology. Washington D. C.

Hunnell, J. W. and Ordall, Z. J. 1961. Cytological and chemical changes in heat killed and germinated bacterial spores. In Spores II. H. O. Halvorson (Ed.). Pp. 101-112 Burgess Publishing Co. Minneapolis.

Keynan, A., Evenchik, Z., Halvorson, H. O., and Hastings, J. W. 1964. Activation of bacterial endospores. J. Bacteriol. 88:313-318.

Kim, J. and Foegeding, P. M. 1993. Principles of control. In Clostridium botulinum: ecology and control. H. W. Hauschild and K. L. Dodds (Ed.). Marcel Dekker, Inc., New York, N. Y.

Levinson, H. S. and Hyatt, M. T. 1966. Sequence of events during Bacillus megaterium spore germination. J. Bacteriol. 91:1811-1818.

Lewis, J. C., Snell, N. S., and Burr, H. K. 1960. Water permeability of bacterial spores and the concept of a contractile cortex. Science 132:544.

Liu, W. and Hansen, J. N. 1990. Some chemical and physical properties of nisin, a small protein antibiotic produced by Lactococcus lactis. Appl. Environ. Microbiol. 56:2551-2558.

Liu, W. and Hansen, J. N. 1993. The antimicrobial effect of a structural variant of subtilin against outgrowing Bacillus cereus T spores and vegetative cells occurs by different mechanisms. Appl. Environ. Microbiol. 59:648-651.

Mayall, B. H. and Robinow, C. 1957. Observations with electron microscope on the organization of the cortex of resting and germinating spores of Bacillus megaterium. J. Appl. Bacteriol. 20:333

Mazzotta, A. S. and Montville, T. J. 1999. Characterization of fatty acid composition, spore germination, and thermal resistance in a nisin-resistant mutant of Clostridium botulinum 169B and in the wild-type strain. Appl. Environ. Microbiol. 65:659-664.

Mikolajcik, E. M., Reeves, C. B., and Harper, W. J. 1965. Efficacy of nisin as a sporicidal agent in the presence of L-alanine. J. Dairy Sci. 48:1522-1524.

Morris, S. L., Walsh, R. C., and Hansen, J. N. 1984. Identification and characterization of some bacterial membrane sulfhydryl groups which are targets of bacteriostatic and antibiotic action. J. Biol. Chem. 259:13590-13594.

115 Rode^L. and Foster^ J. W. 1960.j amines. Nature 188:1132-1134.

Ross, K. F. A. and Billing, E. 1957. Water and solid content of living bacterial spores and vegetative ceils as indicated by refractive index measurements. J. Gen. Microbiol. 16:418

Scott, V. N. and Taylor, S. L. 1981. Effect of nisin on the outgrowth of Clostridium botulinum spores. J. Food Sci. 46:117-120.

Somers, E. R. and Taylor, S. L. 1981. Further studies on the antibotulinal effectiveness of nisin in acidic media. J Food Sci. 46:1972-1973.

Uehara, M. and Frank, H. A. 1967. Sequence of events during germination of putrefactive anaerobe 3679 spores. J. Bacteriol. 94:506-511.

Warth, A. D. and Strominger, J. L. 1972. Structure of the peptidoglycan from spores of Bacillus subtilis. Biochem. 11:1389-1396.

Widdus, R. and Busta, F. F. 1982. Antibotulinal alternatives to the current use of nitrite in foods. Food Technol. 36(12):105-106.

116 Spore suspension 1 Heat 85°C 90 min Heat-activated spore

Incubation at 37°C 1 in the presence of germinants Partially germinated spore population

Heat 80°C for 30 minto Phase-change measure loss of observation heat resistance

Plating on % Phase-dark TPGY agar spores

Ungerminated spores form colonies on agar

Figure 4.1: Monitoring germination by determining loss of heat resistance and observing phase-darkening of spores

117 I Li. O o

10

8

6

4

2

0 0 5 10 15 20 25 30 35 40 Time (Hours) Figure 4.2: Behavior of vegetative cells and spores of Clostridium botulinum in TPGY broth at 37°C in the presence of nisin. (a) 25 pg/ml; (b) 2.5 pg/ml; (c) 0.25 pg/ml. (■ ) vegetative cell; (□ ) vegetative cell + nisin; (# ) spore; ( O) spore + nisin. # indicates less than 10 CFU/ml (detection level). Data points shown are average values of 2 individual experiments.

118 12 -

10 -

52 3 O X R'' = 0.9868 i h-

0.1 1 10 100 Nisin concentration (ug/ml)

Figure 4.3: Lag time of growth from spore inocula, produced by increasing nisin concentrations in the growth media

119 50

nisin added

control nisin i0 Q. CO ■s 1 (0 . c Q.

40 nisin added

Gemination incubation ictivation

0 1 2 3 45 6 7

Time (Hours)

Figure 4.4: Monitoring germination of C. botulinum spores. Spore suspensions were heat activated at 85°C for 90 min in the presence of nisin (250 pg/ml). (a) activated at pH 2.8 in the presence of Nisaplin; (b) activated at pH 3.4 in the presence of pure nisin. Activated spores were transferred to the germination medium and incubated at 37”C. At intervals, phase-change of spores was observed. Each data points indicate the average values of at least two individual experiments. 120 control nisin

nisi oI added Q. CO Ü o

added *

ActwatiorC Germination incubation

2 3 4 5 Time (Hours)

Figure 4.5: Monitoring germination of C. botulinum spores. Spore suspensions were heat activated at 85°C for 90 min in the presence of nisin (250 pg/ml). (a) activated at pH 2.8 in the presence of Nisaplin; (b) activated at pH 3.4 in the presence of pure nisin. Activated spores were transferred to the germination medium and incubated for germination. At intervals, samples were heated at 80°C for 30 min, and enumerated on TPGY agar. Each data points indicate the average values of at least two individual experiments.

121 70 control 6 0 . nisin 50 . 40 . 30 . i 0 2 0 . 8- ■ nisin ■s added (0 90 . 1 80 . 70 . 60 . 50 . 40 . 30 . nisin 2 0 . added Activation 10 , Germination incubation

0 1 2 3 4 5 6 Time (Hours)

Figure 4.6: Monitoring germination of C. botulmum spores in the presence of nisin (256 pg/ml) by observing phase-contrast change after heat-activation at 85°C for 30 min. (a) at pH 2.8 in the presence of Nisaplin; (b) at pH 3.4 in the presence of pure nisin. Each data points indicate the average values of at least two individual experiments.

122 8.6

8.4

8.2 nisin added

control 7.8 nism 7.6

i 7.4 o Q. co 7.2 o o 8.6 8.4 - ■ nisin added

8.2 -

7.8 7.6 - 7.4 -

7.2 - Activation Germination incubation

01 2 3 4 5 6 7 Time(Hours>

Figure 4.7: Monitoring germination of C. botulinum spores in the presence of nisin (250 pg/ml) by plate count method preceded by heat treatment (80“C, 30 min) after heat- activation at 85“C for 30 min. (a) at pH 2.8; (b) at pH 3.4. Each data points indicate the average values of at least two individual experiments.

123 45 n

40 S

o> 2 I 2 25 - -■-control -O -n is in

0 5 10 15 20 Time (Days)

Figure 4.Î: Effect of nîsm (25(T ug/inl) on outgrowth stage o fC botulinum spores when added after 3-h germination incubation at 37**C.

124 LIST OF REFERENCES

Abee, T., BClaenhammer, T. R., and Letellier, L. 1994a. Kinetic studies of action of lactacin F, a bacteriocin produced by Lactobacillus Johnsonii that forms poration complexes in the cytoplasmic membrane. Appl. Environ. Microbiol. 69:1006-1013.

Abee, T., Rombouts, F. M., Hugenholtz, J., Guihard, G., and Letellier, L. 1994b. Mode of action of nisin Z against Listeria monocytogenes Scott A grown at high and low temperatures. Appl. Environ. Microbiol. 60:1962-1968.

Abee, T., Krockel, L., and Hill, C. 1995. Bacteriocins: mode of action and potentials in food preservation and control of food poisoning. Int. J. Food Microbiol. 28:169-185.

All, D., Lacroix, C., Thuault, D., Bourgeois, C. M., and Simard, R. E. 1995. Characterization of diacetin B, a bacteriocin from Lactococcus lactis subsp. lactis bv. diacetylactis UL720. Can. J. Microbiol. 41:832-841.

Arihara, K., O ^ ara , S., Mukai, T., Itoh, M., and Kondo, Y. 1996. Salivacin 140, a novel bacteriocin from Lactobacillus salivarius subsp. salicinius T140 active against pathogenic bacteria. Lett. Appl. Microbiol. 22:420-424.

Anonymous. 1985. Chopped garlic in soybean oil product linked to botulism cases in Canada. Food Chemical News 27:39-40.

Anonymous. 1994. Foodbome outbreaks in California. Dairy, Food Environ. Sanit. 15:611-615.

Austin, J. W., Dodds, K. L., Blanchfreld_ B., and Farber_LM. 1998. Growth and toxin production by Clostridium botulinum on inoculated fresh-cut packaged vegetables. J. Food Prot. 61:324-328. ^

Barefoot, S. F. and Klaenhammer, T. R. 1984. Purification and characterization of the Lactobacillus acidophilus bacteriocin lactacin B. Antimicrob. Agents Chemother. 26:328-334.

Barker, A. N. and Wolf, J. 1971. Effects of thioglycoilate on the germination and growth of some clostridia. In Spore Research. Vol. I. P^.95-109.

125 Baumgart^J, I9&7. Occunenc&and.grawth.o£CZas/r£e/t:mz. hotuUman. in. vacuum-packed raw and pasteurized potatoes and potato salad. Chemie Mikrobiologie Technologie der Lebensmittel. 11(3):74.

Bell, R. G. and De Lacy, K. M. 1986. Factors influencing the determination of nisin in meat products. J. Food Technol. 21:1-7.

Bhugaloo-Vial, P., Dousset, X., Metivier, A., Sorokine, O., Anglade, P., Boyaval, P., and Marion, D. 1996. Purification and amino acid sequences of piscicocins Via and Vlb. Two class Ha bacteriocins secreted by Camobactervum piscicola VI that display significantly different levels of specific inhibitory activity. Appl. Environ. Microbiol. 62:4410.

Bhunia, A. K., Johnson, M. C., and Ray, B. 1988. Purification, characterization and antimicrobial spectrum of a bacteriocin produced by Pediococcus acidilactici. J. Appl. Bacteriol. 65:261-268.

Billon, C.M.-P., McKirgan, C. J., McClure, P. J., and Adair, C. 1997. The effect of temperature on the germination of single spores of Clostridium botulinum 62A. J. Appl. Microbiol. 82:48-56.

Blocher, J. C. and Busta, F. F. 1985. Inhibition of germinant binding by bacterial spores in acidic environments. Appl. Environ. Microbiol. 50:274-279.

Brett, M. 1999. Botulism in the United Kingdom. Eurosurvellance 4:9-11.

Brink, B., Minekus, M., van der Vossen, J. M. B. M., Leer, R. J., and Huis in’t Veld, J. H. J. 1994. Antimicrobial activity of lactobacilli: preliminary characterization and optimization of production of acidocin B, a novel bacteriocin produced by Lactobacillus acidophilus M46. J. Appl. Bacteriol. 77:140-148.

Bruno, M. E. C., Kaiser, A., and Montville, T. J. 1992. Depletion of proton motive force by nisin in Listeria monocytogenes cells. Appl. Environ. Microbiol. 58:2255-2259.

Bruno, M. E. C. and Montville, T. J. 1993. Common mechanistic action of bacteriocins firom lactic acid bacteria. Appl. Environ. Microbiol. 59:3003-3010.

Brusin, S. and Salmaso, S. 1998. Botulism associated with home-preserved mushrooms. Eurosurveillance weekly. 2:980430

Busta, F. F. and Ordal, Z. J. 1963. Heat-activation kinetics of endospores of Bacillus subtilis. J. Bacteriol. 86:345-352.

126 Carlin F. and PecL M. W. 19%. Growth of and toxin production by nonproteolytic Clostridium botulinum in cooked pureed vegetables at refirigeration temperatures. Appl. Environ. Microbiol. 62:3069-3072.

Campbell, L. L. and O’Brian, R. T. 1955. Antobiotics in food preservation. Food Technol. 9:461-465.

Campbell, L. L. and Sniff, E. E. 1959. Effect of subtilin and nisin on the spores of Bacillus coagulans. J. Bacteriol. 77:766-770.

Centers for Disease Control. 1969. Common-source outbreak, type A botulism. Morbid. Mortal. Week. Rep. 18:121-122.

Centers for Disease Control, 1978. Botulism-New Mexico. Morbid. Mortal. Week. Rep. 27:138.

Centers for Disease Control. 1984. Foodbome botulism - Illinois. Morbid. Mortal. Week. Rep. 33:22-23.

Centers for Disease Control. 2000. Foodbome botulism from eating home-pickled eggs- Illinois, 1997. Morbid. Mortal. Week. Rep. 49(34):778-780.

Centers for Disease Control. 2001. Botulism outbreak associated with eating fermented food. Morbid. Mortal. Week. Rep. 50(32)

Cemy, G., Fink, A., and Pecher, A. 1990. Thermal inactivation of bacterial spores in particulate foods. Rassegna dell’Imballagio e Confezionamento. 11(6): 17.

Chaibi, A., Ababouch, L. H., and Busta, F. F. 1996. Inhibition by monoglycerides of L- alanine-triggered Bacillus cereus and Clostridium botulmum spore germination and outgrowth. J. Food Prot. 59:832-837.

Chaibi, A., Ababouch, L. H., Belasri, K., Boucetta, S., and Busta, F. F. 1997. Inhibition of germination and vegetative growth of Bacillus cereus T and Clostridium botulinum 62A spores by essential oils. Food Microbiol. 14:161-174.

Chikindas, M. L., Garcia-Garcera, M. J., Driessen, A. M. J., Ledeboer, A. M., Nissen- Meyer, J., Nes, I. F., Abee, T., Konings, W. N., and Venema, G. 1993. Pediocin PA-1, a bacteriocin from Pediococcus acidilactici PAC 1.0, forms hydrophilic pores in the cytoplasmic membrane of target cells. Appl. Environ. Microbiol. 59:3577-3584.

Christensen, D. P. and Hutkins, R. W. 1992. Collapse of the proton motive force in Listeria monocytogenes caused by a bacteriocin produced by Pediococcus acidilactici. Appl. EnviroiL Microbiol. 58:3312-3315.

127 Cintas^L. Rodriguez, J. M.:^Femand(^ F.^ Sletten^K,^ Nes^ I. P., Hernandez» P. E., and Holo, H. 1995. Isolation and characterization of pediocin L50, a new bacteriocin from Pediococcus acidilactici with a broad inhibitory spectrum. Appl. Environ. Microbiol. 61:2643-2648.

Cintas, L. M., Casaus, P., Havarstein, L. S., Hernandez, P. E., and Nes, I. F. 1997. Biochemical and genetic characterization of enterocin P, a novel ^ec-dependent bacteriocin from Enterococcus faecium PI3 with a broad antimicrobial spectrum. Appl. Environ. Microbiol. 63:4321-4330.

Conner, D. E., Scott, V. N., Bernard, D. T., and Kautter, D. A. 1989. Potential Clostridium botulinum hazards associated with extended shelf life refrigerated foods: A review. J. Food Safety. 10:131-153.

Coventry, M. J., Wan, G., Gordon, J. B., Mawson, R. P., and Hickey, M. W. 1996a. Production of brevicin 286 by Lactobacillus brevis VB286 and partial characterization. J. Appl. Bacteriol. 80:91-98.

Coventry, M. J., Gordon, J. B., Alexander, M., Hickey, M. W., and Wan, J. 1996b. A food-grade process for isolation and partial purification of bacteriocins of lactic acid bacteria that uses diatomite calcium silicate. Appl. Environ. Microbiol. 62:1764-1769.

Crandall, A. D. and Montville, T. J. 1993. Inhibition of Clostridium botulmum growth and toxigenesis in a model gravy system by coinoculation with bacteriocin-producing lactic acid bacteria. J. Food Prot. 56:485-488,492.

Crupper, S. s. and landolo, J. J. 1996. Purification and partial characterization of a novel antibacterial agent (Bad 829) produced by Staphylococcus aureus KSI1829. Appl. Environ. Microbiol. 62:3171.

Curran, H. R. and Evans, F. R. 1945. Heat activation inducing germination in the spores of thermotolerant and thermophilic aerobic bacteria. J. Bacteriol. 49:335-346.

Daeschel, M. A. and Klaenhammer, T. R. 1985. Association of a 13.6-megadalton plasmid in Pediococcus pentosaceus with bacteriocin activity. Appl. Environ. Microbiol. 50:1538-1541.

Daw, M. A. and Falkiner, F. R. 1996. Bacteriocins: nature, function and structure. Micron 27:467-479.

Dean, J. P. and Zottola, E. A. 1996. Use of nisin in ice cream and effect on the survival of Listeria monocytogenes. J. Food Prot 59:476-480.

128 Defour^A. D.^ Thuault^D.^ BouIliQU^A^v Bourgeois^ and Le Pennec^I^-1991, Plasmid-encoded determinants for bacteriocin production and im m unity in a Lactococcm lactis strain and purification of the inhibitory peptide. J. Gen. Microbiol. 137:2423-2429.

Degnan, A. J., Buyong, N., and Luchansky, J. B. 1993. Antilisteriai activity of pediocin AcH in model food systems in the presence of an emulsifier or encapsulated within liposomes. Int. J. Food. Microbiol. 18:127.

Denny, C. B., Sharpe, L. E., and Bohrer, C. W. 1961. Effects of tylosin and nisin on canned food spoilage bacteria. Appl. Microbiol. 9:108-110.

DeVos, W. M., Mulders, J. W. M., Siezen, R. J., Hugenholtz, J., and FCuipers, O. P. 1993. Properties of nisin Z and distribution of its gene, nis Z, in Lactococcus lactis. Appl. Environ. Microbiol. 59:213-218.

De Vos, W. M., Kuipers, O. P., van de Meer, J. R., and Siezen, R. J. 1995. Maturation pathway of nisin and other lantibiotics: post-translationally modified antimicobial peptides exported by Gram-positive bacteria. Mol. Microbiol. 17:427-437.

De Vuyst, L. and Vandamme, E. J. 1994. Antimicrobial potential of lactic acid bacteria. In Bacteriocins of Lactic Acid Bacteria. L. De Vuyst, and E. J. Vandamme (Ed.). Pp.91- 142. Blackie Academic and Professional, Glasgow.

Dignan, D. M. 1985. Evaluation of the botulism hazard fi-om diced raw potatoes preserved by gas exchange. Dissertation Abstracts International B46 (4):1011.

Dodd, H. M., Horn, N., and Gasson, M. J. 1990. Analysis of the genetic determinant for the production of the peptide antibiotic nisin. J. Gen. Microbiol. 136:555-566.

Dodd, H. M. and Gasson, M. J. 1994. Bacteriocins of lactic acid bacteria. In Genetics and Biotechnology of Lactic Acid Bacteria. M. J. Gasson and W. M. De Vos (Ed.). Blackie Academic and Professional.

Dodds, K. L. 1989. Combined effect of water activity and pH on inhibition of toxin production by Clostridium botulinum in cooked, vacuum-packed potatoes. Appl. Environ. Microbiol. 55:656-660.

Dodds, K. L. 1990. Restaurant-associated botulism outbreaks in North America. Food Control. 1:139-141.

Doyle, M. P. 1991. Evaluating the potential risk from extended-shelf-life refiigerated foods by Clostridium botulinum inoculation studies. Food Technol. 45(4): 154-156.

129 Ennahar^S.,. Aoudfc-Wemer^DM. Sorokine^O^ vaiLPorsselaer^ BringeL E^Hubert^ J., and Hasselmann, C. 1996. Production of pediocin AcH by Lactobacillus plantarum WHE 92 isolated from cheese. Appl. Environ. Microbiol. 62:4381-4387.

Ennahar, S., Assobhei, O., and Hasselmann, C. 1998. Inhibition of Listeria monocytogenes in a smear-surface soft cheese by Lactobacillus plantarum WHE 92, a pediocin AcH producer. J. Food Prot. 61:186-191.

Evans, F. R. and Curran, H. R. 1943. The accelerating effect of sublethal heat on spore germination in mesophilic aerobic bacteria. J. Bacteriol. 46:513-523.

Evans, R. 1., Russell, N. J., Gould, 0. W,, and McClure, P. J. 1997. The germinability of spores of a psychrotolerant, non-proteolytic strain of Clostridium botulinum is influenced by their formation and storage temperature. J. Appl. Microbiol. 83:273-280.

FAO/WHO Expert Committee on Food Additives. 1969. Specifications for identity and purity of some antibiotics. 12* Report. WHO Technical Report Series, No. 430.

Federal Register. 1988. Nisin preparation: Affirmation of GRAS status as a direct human ingredient. Federal register. 53:11247-11251.

Foerster, H. F. 1985. The effects of alterations in the suspending medium on low temperature activation of spores of Bacillus stearothermophilus NGBIOI. Arch. Microbiol. 142:185-189.

Food chemical news. 1989. March 6 . P.15. Morbid. Mortal. Week. Rep. 27:138

Foster, S. J. and Johnston, K. 1989. The trigger mechanism of bacterial spore germination. In Regulation of Prokaryotic Development. I. Smith, R. A. Slepecky, and P. Setlow (Ed.). Pp. 89-108. Washington, DC. American Society for Microbiology.

Franz, C. M. A. P., Toit, M. D., Olasupo, N. A., Schillinger, U., and Holzaphfel, W. H. 1998. Plantaricin D, a bacteriocin produced by Lactobacillus plantarum BFE 905 from ready-to-eat salad. Lett. Appl. Microbiol. 26:231-235.

Ganzle, M. G., Weber, S., and Hammes, W. P. 1999. Effect of ecological factors on the inhibitory spectrum and activity of bacteriocins. Int. J. Food Microbiol. 46:207-217.

Giannuzzi, L. and Zaritzky, N. E. 1993. Chemical preservatives action on microbial growth in a model system of refrigerated prepeeled potatoes. J. Food Prot. 56:801-807.

Gibbs, P. A. 1964. Factors affecting the germination of spores of Clostridium bifermentans. J. Gen. Microbiol. 37:41-48.

Gibbs, P. A. 1967. The activation of spores of Clostridium bifermentans. J. Gen. Microbiol. 46:285-291. 130 Gombas, D. E. 1989. Biological competition as a preserving mechanism. J. Food Safety. 10:107-117.

Gonzalez, C. P. and Kunka, B. S. 1987. Plasmid-associated bacteriocin production and sucrose fermentation in Pediococcus acidilactici. Appl. Environ. Microbiol. 53:2534- 2538.

Gould, G. W. and Hitchins, A. D. 1963. Sensitization of bacterial spores to lysozyme and to hydrogen peroxide with agents which rupture disulfide bonds. J. Gen. Microbiol. 33:413-423.

Gould, G. W. 1999. Bacterial endospores. In Encyclopedia of Food Microbiology. R. K. Robinson, C. A. Batt, and P. D. Patel (Ed.). Vol. I. Pp. 168-173. Academic press, London

Grinstead, D. A. and Barefoot, S. F. 1992. Jenseniin G, a heat-stable bacteriocin produced by Propionibacterium jensenii PI26. Appl. Environ. Microbiol. 58:215-220.

Gross, E. and Morrell, J. L. 1971. The presence of dehydroalanine in the antibiotic nisin and its relationship to activity. J. Amer. Chem. Soc. 93:4634-4635.

Hanlin, M. B., Kalchayanand, N., Ray, P., and Ray, B. 1993. Bacteriocins of lactic acid bacteria in combination have greater antibacterial activity. J. Food Prot. 56:252-255.

Hao, Y. Y., Brackett, R. E., Beuchat, L. R., and Doyle, M. P. 1998. Microbiological quality and the inability of proteolytic Clostridium botulinum to produce toxin in film- packaged fresh-cut cabbage and lettuce. J. Food Prot. 61:1148-1153.

Hashimoto, H. and Parko, K. L. 1998. Botulism from peyote. New Engl. J. Med. 339: (3).

Hauschild, A. H. W. 1989. Clostridium botulinum. FoodbomeIn Bacterial Pathogens. M. P. Doyle (Ed.). P. 111. Marcel Dekker, New York.

HPB. Food directorate. Bureau of Microbial Hazards. Botulism Reference Service. Ottawa, Canada, 1997

Health and Welfare Canada. 1986. Botulism in Canada - summary for 1985. Can. Dis. Week. Rep. 12:53-54.

Health and Welfare Canada. 1987. Restaurant-associated botulism from in-house bottled mushrooms - British Columbia. Can. Dis. Week. Rep. 13:35-36.

131 Vods^L^ and Stumbo^C. EL 1965. Use of nisin m processing, food products. Food Technol. 19(4):160-164.

Henderson, J. T., Chopko, A. L., and van Wassenaar, P. D. 1992. Purification and primary structure of pediocin PA-1 produced by Pediococcus acidilactici PAC-1.0. Arch. Biochem. Biophys. 295:5-12.

Hirsch, A. and Grinsted, E. 1959. Methods for the growth and enumeration of anaerobic spore-formers firom cheese, with observations on the effect of nisin. J. Dairy Research 21:101- 110.

Holland, D., Barker, A. N., and Wolf, J. 1969. Factors affecting germination of Clostridia. In Spores IV. L. L. Campbell (Ed.). P. 317. Bethesda, Maryland. American Society for Microbiology

Holzaphel, W. H., Geisen, R., and Schillinger, U. 1995. Biological preservation of foods with reference to protective cultures, bacteriocins and food-grade enzymes. Int. J. Food Microbiol. 24:343-362.

Horn, N., Swindell, S., Dodd, H. M., and Gasson, M. J., 1991. Nisin biosynthesis genes are encoded by a novel conjugative transposon. Mol. Gen. Genet. 228:129-135.

Hotchkiss, J. H., Banco, M. J., Busta, F. F., Genigeorgis, C. A., Kociba, R, Rheaume, L., Smoot, L. A., Schuman, J. D., and Sugiyama, H. 1992. The relationship between botulinal toxin production and spoilage of fiesh tomatoes held at 13 and 23°C under passively modified and controlled atmospheres and air. J. Food Prot. 55:522-527.

Hsieh, H. and Glatz, B. A. 1996. Long-term storage stability of the bacteriocin propionicin PLG-1 produced by Propionibacterium thoenii and potential as a food preservative. J. Food Prot. 59:481-486.

Hutton, M. T., Chehak, P. A., and Hanlin, J. H. 1991. Inhibition of botulinum toxin production by Pediococcus acidilactici in temperature abused refiigerated foods. J. Food Safety. 11:255-267.

HuxsoU, C. C. and Bolin, H. R. 1989. Processing and distribution alternatives for minimally processed fimits and vegetables. Food Technol. 43(2):124-128.

Jack, R. W., Tagg, J. R., and Ray, B. 1995. Bacteriocins of Gram-positive bacteria. Microbiol. Rev. 59:171-200.

Jager, K. and Harlander, S. 1992. Characterization of a bacteriocin firom Pediococcus acidilactici PC and comparison of bacteriocin-producing strains using molecular typing procedures. Appl. Microbiol. Biotechnol. 37:631-637.

132 Jimenez-Diaz^ R.^ RiQS-Ssanchez^R^M^DesmazeaiuUM^RuizrBarba, J. L^an&PiarcL J. C. 1993. Plantaricin S and T, two new bacteriocins produced by Lactobacillus plantarum LPCOIO isolated from a green olive fermentation. Appl. Environ. Microbiol. 59:1416-1424.

Johnson, C. E. 1979. Behavior of Clostridium botulinum in vacuum-packed fresh celery. J. Food Prot. 42:49-50.

Johnson, E. A. 2000. Clostridium botulinum Encyclopedia In of Food Microbiology. R. K. Robinson, C. A. Batt, and P. D. Patel (Ed.). Vol. I. Academic Press.

Joosten, H. M. L. J., Nunez, M., Devreese, B., Beeumen, J. V., and Marugg, J. D. 1996. Purification and characterization of enterocin 4, a bacteriocin produced by Enterococcus faecalis INIA4. Appl. Environ. Microbiol. 62:4220.

June)a, V. K. and Marks, H. M. 1999. Proteolytic Clostridium botulinum growth at 12- 48“C simulating the cooling of cooked meat: development of a predictive model. Food Microbiol. 16:583-592.

Jung, D. S., Bodyfelt, F. W., and Daeschel, M. A. 1992. Influence of fat and emulsifiers on the efficacy on nisin inhibiting Listeria monocytogenes in fluid milk. J. Dairy Sci. 75:387.

Kaffezakis, J. G., Palmer, S. J., and Kramer, A. 1969. Microbiology of fresh apple and potato plugs preserved by gas exchange. J. Food Sci. 34:426-429.

Keynan, A., Evenchik, Z., Halvorson, H. O., and Hastings, J. W. 1964. Activation of bacterial endospores. J. Bacteriol. 88:313-318.

King, A. D. and Bolin, H. R. 1989. Physiological and microbiological storage stability of minimally processed firuits and vegetables. Food Technol. 43(2): 132-135.

Klaenhammer, T. R. 1993. Genetics of bacteriocins produced by lactic acid bacteria. FEMS Microbiol. Rev. 12:39-86.

Ku, W. L. 1993. Partial purification of a bacteriocin-like substance produced by Bacillus subtilis. A master’s thesis. The Ohio State University.

Kvenberg, J. E. 1990. Microbiological criteria and regulatory aspects of minimally processed refiigerated foods. J. Food Prot 53:1990.

Lewis, J. C., Michener, H. D., Stumbo, C. R., and Titus, D. S. 1954. Additives accelerating death of spores by moist heat Agric. Food Chem. 2:298-302.

133 LewuSt C. Kaiser^ A... and Montville^T. J. 199L Inhibition of food-home bacterial pathogens by bacteriocins firom lactic acid bacteria isolated firom meat. Appl. Environ. Microbiol. 57:1683-1688.

Lewus, C. B., Sun, S., and Montville, T. J. 1992. Production of an amylase-sensitive bacteriocin by an atypical Leuconostoc paramesenteroides strain. Appl. Environ. Microbiol. 58:143-149.

Liao, C. C., Yousef, A. E., Chism, G. W., and Richter, E. R. 1994. Inhibition of Staphylococcus aureus in buffer, culture media and foods by lacidin A, a bacteriocin produced by Lactobacillus acidophilus OSU133. J. Food Safety 14:87-101.

Lilly, T., Solomon, H. M., and Rhodehamel, E. J. 1996. Incidence of Clostridium botulinum in vegetables packaged under vacuum or modified atmosphere. J. Food Prot. 59:59-61.

Lipinska, E. 1977. Nisin and its applications. In Antibiotics and Antibiosis in Agriculture. M. Woodbine (Ed.). P. 103. Butterworths, London.

Liu, W. and Hansen, J. N. 1990. Some chemical and physical properties of nisin, a small protein antibiotic produced by Lactococcus lactis. Appl. Environ. Microbiol. 56:2551-2558.

Liu, W. and Hansen, J. N. 1993. The antimicrobial effect of a structural variant of subtilin against outgrowing Bacillus cereus T spores and vegetative cells occurs by different meahanisms. Appl. Environ. Microbiol. 59:648-651.

Lynt, R. K., Kautter, D. A., and Solomon, H. M. 1982. Differences and similarities among proteolytic and nonproteolytic strains of Clostridium botulmum type A, B, E, and F: A review. J. Food Prot. 45:466

Lyon, W. J., Sethi, J. K., and Glatz, B. A. 1993. Inhibition of psychrotrophic organisms by propionicin PLG-1, a bacteriocin produced by Propionibacterium thoenii. J. Dairy Sci. 76:1506-1513.

Lund, B. M., Graham, A. F., and George, S. M. 1988. Growth and formation of toxin by Clostridium botuliman in peeled, inoculated, vacuum-packed potatoes after a double pasteurization and storage at 25°C. J. Appl. Bacteriol. 64:241-246.

Lund, B. M. and Notermans, S. H. W. 1993. Potential hazards associated with REPFEDS. In Clostridium botulinum. Ecology and Control in Foods. A. W. Hauschild and K. L. Dodds (Ed.). Pp. 279-303. Marcel Dekker, Inc., New York, NY.

MacDonald, K. L., Spengler, R. F., Hatheway, C. L., Hargrette, N. T., and Cohen, M. L. 1985. Type A botulism firom sautéed onions. JAMA 253:1275-1278

134 MacDonald, K. L., Cohen, M. L., and Blake, P. A. 1986. The changing epidemiology of adult botulism in the United States. Amer. J. Epidemiol. 124:794-799.

Malizio, C. J. and Johnson, E. A. 1991. Evaluation of botulism hazard from vacuum- packaged Enoki mushrooms (Flammulina velutipes). J. Food Prot. 54:20-21, 27.

Mathieu, P., Michel, M., Lebrihi, A., and Lefebvre. G. 1994. Effect of the bacteriocin camocin CP5 and of the producing strain Carnobacterium piscicola CPS on the viability of Listeria monocytogenes ATCC 15313 in salt solution, broth and skimmed milk at various incubation temperatures. Int. J. Food Microbiol. 22:155.

Mattick, A. T. R. and Hirsch, A. 1947. Further observations on an inhibitory substance (nisin) from lactic streptococci. Lancet 2:5-7.

Mazzotta, A. S. and Montville, T. J. 1999. Characterization of fatty acid composition, spore germination, and thermal resistance in a nisin-resistant mutant of Clostridium botulinum 169B and in the wild-type strain. Appl. Environ. Microbiol. 65:659-664.

Meghrous, J., Lacroix, C., and Simard, R. E. 1999. The effects on vegetative cells and spores of three bacteriocins from lactic acid bacteria. Food Microbiol. 16:105-114.

McAuliffe, 0., Ryan, M. P., Ross, R. P., and Hill, C., Breeuwer, P., and Abee, T. 1998. Lacticin 3147, a broad-spectrum bacteriocin which selectively dissipates the membrane potential. App. Environ. Microbiol. 64:439-445.

Mikolajcik, E. M., Reeves, C. B., and Harper, W. J. 1965. Efficacy of nisin as a sporicidal agent in the presence of L-alanine. J. Dairy Sci. 48:1522-1524.

Montville, T. J. 1981. Effect of plating medium on heat activation requirement of Clostridium botulinum spores. Appl. Environ. Microbiol. 42:734-736.

Montville, T. J. and Bruno, M. E. C. 1994. Evidence that dissipation of proton motive force is a common mechanism of action for bacteriocins and other antimicrobial proteins. Int. J. Food Microbiol. 24:53-74.

Montville, T. J., Rogers, A. M., and Okereke, A. 1992. Differential sensitivity of Clostridium botulinum strains to nisin is not biotype-associated. J. Food Prot. 55:444- 448.

Montville, T. J., Winkowski, K., and Ludescher, R. D. 1995. Models and mechanisms for bacteriocin action and application. Int. Dairy J. 5:797-814.

135 Morris^ s. WWalsh^IL and.Hansen^LN. 1984^] of some bacterial membrane sulfhydryl groups which are targets of bacteriostatic and antibiotic action. J. Biol. Chem. 259:13590-13594.

Muriana, P. M. 1996. Bacteriocins for control of Listeria spp. in food. J. Food Prot. Suppl. Pp. 54-63.

Murinda, S. E., Roberts, R. P., and Wilson, R. A. 1996. Evaluation of colicins for inhibitory activity against diarrheagenic Escherichia coli strains, including serotype 0157:H7. Appl. Environ. Microbiol. 62:3196.

Murrell, W. G. 1969. Chemical composition of spores and spore structures. In The Bacterial Spore. G. W. Gould and A. Hurst (Ed.). Pp. 215-273. Academic Press, London.

Nettles, C. G. and Barefoot, S. F. 1993. Biochemical and genetic characteristics of bacteriocins of food-associated lactic acid bacteria. J. Food Prot. 56:338-356.

Notermans, S., Dufienne, J., and Keiybets, M. J. H. 1981. Vacuum-packed cooked potatoes: Toxin production by Clostridium botulinum and shelf life. J. Food Prot. 44:572-575.

Notermans, S., Dufireime, J., and Keiybets, M. J. H. 1985. Use of preservatives to delay toxin formation by Clostridium botulinum (Type B, strain okra) in vacuum-packed, cooked potatoes. J. Food Prot. 48:851-855.

Notermans, S., Dufrenne, J., and Lund, B. M. 1990. Botulism risk of refrigerated, processed foods of extended durability. J. Food Prot. 53:1020-1024.

O’Brien, R. T., Titus, D. S., Devlin, K. A., Stumbo, C. R., and Lewis, J. C. 1956. Antibiotics in food preservation. U. Studies on the influence of subtilin and nisin on the thermal resistance of food spoilage bacteria. Food Technol. 10(8):352-355.

Okereke, A. and Montville, T. J. 1991a. Bacteriocin inhibition of Clostridium botulinum spores by lactic acid bacteria. J. Food Prot 54:349-353.

Okereke, A. and Montville, T. J. 1991b. Bacteriocin-mediated inhibition of Clostridium botulinum spores by lactic acid bacteria at refrigeration and abuse temperatures. Appl. Environ. Microbiol. 57:3423-3428.

Okereke, A. and Montville, T. J. 1992. Nisin dissipates the proton motive force of the obligate anaerobe Clostridiian sporogenes PA 3679. Appl. Environ. Microbiol. 58:2463-2467.

136 Peck^NL w. 1997. Closttîdium. 6 ofM/w«ot,ancLthfcsafety^o£xefidgerated. processed foods, of extended durability. Trends Food Sci. Technol. 8:186-192.

Petran, R. L., Sperber, W. H., and Davis, A. B. 1995. Clostridium botulinum toxin formation in romaine lettuce and shredded cabbage: Effect of storage and packaging conditions. J. Food Prot. 58:624-627.

Pierson, M. D. and Reddy, N. R. 1988. Bacteria associated with foodbome diseases- Clostridium botulinum. Food Technol. 42:181.

Piard, J. C. and Desmazeaud, M. 1992. Inhibiting factors produced by lactic acid bacteria. 2. Bacteriocins and other antibacterial substances. Lait 72:113-142.

Piva, A. and Headon, D. R. 1994. Pediocin A, a bacteriocin produced by Pediococcus pentosaceus FBB61. Microbiol. 140:697-702.

Powell, J. F. and Hunter, J. R. 1955. Spore germination in the genus Bacillus: the modification of germination requirements as a result of preheating. J. Gen. Microbiol. 13:59-67.

Pridmore, D., Rekhif, N., Pittet, A. C., Suri, B., and Mollet, B. 1996. Variacin, a new lanthionin-containing bacteriocin produced by Micrococcus varions: Comparison to lacticin 481 of Lactococcus lactis. Appl. Environ. Microbiol. 62:1799.

Pucci, M. J., Vedamuthu, E. R., Kunka, B. S., and Vandenbergh, P. A. 1988. Inhibition of Listeria monocytogenes by using bacteriocin PA-1 produced by Pediococcus acidilactici PAC 1.0. Appl. Environ. Microbiol. 54:2349-2353.

Rayman, K., Malik, N., and Hurst, A. 1983. Failure of nisin to inhibit outgrowth of Clostridium botulinum in a model cured meat system. Appl. Environ. Microbiol. 46:1450-1452.

Ray, S. K., Kim, W. J., Johnson, M. C., and Ray, B. 1989. Conjugal transfer of a plasmid encoding, bacteriocin production and immunity in Pediococcus acidilactici H. J. Appl. Bacteriol. 66:393-399.

Ray, B. and D. G. Hoover. 1993. Pediocins. In Bacteriocins of Lactic Acid Bacteria. D. G. Hoover and L. R. Steenson (Ed.). Pp. 181-206. Academic Press, San Diego, CA.

Rekhif, N., Atrih, A., and Lefebvre, G. 1995. Activity of plantaricin SA 6, a bacteriocin produced by Lactobacillus plantarum SA6 isolated firom fermented sausage. J. Appl. Bacteriol. 78:349-358.

Rhodehamel, E. J. 1992. FDA’s concerns with sous vide processing. Food Technol. 46(12):73-76.

137 Rogers, L. A. and Whittier, E. O. 1928. Limiting factors in lactic fermentation. J. Bacteriol. 16:211-229

Rogers, A. M. and Montville, T. J. 1994. Quantification of factors which influence nisin's inhibition of Clostridium botulinum 56A in a model food system. J. Food Sci. 59:663.

Rose, N. L., Spoms, P., Stiles, M. E., and McMullen, L. M. 1999. Inactivation of nisin by gluthathione in fresh meat. J. Food Sci. 64:759-762.

Rowley, D. B. and Feeherry, F. 1970. Conditions affecting germination of Clostridium botulinum 62A spores in a chemically defined medium. J. Bacteriol. 104:1151-1157.

Ruhr, E. and Sahl, H. G. 1985. Mode of action of the peptide antibiotic nisin and influence on the membrane potential of whole cells and on cytoplasmic and artificial membrane vesicles. Antimicrob. Agents Chemother. 27:841-845.

Ryan, D. W. and Cerington, M. 1971. Human type A botulism. JAMA. 216:513-514

Ryan, M. P., Rea, M. C., Hill, C., and Ross, R. P. 1996. An application in cheddar cheese manufacture for a strain of Lactococcus lactis producing a novel broad-spectrum bacteriocin, lacticin 3147. Appl. Environ. Microbiol. 62:612-619.

Ryan, M. P., Meaney, W. J., Ross, R. P., and Hill, C. 1998. Evaluation of lacticin 3147 and a teat seal containing this bacteriocin for inhibition of mastitis pathogens. Appl. Environ. Microbiol. 64:2287-2290.

Schved, F., Lalazar, A., Henis, Y., and Juven, B. J. 1993. Purification, partial characterization and plasmid-linkage of pediocin SJ-I, a bacteriocin produced by Pediococcus acidilactici. J. Appl. Bacteriol. 74:67-77.

Schillinger, U. 1990. Bacteriocins of lactic acid bacteria. In Biotechnology and Food Safety. D. D. Bills and S. D. Kung (Ed.). Pp. 55-74. Butterworth-Heinemarm, Boston.

Scott, V. N. and Taylor, S. L. 1981a. Effect of nisin on the outgrowth of Clostridium botulinum spores. J. Food Sci. 46:117-120,126.

Scott, V. N. and Taylor, S. L. 1981b. Temperature, pH, and spore load effects on the ability of nisin to prevent the outgrowth of Clostridium botulinum spores. J. Food Sci. 46:121-126.

Seals, J. E., Snyder, J. D., Edell, T. A., Hatheway, C. L., Johnson, C. J., Swanson, R. C., and Hughes, J. M. 1981. Restaurant-associated Type A botulism: Transmission by potato salad. Amer. J. Epidemiol. 113:436-444.

138 Setlow, P. and Johnson, E. A. 1997 Spores and their significance. In Food Microbiology. Fundamentals and Frontiers. M. P. Doyle, L. R. Beuchat, and T. J. Montville (Ed.). Pp. 30-65. ASM press. Washington D. C.

Simpson, M. V., Smith, J. P., Dodds, K., Ramaswamy, H. S., Blanchfield, B., and Simpson, B. K. 1995. Challenge studies with Clostridium botulinum in a sous-vide spa^etti and meat-sauce product. J. Food Prot. 58:229-234.

Skinner, G. E., Solomon, H. M., and Fingerhut, G. A. 1999. Prevention of Clostridium botulinum type A, proteolytic type B and E toxin formation in refrigerated pea soup by Lactobacillus plantarum ATCC 8014. J. Food Sci. 64:724-727.

Solomon, H. M. and Kautter, D. A. 1986. Growth and toxin production by Clostridium botulinum in sautéed onions. J. Food Prot 49:618-620.

Solomon, H. M., Kautter, D. A., Lilly, T., and Rhodehamel, E. J. 1990. Outgrowth of Clostridium botulinum in shredded cabbage at room temperature imder a modified atmosphere. J. Food Prot. 53:831-833.

Solomon, H. M., Rhodehamel, E. J., and Kautter, D. A. 1994. Growth and toxin production by Clostridium botulinum in sliced raw potatoes under vacuum with and without sulfite. J. Food Prot. 57:878-881.

Somers, E. B. and Taylor, S. L. 1981. Further studies on the antibotulinal effectiveness of nisin in acidic media. J. Food Sci. 46:1972-1973.

Somers, E. B. and Taylor, S. L. 1987. Antibotulinal effectiveness of nisin in pasteurized process cheese spreads. J. Food Prot. 50:842-848.

Spelhaug, S. R. and Harlander, S. K. 1989. Inhibition of foodbome bacterial pathogens by bacteriocins from Lactococcus lactis and Pediococcus pentosaceus. J. Food Prot. 52:856-862.

Sperber, W. H. 1982. Requirements of Clostridium botulinum for growth and toxin production. Food Technol. 36(12):89-94

Stiles, M. E. 1996. Biopreservation by lactic acid bacteria. Antonie van Leeuwenhoek. 70:331-345.

Stiles, M. E. and Hastings, J. W. 1991. Bacteriocin production by lactic acid bacteria. Trends Food Sci. Technol. Oct:247-251.

139 Sugiyama^ ïL and Yang». BL tL, 1975^ GrawtL potential of Clo&tridium. botidimun. in. firesh mushrooms packaged in semipermeable plastic film. Appl. Microbiol. 30:964- 969.

Sugiyama, H., Woodbum, M., Yang, K. H., and Movroydis, C. 1981. Production of botulinum toxin in inoculated pack studies of foil-wrapped baked potatoes. J. Food Prot. 44:896-898.

Tagg, J. R., Dajani, A. S. and Wannamaker, L. W. 1976. Bacteriocins of gram-positive bacteria. Bacteriol. Rev. 40:722-756.

Tahara, T., Oshimura, M., Umezawa, C., and Kanatani, K. 1996. Isolation, partial characterization, and mode of action of acidocin J1132, a two-component bacteriocin produced by Lactobacillus acidophilus JCM 1132. Appl. Environ. Microbiol. 62:892- 897.

Tamminga, S. K., Beumer, R. R., Kampelmacher, E. H., and Keijbets, M. J. H. 1980. Microbial spoilage of fully or partially cooked vacuum packaged potatoes. Voedingsmiddelentechnologie. 13 (20) : 12.

Taylor, S. L. and Somers, E. B. 1985. Evaluation of the antibotulinal effectiveness of nisin in bacon. J. Food Prot. 48:949-952.

Taylor, S. L., Somers, E. B., and Krueger, L. A. 1985. Antibotulinal effectiveness of nisin-nitrite combinations in culture medium and chicken firankfurter emulsions. J. Food Prot. 48:234-239.

Terranova, W., Breman, J. G., Lacey, R. P., and Speck, S. 1978. Botulism type B: epidemiologic aspects of an extensive outbreak. Am J. Epidemiol. 108:150-156.

Thompson, J. K., Collins, M. A., and Mercer, W. D. 1996. Characterization of a proteinaceous antimicrobial produced by Lactobacillus helveticus CNRZ450. J. Appl. Bacteriol. 80:338-348.

Tipper, D. J. and Gauthier, J. J. 1972. Structure of the bacterial endospore. In Spores V. H. O. Halvorson, R. Hansen, and L. L. Campnell (Ed.). Pp. 3-12. American Society for Microbiology, Washington D.C.

Treadwell, P. E., Jann, G. J., and Salle, A. J. 1958. Studies on factors affecting the rapid germinations of spores of Clostridium botulinum. J. Bacteriol. 76:549-556.

Trevejo, R. T. 1995. Foodbome outbreaks in California. Dairy, Food Environ. Sanit. 15:611-615.

140 Valde&Stauber^ N- and Scberev Nucleotide-sequence and tæconomicat distribution of the bacteriocin gene lin cloned from Brevibacterium linens M l8 . Appl. Environ. Microbial. 62:1283.

Van de Meer, J. R., Polman, J., Berrthuyzen, M. M., Siezen, R. J., Kuipers, O. P., and De Vos, W. 1993. Characterization of the Lactococcus lactis nisin A operon genes nisP, encoding a subtilisin-Iike serine protease involved in precursor processing, and nisR, encoding a regulatory protein involved in nisin biosynthesis. J. Bacteriol. 175:2578- 2588.

Venema, K., Abee, T., Haandrikman, A. J., Leenhouts, K. J., Kok, J., Konings, W. N., and Venema, G. 1993. Mode of action of lactococcin B, a thiol-activated bacteriocin from Lactococcus lactis. Appl. Environ. Microbiol. 59:1041-1048.

Venema, K., Kok, J., Maragg, J. D., Toonen, M. Y., Ledeboer, A. M., Venema, G., and Chikindas, M. L. 1995. Functional analysis of the pediocin operon of Pediococcus acidilactici PACl.O: PedB is the immunity protein and PedD is the precursor processing enzyme. Mol. Microbiol. 17:515-522.

Villani, P., Pepe, O., Mauriello, G., Salzano, G., Moschetti, G. and Coppola, S. 1995. Antilisterial activity of thermophilin 347, a bacteriocin produced by Streptococcus thermophilus. Int. J. Food Microbiol. 25:179.

Villar, R. G., Shapiro, R. L., Busto, S., Riva-Posse, C., Verdejo, G., Farace, M. I., Rosetti, P., San-Juan, J. A., Julia, C. M., Becher, J., Maslanka, S. E., and Swerdlow, D. L. 1999. Outbreak of Type A botulism and development of a botulism surveillance and antitoxin release system in Argentina, JAMA 281:1334-1338,1340

Winkowski, K. D., Bruno, M. E. C., and Montville, T. J. 1994. Correlation of bioenergetics parameters with cell death in Listeria monocytogenes cells exposed to nisin. Appl. Environ. Microbiol. 60:4186-4188.

Van, T. R. and Lee, C. S. 1997. Characterization of a partially purified bacteriocin, l.LetL 19:741-744.

Yildirim, Z and Johnson, M. G. 1998. Detection and characterization of a bacteriocin produced by Lactococcus lactis subsp. cremoris R isolated from radish. Lett. Appl. Microbiol. 26:297-304.

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