IDENTIFICATION AND CHARACTERIZATION OF BACTERIA FROM THE

SKIN OF JACKFRUIT

CHITRA A/P SELVARATNAM

A dissertation submitted in partial fulfilment of the

requirements for the award of the degree of

Master of Science (Biotechnology)

Faculty of Biosciences and Medical Engineering

Universiti Teknologi Malaysia

JUNE 2014

iii

To my beloved family

iv

ACKNOWLEDGEMENT

I would like to forward my sincere appreciation and deep gratitude to my

supervisor, Dr. Chong Chun Shiong for his encouragement, guidance, advices,

motivation and critics throughout this dissertation project.

I would like to take this opportunity to show my greatest appreciation and

gratitude to all my lab members for their guidance, and assistance on handling

equipment and chemical used in this project. Without their continued support and

guidance, this report would not have been the same as presented here. In addition,

I’m very thankful to Universiti Teknologi Malaysia for providing good facilities in

the campus.

My sincere appreciation also extends to all my fellow friends and others who

have provided assistance at various occasions. Their views and tips are useful indeed.

Thank you for the time sacrificed to accompany me. And last but not least, I am

grateful to all my family members.

v

ABSTRACT

Fresh-cut fruits industry is developing extensively in this global era due to

busy lifestyle. However, the fresh-cut processing could be contaminated by

microorganisms; resulting in food poisoning outbreaks. This study presented about

the characterization and inhibition of bacteria isolated from the fresh skin of

jackfruit. The isolation of single colonies was done by spread plate and streak plate

method. The isolates were identified by 16S rRNA analysis. Gram staining, growth

profiling and 15 biochemical tests were carried out to characterize each isolates.

Lastly, the isolates were treated with various antimicrobial agents by disc agar

diffusion technique and the fresh skin of jackfruit was treated with antimicrobial

agent, XY-12 with different conditions by spread plate technique to study the effects

of antimicrobial agents on microbial growth. Experimental results demonstrated that

four types of bacterial single colonies were isolated with different morphology and

designated as CE1, CE2, CE3 and CE4. The 16S rRNA analysis showed that the

isolates of CE1 and CE4 were Bacillus sp. strain CY-b33. Isolates of CE2 and CE3

were identified as Bacillus pumilus strain SBTBP-008 and Bacillus thuringiensis

strain EA26.1 respectively. All the isolates were Gram positive and in rod shaped.

The growth kinetics of CE3 was highest (0.2280 h-1

) compared to CE1 (0.1317 h-1

)

which was the lowest. Bacteria CE1 and CE2 have the highest sensitivity against

XY-12 (0.6 mL/L) and Kanamycin Sulfate respectively. Bacteria CE3 and CE4 have

the highest sensitivity against Ampicillin Trihydrate and Tetracycline Hydrochloride

respectively. The fresh skin of jackfruit treated with XY-12 (0.6 mL/L) and

incubated at 4°C revealed high efficiency in microbial reduction at day 2 (100%) and

4 (99.72%).

KEYWORDS: 16S rRNA analysis, isolates, Gram staining, XY-12, Bacillus sp.,

Bacillus pumilus, Bacillus thuringiensis.

vi

ABSTRAK

Industri buah-buahan hirisan segar telah membangun maju dalam era global

ini disebabkan oleh gaya hidup yang sibuk. Walau bagaimanapun, pemprosesan

hirisan segar boleh menyebabkan kerosakan oleh mikroorganisma; mengakibatkan

wabak keracunan. Kajian ini membentangkan tentang pengenalan, pencirian dan

seterusnya perencatan bakteria yang diasingkan daripada kulit segar nangka.

Pengasingan bakteria koloni tunggal telah dilakukan dengan kaedah piring sebaran

dan piring garis jalur. Bakteria yang diasingkan dikenal pasti melalui analisis 16S

rRNA. Pewarnaan Gram, profil pertumbuhan dan 15 ujian biokimia telah dijalankan

untuk mencirikan setiap koloni. Kesemua bakteria telah dirawat dengan pelbagai

agen antimikrob menggunakan teknik penyebaran disk agar dan kulit segar nangka

telah dirawat dengan agen antimikrob, XY-12 dengan syarat-syarat yang berbeza

melalui teknik piring sebaran untuk mengkaji kesan agen antimikrob pada

pertumbuhan mikroorganisma. Keputusan eksperimen menunjukkan bahawa empat

jenis koloni bakteria tunggal telah diasingkan dengan morfologi yang berbeza dan

ditetapkan sebagai CE1, CE2, CE3 dan CE4. Keputusan 16S rRNA menunjukkan

bahawa bakteria CE1 dan CE4 adalah Bacillus sp. jenis CY-b33. Manakala, CE2

adalah Bacillus pumilus jenis SBTBP-008 dan CE3 menunjukkan Bacillus

thuringiensis jenis EA26.1. Semua bakteria yang diperoleh adalah Gram positif dan

berbentuk rod. Kinetik pertumbuhan CE3 adalah tertinggi (0.2280 h-1

) berbanding

CE1 (0.1317 h-1

) yang paling rendah. Bakteria CE1 dan CE2, masing-masing

mempunyai sensitiviti yang tertinggi terhadap XY-12 (0.6 mL/L) dan Kanamycin

Sulfat. CE3 mempunyai sensitiviti yang tertinggi terhadap Ampicillin Trihydrate

manakala CE3 terhadap Tetracycline Hydrochloride. Kulit segar nangka dirawat

dengan XY-12 (0.6 mL/L) dan disimpan pada suhu 4°C mendedahkan kecekapan

tinggi dalam pengurangan mikroorganisma pada hari ke-2 (100%) dan ke-4

(99.72%).

KATA KUNCI: analisis 16S rRNA, koloni tunggal, pewarnaan Gram, XY-12,

Bacillus sp, Bacillus pumilus, Bacillus thuringiensis.

vii

TABLE OF CONTENTS

CHAPTER TITLE PAGE

DECLARATION ii

DEDICATION iii

ACKNOWLEDGEMENT iv

ABSTRACT v

ABSTRAK vi

TABLE OF CONTENTS vii

LIST OF TABLES xii

LIST OF FIGURES xiv

LIST OF ABBREVIATIONS xviii

LIST OF APPENDICES xix

1 INTRODUCTION 1

1.1 Research Background 1

1.2 Problem Statement/ Significance of Research 2

1.3 Research Objectives 3

1.4 Scope of Research 3

2 LITERATURE REVIEW 4

2.1 Fresh-cut Fruits 4

2.2 Microbial Contamination of Fresh-cut Fruits 6

2.3 Factors Affecting the Microbial Contamination 7

2.3.1 Intrinsic Factors of Microbial Growth 8

viii

2.3.1.1 Chemical Characteristics of Intrinsic

Factors 8

2.3.1.2 Physical Characteristics of Intrinsic

Factors 10

2.3.1.3 Biological Characteristics of Intrinsic

Factors 11

2.3.2 Extrinsic Factors of Microbial Growth 12

2.4 Microorganisms that are Found in Fresh-cut Fruits 13

2.5 Negative effects of Spoilage Microorganisms in

Fresh-cut Fruits 17

2.5.1 Outbreaks of Foodborne Illness Caused by

Spoilage Bacteria Associated with Fresh-cut

Fruits

19

2.6 Innovation in Fresh-Cut Fruits to Ensure the Quality

and Safety 20

2.7 Antimicrobial Agents 25

2.7.1 Non-Residue-Producing Antimicrobial Agents 26

2.7.1.1 Alcohols 26

2.7.1.2 Aldehydes 26

2.7.1.3 Peroxides 27

2.7.1.4 Ozone Treatment 27

2.7.1.5 Ethylene Oxide 28

2.7.1.6 Chlorine Compound 28

3 METHODOLOGY 30

3.1 Experimental Design 30

3.2 Flow Chart 31

3.3 Media Preparation 32

3.3.1 Nutrient Agar 32

3.3.2 Nutrient Broth 32

3.3.3 Mueller-Hinton Agar 32

3.4 Preparation of Sample 33

3.5 Isolation of Bacteria 33

ix

3.5.1 Sample Dilution 33

3.5.2 Spread Plate Method 33

3.5.3 Streak Plate Method 34

3.6 Identification of Bacteria 34

3.6.1 Bacterial Genomic DNA Extraction 34

3.6.2 Gel Electrophoresis 35

3.6.3 DNA Amplification by Polymerase Chain

Reaction (PCR) 35

3.6.4 PCR Product Purification 36

3.6.5 16S rRNA Sequence Analysis 37

3.7 Characterization of Bacteria 38

3.7.1 Gram Staining 38

3.7.2 Growth Profiling 39

3.7.3 Biochemical Tests 39

3.7.3.1 Oxidase Test 39

3.7.3.2 Catalase Test 40

3.7.3.3 Indole Test 40

3.7.3.4 Citrate Utilization Test 40

3.7.3.5 Triple Sugar Ion Agar Test 41

3.7.3.6 Nitrate Reduction Test 41

3.7.3.7 Oxidative-Fermentative (OF) Glucose

Test 41

3.7.3.8 Urease Test 42

3.7.3.9 Methyl Red Test 42

3.7.3.10 Starch Hydrolysis Test 43

3.7.3.11 Mac Conkey Agar Test 43

3.7.3.12 Mannitol Salt Agar Test 44

3.7.3.13 Hektoen Enteric Agar Test 44

3.7.3.14 Xylose-Lysine Deoxycholate (XLD)

Agar Test 44

3.7.3.15 Motility Test 45

x

3.8 Effects of Antimicrobial Agents on Bacterial Growth 45

3.8.1 Disc Agar Diffusion Technique 45

3.8.2 Treatment of jackfruit skin with antimicrobial

agent (XY-12) 46

4 RESULTS AND DISCUSSION 47

4.1 Isolation of Pure Bacterial Single Colonies 47

4.2 Identification of Bacteria using 16S rRNA Gene

Analysis 51

4.2.1 Genomic Extraction of Bacterial DNA 51

4.2.2 PCR Amplification of 16S rRNA Gene 52

4.2.3 Purification of PCR Product 53

4.2.4 Analysis of 16S rRNA 55

4.2.5 Study of Phylogenetic Tree of Bacillus Species 56

4.2.6 Potential Roles of Bacillus spp. from the Fresh

Skin of Jackfruit 57

4.3 Characterization of Bacteria 58

4.3.1 Gram Staining 58

4.3.2 Growth Profile of Isolated Bacteria 60

4.3.2.1 Growth Profile of Bacterial strain of

CE1 61

4.3.2.2 Growth Profile of Bacterial strain of

CE2 62

4.3.2.3 Growth Profile of Bacterial strain of

CE3 63

4.3.2.4 Growth Profile of Bacterial strain of

CE4 64

4.3.3 Biochemical Tests 65

4.3.3.1 Oxidase Test 65

4.3.3.2 Catalase Test 67

4.3.3.3 Indole Test 68

4.3.3.4 Citrate Utilization Test 69

4.3.3.5 Triple Sugar Ion (TSI) Agar Test 70

xi

4.3.3.6 Nitrate Reduction Test 71

4.3.3.7 Oxidative-Fermentative (OF) Glucose

Test 72

4.3.3.8 Urease Test 73

4.3.3.9 Methyl Red Test 74

4.3.3.10 Starch Hydrolysis Test 75

4.3.3.11 Mac Conkey Agar Test 76

4.3.3.12 Mannitol Salt Agar Test 77

4.3.3.13 Hektoen Enteric Agar Test 78

4.3.3.14 Xylose-Lysine Deoxycholate (XLD)

Agar Test 79

4.3.3.15 Motility Test 80

4.4 Effects of Antimicrobial Agents on Bacterial Growth 81

4.4.1 Effects of Antimicrobial Activity by Disc Agar

Diffusion Technique 81

4.4.2 Effects of Antimicrobial Activity by Spread

Plate Technique 84

4.4.2.1 Effects of Antimicrobial Activity of

Treated Skin of Jackfruit at Day 2 84

4.4.2.2 Effects of Antimicrobial Activity of

Treated Skin of Jackfruit at Day 4 86

5 CONCLUSION 89

5.1 Conclusion 89

5.2 Future Work 91

REFERENCES 92

APPENDICES 107

xii

LIST OF TABLES

TABLE NO. TITLE

PAGE

2.1 Outbreaks of human foodborne disease from various

microorganisms associated with fresh-cut fruits (Danyluk et

al., 2012) 20

3.1 Components of PCR 36

4.1 Viable count of colonies using spread plate technique after

serial dilution 49

4.2 Isolation of single pure culture of four different colonies by

streaking method 50

4.3 Concentration of DNA for all four bacterial strains by using

Nanodrop (DNA Spectophotometric) 54

4.4 The Gram staining results of four of the bacteria 59

4.5 The oxidase test was carried out to study the cytochrome

oxidase activity in all the four bacterial strains 66

4.6 Detection of catalyse enzymes in isolated bacteria was

performed by catalase test 67

4.7 Observation of carbohydrate utilization in isolated bacteria

by performing Triple Sugar Ion agar test 70

4.8 The nitrate reducing bacteria was identified by performing

nitrate reduction test 71

xiii

4.9 Oxidative-fermentative (OF) glucose test was used to study the

ability of microorganisms to ferment glucose 73

4.10 The average microbial load (CFU/mL) on fresh skin of jackfruit

treated with different concentration of chlorine based

antimicrobial agent XY-12 at day 2 85

4.11 The percentage of microbial load reduction (%) of treated

jackfruit skin with different concentration of chlorine based

antimicrobial agent XY-12 compared to untreated of control at

day 2 85

4.12 The average microbial load (CFU/mL) on fresh skin of jackfruit

treated with different concentration of chlorine based

antimicrobial agent XY-12 at day 4 86

4.13 The percentage of microbial load reduction (%) of treated

jackfruit skin with different concentration of chlorine based

antimicrobial agent XY-12 compared to untreated of control at

day 4 87

xiv

LIST OF FIGURES

FIGURE NO.

TITLE

PAGE

2.1 Steps in fresh-cut processing (Source: Vasantha and Li

Juan Yu, 2013) 5

2.2 Local fruit of jackfruit and its interior fruit (Source:

Gayatri, 2014) 6

2.3 Main factors affecting microbial contamination (Source:

FDA, U.S. Food and Drug Administration) 7

2.4 Three main characteristics of intrinsic factors 8

2.5 Extrinsic factors that affecting the growth of

microorganisms 12

2.6 Pseudomonas graminisca causing foodborne disease

(Source: Lauren Paxman, 2012) 15

2.7 Microscopic images of Leuconostoc mesenteroides

(Source: Máté Borsos, 2011) 15

2.8 Salmonella bacteria triggers food poisoning (Source:

Katie Dawson, 2011) 16

2.9 Escherichia coli via micrographic images (Source:

Escherichia coli (E. coli) bacteria, 2010) 16

2.10 Four types of Clostridium bacteria (Source: Siven Benson,

2012) 16

xv

2.11 Species of Listeria monocytogenes bacterium (Source:

Listeriosis, 2014) 17

2.12 Negative impacts in fresh-cut fruits due to spoilage microbes 18

2.13 Untreated and treated of fresh-cut apples with anti-browning

agent (Source: Victor et al., 2009) 22

2.14 Packaging system of active modified atmosphere (Source:

Mehyar and Han, 2010) 23

2.15 Treated and untreated fresh pomegranate arils with active

modified atmosphere technology (Source: Victor et al., 2009) 23

2.16 Variable fresh-cut melons via cultivars technology (Source:

Victor et al., 2009) 24

2.17 Process of edible coating layer by layer (Source: Elena, 2014) 25

2.18 Untreated and treated fresh-cut melons with edible coating

(Source: Elena, 2014) 25

3.1 Flow chart of project which focused on five major steps 31

4.1 The extracted genomic DNA of four of the bacterial with the

standard labeled size of 1 kb DNA marker on gel

electrophoresis viewed under UV light 51

4.2 The 1.5kb of PCR product from the 16S rRNA fragment

obtained using 27F and 1525 R primers 52

4.3 The purified PCR amplification product of 16S rRNA gene

with the standard labeled size of DNA marker on gel

electrophoresis observed under UV light 54

xvi

4.4 A phylogenetic tree showing position and the evolutionary

relationships between the four bacterial strain CE1, CE2, CE3

and CE4 with other Bacillus species 56

4.5 Growth curve of bacterial strain of CE1 grown in nutrient broth

at 37°C 61

4.6 Growth curve of bacterial strain of CE2 grown in nutrient broth

at 37°C 62

4.7 Growth curve of bacterial strain of CE3 grown in nutrient broth

at 37°C 63

4.8 Growth curve of bacterial strain of CE4 grown in nutrient broth

at 37°C 64

4.9 The Indole test was carried out to detect the presence of

tryptophanase enzyme in the isolated bacteria 68

4.10 The citrate utilization test was performed to identify the ability

of the isolated bacteria to utilize citrate 69

4.11 The presence of urea degrading bacteria were very lack in all

the isolated bacterial strains 74

4.12 Three isolated bacterial strains were able to ferment sugars and

identified by methyl red test 75

4.13 Starch degrading bacteria were identified by performing starch

hydrolysis test 76

4.14 No isolated Gram-negative bacteria strain was identified in this

Mac Conkey agar test 77

4.15 High salt-tolerant bacteria were identified by conducting

Mannitol salt agar test 77

xvii

4.16 The isolated bacteria were identified as not enteric pathogen

bacteria because the bacterial colonies were not formed on the

agar 78

4.17 The bacterial strains were not able to grow on XLD agar due to

the isolated bacteria were Gram-positive 79

4.18 All the bacterial strains showed their ability of motility 80

4.19 Average zone of inhibition was measured by using different

antimicrobial agents 82

xviii

LIST OF ABBREVIATIONS

CFU/mL - Colony Forming Units per millilitre

kb - kilobytes

PCR - Polymerase Chain Reaction

16S rRNA - 16 Subunit Ribosomal Ribonucleic Acid

bp - base pair

ng/µL - Nanogram per microlitre

°C - degree Celsius

µ - micro (unit of growth rate)

g - unit of generation time

h-1

- per hour

h - hour

nm - nanometre

mL/L - mililitre per litre

ppm - parts per milion

xix

LIST OF APPENDICES

APPENDIX

TITLE

PAGE

A Sequences of isolates in Fasta format 107

B Growth profile of isolates 111

C Effects of antimicrobial activity by

different techniques

116

1

CHAPTER 1

INTRODUCTION

1.1 Research Background

Nowadays, the markets of fresh-cut fruits have shown a marked upward trend

in Malaysia and also in international markets. Occasionally, fresh fruits can become

contaminated with harmful microorganisms (Peggy VL et al., 2001). The fresh fruits

are exposed to microbial contamination through a variety of sources. Environmental

contamination such as water quality, soil fertility management, equipment sanitation

and many other factors contribute to the risk of microbial contamination. In addition,

the microbial contamination with fresh produce could be associated with human or

animal feces (Pradnya and Sonali, 2008).

According to survey, the total production of the fruit in the world is around

27x106 ton per year (Hathaitip et al., 2013). A survey in United Kingdom shows that

15-20% of fresh-cut fruits are lost each year due to the microbial contamination

(Bond et al., 2013). Recently, the Federal Food and Drug Administration from

United States has reported the outbreak of listeria infection from cantaloupe which

has killed 33 people (Reuters, 2013). Listeriosis, a serious bacterial infection usually

caused by eating food or fruits contaminated with the Listeria monocytogenes, is an

important public health problem in the United States (Centers for Disease Control

and Prevention, 2013).

Jackfruit is rich in vitamins A, C and B-complex with abundant of fiber,

minerals and energy. Nowadays, people who live a busy lifestyle are interested in

2

fresh-cut fruits, which jackfruit is one of them. During the cutting process of fresh

jackfruit, the microorganisms on the surface of a skin of jackfruit may be transferred

into the flesh of fruit. These bacteria utilize the host via extracellular lytic enzymes

that hydrolyzed these polymers to release water and the other intracellular

constituents for use as nutrients for their growth (Margaret B et al., 2009). As a

result, the quality of the fruit is affected. To prevent the microbial spoilage, effective

preventive measures should be taken to prevent microbial spoilage on fresh-cut

fruits.

In this project, bacteria from the skin of jackfruit were isolated and the

diversity of the bacterial population were studied. These bacteria were morphological

and biochemically characterized. The bacteria were identified using 16S rRNA gene

analysis. The effectiveness of XY-12, a commercial available antimicrobial agent,

was used to test its efficiency in retarding the growth of these bacteria.

1.2 Problem Statement/ Significance of Research

The processing steps of cleaning, trimming, coring, slicing, shredding,

washing, centrifugal drying and packaging for production of fresh-cut fruits have

been well developed (Chung CC et al., 2011). However, the processes are sometimes

not hygienic enough and could cause a numerous fruits contamination and poisoning

outbreaks. The presence of bacteria on the cut-fruits could proliferate and thus

decrease the shelf-life of the products. Attempt to isolate and characterize the

bacteria from the skin of the jackfruit, and the studies of the effect of antibacterial

agent on these bacteria are crucial because these findings provide useful information

on the improvement of antimicrobial steps to produce a good quality of fresh cut-

fruits with better shelf-life.

3

1.3 Research Objectives

Followings are the objectives of this research:

1.3.1 To isolate bacteria from the skin of jackfruit.

1.3.2 To identify the bacteria using 16S rRNA analysis.

1.3.3 To characterize bacteria using biochemical tests and antimicrobial agents.

1.4 Scope of Research

In this study, the bacterial diversity on the skin of jackfruit was investigated.

The bacteria were isolated by using serial dilution and spread plate technique. Then,

these bacteria were indentified based on 16S rRNA analysis. The bacteria were

morphologically and biochemically characterized. The effect of chlorine based

antimicrobial agent (XY-12) and temperature in inhibiting the growth of bacteria

from jackfruit skin were investigated by measuring the population of bacteria on the

skin after being treated with XY-12 at different temperature.

REFERENCES

16S rDNA Sequencing to Identify Unknown Microorganisms. (1-5). Retrieved April

21, 2014 from

http://delliss.people.cofc.edu/virtuallabbook/LabReadings/16s%20rDNA%20/16s

%20rDNA%20SeqAnal.pdf

Abdulsudi, I. Z., Yoshinori, K., Muhimbula, H. S. and Bernadette, K. N. (2010). A

Review of Microbiological Safety of Fruits and Vegetables and the Introduction

of Electrolyzed Water as An Alternative to Sodium Hypochlorite Solution. Afr. J.

Food Sci. 4(13): 778 – 789.

Acumedia Manufacturers. (2008). XLD Agar (7166). Neogen Corporation. 1-3.

Acumedia Manufacturers. (2011). Simmons Citrate Agar (7156). Neogen

Corporation. 1-2.

Adams, M. R., Hartley, A. D. and Cox, L. J. (1989). Factors Affecting the Efficacy

of Washing Procedures Used in the Production of Prepared Salads. Food Microb.

6:69–77.

Alegre I., Viñas I., Usall J., Teixidó N., Figge M. J., Abadias M. (2013). Control of

foodborne pathogens on fresh-cut fruit by a novel strain of Pseudomonas

graminis. Food Microbiology. 34: 390-399.

Ana, T. C., Sonia, S. J., Santos A. J. M. and Carlos, R. J. (2006). Antimicrobial

Activity of Steady-State Cultures of Bacillus sp. CCMI 1051 Against Wood

Contaminant Fungi. Biochem. Eng. J. 30: 231–236.

Angela, O. E., Ibukunoluwa, A. O. and Oranusi, U. S. (2010). Microbial Quality of

Fruits and Vegetables Sold in Sango Ota, Nigeria. Afr. J. Food Sci. 4(5): 531-535.

93

Ankit, K., Anil, P. and Johri, B.N. (2011). Chapter 2: Bacillus as PGPR in Crop

Ecosystem. Springer-Verlag Berlin Heidelberg. 37-59.

Anna, K., Tomasz, Z., Beata, N. and Helena, K. (2006). Influence of Additives and

Storage Temperature on Physicochemical and Microbiological Properties of Eye

Drops Containing Ceftazidime. Acta Poloniae Pharmaceutica - Drug Research,

63(6): 507-513

Anne Hanson. (2013). Oxidative-Fermentative Test Protocol. American Society for

Microbiology. 64-75.

Anne Y. Tsang. (2013). Mannitol Salt Agar Plates Protocol. American Society for

Microbiology. 79-81.

Anthony J. Fontana. (2008). Understanding the Importance of Water Activity in

Food. Innovations in Food Technology, UK. 116-117.

Anthony Shelton. (2001). Biological Control: A Guide to Natural Enemies in North

America. College of Agriculture and Life Science, Cornell University. 1-3.

A System Approach for Produce Safety. (2011). Acidified Sodium Chlorite as

Disinfectant. University of Georgia.

Australian Government. (2014). Hypochlorite Solutions. Commonwealth of

Australia.

Avian Biotech International. (2009). Clostridium. Animal Genetics, Inc. Tallahassee.

Awofisayo Oladoja Abosede and Igbeneghu Oluwatoyin Abimbola. (2013). The

Effect of Chloroquine Phosphate on the Pharmacodyanmic Activity of Ampicillin

Trihydrate Against Staphylococcus aureus. J. Microbiol. Antimicrob. 5(11): pp.

124.

Ayesha Patela and Anthony Ouelletteb. (2011). 24 Interactions Among Bacilli.

Jacksonville University. 25-36.

Badiaa, E., Mohammed, B., Haissam, J., Abdellatif, B. and Najla, S. Z. (2009). High

Salt-Tolerant Protease From A Potential Biocontrol Agent Bacillus pumilus M3-

16. Annals of Microbiology. 59 (3) 553-558.

94

BD. (2010). Triple Sugar Iron Agar • TSI Agar. Difco™ & BBL™. 2:1-2.

Benita Brink. (2013). Urease Test Protocol. American Society for Microbiology. 25-

28.

Beth Ann Crozier-Dodson. (2005). Formulating Food Safety: An Overview of

Antimicrobial Ingredients. Food Safety Magazine. December 2004/January 2005:

58-63.

Beuchat, L. R., Nail, B. V., Adler, B. B. and Clavero, M. R. S. (1998). Efficacy of

Spray Application of Chlorinated Water in Killing Pathogenic Bacteria on Raw

Apples, Tomatoes and Lettuce. J. Food Prot. 61: 1305–1311.

Beveridge, T. J. (2001). Use of the Gram Stain in Microbiology. Biotech Histochem.

76(3):111-8.

Bielinski, M. S. and Teresa, P. S. (2004). Protected Culture for Vegetable and Small

Fruit Crops: The Soilless Trench System. University of Florida. HS1204.

Bizuayehu Mengstie Muche and Vasantha Rupasinghe. (2011). Natural antimicrobial

agents of cinnamon (Cinnamomum zeylanicum L. and C. cassia) and vanilla

(Vanilla planifola, V. pompona, and V. tahitensis) for extending the shelf-life of

fresh-cut fruits. Ethiop. J. Appl. Sci. Technol. 2(1): 1 – 13.

Bond, M., Meacham, T., Bhunnoo, R. and Benton, T. G. (2013). Food waste within

global food systems. A Global Food Security report (www.foodsecurity.ac.uk).

Brian, C. M., Richard, W. B., David, L. A., Michael, W. A. and Andrew, F. P.

(2001). Analyzing Differences in Bacterial Optical Density Measurements

between Spectrophotometers. Thermo Fisher Scientific. 1730: 1-2.

Buck, J. W., Walcott, R. R. and Beuchat, L. R. (2003). Recent Trends in

Microbiological Safety of Fruits and Vegetables. . Plant Health Progress.

doi:10.1094/PHP-2003-0121-01-RV.

Burt, S. (2004). Essential Oils: Their Antibacterial Properties and Potential

Applications in Foods: A Review. Int. J. Food Microbiol. 94: 223-253. HS1204.

95

Carlos, R., Joã, C. and Bartolomeu, A. (2012). Prospects of UV Radiation for

Application in Postharvest Technology. Emir. J. Food Agric. 24 (6): 586-597.

Cedric, N. B., Samir, V. S., Robert, K. S., Patricia, M. G., David, P., Paul, H. and

Gad, F. (2010). Minireview: Fresh Fruit and Vegetables as Vehicles for the

Transmission of Human Pathogens. Environmental Microbiology. 12(9): 2385–

2397.

Centers for Disease Control and Prevention. (2013). National Center for Emerging

and Zoonotic Infectious Diseases. Available from http://www.cdc.gov/listeria/

Chang, H. K., Sang,Y. C., Hyog, Y. K., Eun, H. K., Hyun, M. K., Jin, S. K., Geum,

C. J., Hee, S. L., Seung, W. K., Jong, M. K., Suhkneung, P. and Dong, K. R.

(2012). Isolation, Characterization and Evaluation of Bacillus thuringiensis

Isolated From Cow Milk. Korean J Vet Res. 52(3): 169-176.

Chapter 12: Fresh-Cut Produce. (2000). Part II: Small-Scale Processing

Technologies for Horticultural Products. 12.1-12.5. RV.

Charles E. Deutch. (1994). Characterization of A Novel Salt-Tolerant Bacillus sp.

From the Nasal Cavities of Desert Iguanas. Elsevier B.V. 121(1): 55-60.

Chung, C. C., Huang, T. C., Yu, C. H., Shen, F. Y. and Chen, H. H. (2011).

Bactericidal effects of fresh-cut vegetables and fruits after subsequent washing

with chlorine dioxide. 2011 International Conference on Food Engineering and

Biotechnology. IPCBEE vol.9:107-112.

Consolacion, Y. R., Karen, J. and John, A. R. (2004). Antimicrobial Compounds

from Artocarpus heterophyllus. Philippine Journal of Science. 133 (2): 97-101.

Cornelis, P. (2008). Pseudomonas: Genomics and Molecular Biology. Int. J. Syst.

Bacteriol. 30: 225-420.

Daniel, F., Colette, G., Mê-Linh, L. and Mona, S. (2011). Effectiveness of

Alternative Antimicrobial Agents for Disinfection. National Collaborating Centre

for Environmental Health.

96

Daniel, N. A. T. and Obed, A. A. (2011). Effect of Increasing Concentration of

Antimicrobial Agent on Microbial Load and Antibiotic Sensitivity Pattern of

Bacterial Isolates from Vegetables. Eur. J. Exp. Biol. 1 (4):12-23.

Danyluk, M. D., Goodrich, G. M., Schneider, K. R., Harris, L. J. and Worobo, R. W.

(2012). Outbreaks of Foodborne Disease Associated with Fruit and Vegetable

Juices, 1922–2010. University of Florida. FSHN12-04.

David, M. I., Eric, J., Kim, C. S. C., Joseph, M. M., Sherri, A. M., Cheyanne, K. and

Michael W. D. (2013). Natural Farming: Lactic Acid Bacteria. College of

Tropical Agriculture and Human Resources. SA.8.

Dawn Merton Boothe. (2012). Tetracyclines. Merck Sharp & Dohme Corp. 1-5.

Devendra, K. C. and Bhavdish, N. J. (2009). Interactions of Bacillus spp. and Plants-

With Special Reference to Induced Systemic Resistance (ISR) Microbiological

Research. 164:493-513.

Dima Al-Sharif. (2011). Handling and Storing Fruits & Vegetables. Understanding

Fruits & Vegetables – Part 2. Dubai.

Dube, S., Dlamini, N. R., Shereni, I. and Sibanda, T. (2012). Extending the Shelf

Life of Fresh Marula (Sclerocarya birrea) Juice by Altering Its Physico-Chemical

Parameters. InTech. 181-196.

EDVOTEK. Principles and Practice of Agarose Gel Electrophoresis. (2003). The

Biotechnology Education Company®. 3-22.

Edward, R. B. M., Brian, J. T., Vitor, A. P. M., Dietmar, H. P., Juan-Luis, R. and

Norberto, J. P. (2006). Nonmedical: Pseudomonas. Prokaryotes. 6:646–703.

Ehimare, A., Marie, S., Mark, G. W., Warren, B. B. and Ebbing, L. (2011).

Cefepime-Resistant: Pseudomonas aeruginosa. Emerging Infectious Diseases.

17(6): 1037-1043.

Elena Poverenov.(2014). Layer by Layer Edible Coating. Israel Agriculture.

Elisabeth, G. and Diane, M. B. (2000). Preservative Treatments for Fresh-Cut Fruits

and Vegetables. University of California. 3-22.

97

Escherichia coli (E. coli) bacteria. (2010). Escherichia coli (E. coli) bacteria Science

Image. PSmicrographs.

FDA U.S. Food and Drug Administration. (2013). Chapter V. Methods to

Reduce/Eliminate Pathogens from Produce and Fresh-Cut Produce. U.S.

Department of Health & Human services.

Fernandez, J. S., Gomez, R. and Carmona, M. A. (1993). Water Activity in Selected

High-Moisture Foods. J. Food Compos. Anal. 6: 364-369.

Fresh-cut Fruits. (2013). The Packer. Vance Publishing Corporation.

Galal, A. A., El-Bana, A. A. and Janse, J. (2006). Bacillus pumilus, A New Pathogen

on Mango Plants. Egypt. J. Phytopathol. 34(1):17-29.

Galgóczy, L., Hevér, T., Orosz, L., Krisch, J., Vágvölgyi, C., Tölgyesi, M. and Papp,

T. (2006). Growth inhibition effect of fruit juices and pomace extracts on the

enteric pathogens Campylobacter jejuni and Salmonella ser. Typhimurium.

Internet Scientific Publications. 7:1.

Gayatri. (2014). 13 Amazing Benefits of Jackfruit. India. Retrieved January 28, 2014

from http://www.stylecraze.com/articles/amazing-benefits-of-jackfruit/#

GC-Rich PCR System. (2004). Roche Applied Science. 1-4.

Geeta, G., Alagawadi, A. R., Krishnaraj, P. U. and Basavana, G. K. (2012).

Characterization of Bacillus thuringiensis Isolates of Western Ghats and their

Insecticidal Activity Against Diamond Back Moth (Plutella xylostella L.).

Karnataka J. Agric. Sci. 25 (2) :199-202.

Geetha, M., Saranraj, P., Mahalakshmi, S. and Reetha, D. (2012). Screening of

Pectinase Producing Bacteria and Fungi for its Pectinolytic Activity Using Fruit

Wastes. Int. J. Biochem. Biotech. Sci. 1: 30-42.

George, A. M. and Reese, H. V. (2006). The Fermentation of Glucose by Bacillus

Stearothermophilus. J. Food Sci. 22(5): 494-500.

98

Gerald M. Sapers. (2001). Efficacy of Washing and Sanitizing Methods for

Disinfection of Fresh Fruit and Vegetable Products. Food Technol. Biotechnol. 39

(4): 305–311.

Glenda Dvorak. (2008). Disinfection 101. Center for Food Security and Public

Health. 6-18.

Gorny, J. R., Cifuentes, R. A., Hess, P. B. and Kader, A. A. (2000). Quality Changes

in Fresh-cut Pear Slices as Affected by Cultivar, Ripeness Stage, Fruit Size, and

Storage Regime. J. Food Sci. 65(3): 541-544.

Harold Eddleman. (1998). Reasons to Study the Bacillus Bacteria. Indiana Biolab. 1-

2.

Harris, L. J., Farber, J. N., Beuchat, L. R., Parish, M. E., Suslow, T. V., Garrett, E.

H. and Busta, F. F. (2003). Outbreaks Associated with Fresh Produce: Incidence,

Growth, and Survival of Pathogens in Fresh and Fresh-Cut Produce.

Comprehensive Reviews in Food Science and Food Safety. 2: 78-141.

Hathaitip, R., Aphirak, P. and Tri, I. W. (2013). Physical properties of impregnated

cantaloupe and apple affected by different pressure levels. Asian J. Agric. Food

Sci. Volume 01– Issue 04, October 2013.

Hua, W., Hao, F. And Yaguang, L. (2006). Dual-Phasic Inactivation of Escherichia

Coli O157:H7 with Peroxyacetic Acid, Acidic Electrolyzed Water and Chlorine

on Cantaloupes and Fresh-Cut Apples. J. Food Saf. 26: 335–347.

Hui, Y. H. (2008). Handbook of Fruits and Fruit Processing. John Wiley & Sons. 10-

15.

Hugo, C. R., Tamara, H., Marco, M., Hafed, H., Elena, P. S., Oliver, D., Philippe, G.

and Dieter, J. (2000). Fermentative Metabolism of Bacillus subtilis: Physiology

and Regulation of Gene Expression. J Bacteriol. 182(11): 3072–3080.

Hyakumachi, M., Nishimura, M., Arakawa, T., Asano, S., Yoshida, S., Tsushima, S.

and Takahashi, H. (2013). Bacillus thuringiensis suppresses bacterial wilt disease

caused by Ralstonia solanacearum with systemic induction of defense-related

gene expression in tomato. Microbes Environ. 28(1):128-34.

99

Hygiene and Environmental Health HEAT Module. (2013). Open Learn Lab Space.

GNU GPL.

Irina, I. and Mohamed, G. (2013). Prevention of Enzymatic Browning in Fruit and

Vegetables. Eur. Sci. J. 9: 1857 – 7881.

Isabelle, V., John, V. C., Frank, D., Kim, V., Quenten, D., Peter, R. and Bruno D. M.

(2008). Effect of Decontamination Agents on the Microbial Population, Sensorial

Quality, and Nutrient Content of Grated Carrots (Daucus carota L). J. Agric.

Food Chem.

James, N. B., Manley, M., and Robert, L. G. (1978). Comparative Zone

Electrophoresis of Enzymes in the Genus Bacillus. Int. J. Sys. Bacteriol. 229-244.

Jan Hudzicki. (2013). Hektoen Enteric Agar Protocol. American Society for

Microbiology. 81-86.

Janie Sigmon. (2011). The Starch Hydrolysis Test. American Society for

Microbiology. 156-160.

Jay, J. M. (2000). Modern food microbiology. Gaithersburg (MD). Aspen. 6: 679.

Jean, C. B., Hodan, F. W., Bruce, A. and Jana M. (2009). Supermarket Loss

Estimates for Fresh Fruit, Vegetables, Meat, Poultry, and Seafood and Their Use

in the ERS Loss-Adjusted Food Availability Data. United States Department of

Agriculture. 1-18.

Jesús, L. S., Juan, A. S. N., Mar, T., Silvia, V., Dolores, S., Francisco, L., Rosa, V.,

Maria, J. P. and Pedro, L. (2008). Nosocomial Outbreaks Caused by Leuconostoc

mesenteroides subsp. mesenteroides. EID Journal. 14:6.

Ji Gang Kim. (2012). Environmental Friendly Sanitation to Improve Quality and

Microbial Safety of Fresh-Cut Vegetables. Biotechnology - Molecular Studies and

Novel Applications for Improved Quality of Human Life. 11: 173-196.

Jim Cooper. (2006). Bacterial Plant Pathogens and Symptomology. WSU County

Extension, SJI 1-7.

100

John, C. B. and James, R. G. (2002). Fresh-cut Fruits. Alexandria, VA: International

Fresh-cut Produce Association.

Jong-Kang Liu and Peter Jurtshuk. (1986). N,N,N’-N’-Tetramethyl-p-

Phenylenediamine-Dependent Cytochrome Oxidase Analyses of Bacillus Species.

Int. J. Syst. Bacteriol. 36(1): 38-46.

Joyce, A. K., and Victor, L. Y. (1991). Minireview: Antimicrobial Agent Therapy for

Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 35(11): 2167-2172.

Kader A. A. (2002). Pre- and Postharvest Factors Affecting Fresh Produce Quality,

Nutritional Value, and Implications for Human Health. Proceedings of the

International Congress Food Production and the Quality of Life. 1: 109-119.

Karen Reiner. (2013). Catalase Test Protocol. American Society for Microbiology. 3-

6.

Katie Dawson. (2011). How Consumers Can Prevent Food Borne Illnesses. Indiana

Public Media.

Kenneth Todar. (2012). The Growth of Bacterial Populations. Academic Journal. 3-

8.

Lansing, M. P., Donald, A. K. and John, P. H. (2002). Online Learning Center:

Microbial Growth. McGraw-Hill Higher Education. 5:6 e.

Larry R. Beuchat. (1998). Surface Decontamination of Fruits and Vegetables Eaten

Raw: A Review. Food Safety Unit World Health Organization.

Lauren Paxman. (2012). Deadly Infection which Killed Four Babies in Belfast Found

in Neo-Natal Ward of Norfolk Hospital. Mail Online.

Liangji Xu. (1999). Use of Ozone to Improve the Safety of Fresh Fruits and

Vegetable. Food Technol. 53(10): 58-62.

Listeriosis. (2014). National Food and Veterinary Risk Assessment Institute.

Retrieved January 29, 2014 from http://www.nmvrvi.lt/lt/zoonozes/listerioze/

Madhumita, M., Hussam A. M., M. Dominika, K., Dave, A. and Catherine, M. K.

(2009). Cell Lysis and DNA Extraction of Gram-Positive and Gram-Negative

101

Bacteria from Whole Blood in a Disposable Microfluidic Chip. The Royal Society

of Chemistry: Lab Chip. 9: 2811–2817.

Margaret, B., Thomas, R. H., Hong, Z. and Frederick, B. (2009). Microbiological

Spoilage of Fruits and Vegetables. Food Microbiology and Food Safety, DOI

10.1007/978-1-4419-0826-1_6.

Maria, I. G., Maria, V. S., Francisco, L. G. and Ana, A. (2009). Fresh-Cut Product

Sanitation and Wash Water Disinfection: Problems and Solutions. Int. J. Food

Microb. 4779:9.

Maria P. MacWilliams. (2013). Citrate Test Protocol. American Society for

Microbiology. 45-63.

Maria P. MacWilliams. (2013). Indole Test Protocol. American Society for

Microbiology. 16-22.

Mary E. Allen. (2013). Mac Conkey Agar Plates Protocol. American Society for

Microbiology. 76-78.

Máté Borsos. (2011). Bacteria Worth Munching On. Lab Adventures: Kickass

Microbes.

Matthew, D. R., Christopher, J. R., Sacha, L., Carmen, P., Arthur, T., Andrew, D. S.

C., Mark, A., Michael, F. S.,b Roy, P. B., József, B., Michael, W. P. and Jay, C.

D. H. (2012). Lag Phase is A Distinct Growth Phase that Prepares Bacteria for

Exponential Growth and Involves Transient Metal Accumulation. J Bacteriol.

194(3): 686–701.

McFeeters, R. F., Hankin, L. and Lacy, G. H. (1992). Compendium of Methods for

the Microbiological Examination of Foods. American Public Health Association.

213-223.

Mehyar, G. F. and Han, J. H. (2010). Active Packaging for Fresh-Cut Fruits and

Vegetables. Novel Packaging Technologies. 267-284.

Melda Zeynep Güray. (2009). Partial Purification and Characterization of Polyphenol

Oxidase from Thermophilic Bacillus sp. İzmir Institute of Technology. 2-37.

102

Meldau, D. G., Long, H. H., and Baldwin, I. T. (2012). A Native Plant Growth

Promoting Bacterium, Bacillus sp. B55, Rescues Growth Performance of an

Ethylene-Insensitive Plant Genotype in Nature. Front Plant Sci. 3:112.

Microorganism. (2013). ScienceDaily. Rockville. 150,461. Retrieved January 25,

2014 from http://www.sciencedaily.com/articles/m/microorganism.htm

Mikal E. Saltveit. (2001). Edible Films, Coatings & Processing Aids. Mann

Laboratory, Department of Plant Sciences. California.

Mohan and Manuselis. (2010). Gram Positive Bacilli. Clinical Microbiol. 2:2-9.

Moosavi-Nasab, M., Alahdad, Z. and Nazemi, S. (2009). Characterization of the

Dextran Produced by Leuconostoc mesenteroides from Date Fruit Extract. Islamic

Republic of Iran Shiraz University. 28:1.

Morton, J. (1987). Jackfruit. West Lafayette: NewCROPTM

.

Mossel, D. A. A., Corry, J. E. L., Struijk, C. B. and Baird, R. M. (1995). Essentials

of the Microbiology of Foods: A Textbook for Advanced Studies. John Wiley and

Sons. Chichester (England). 699 p.

Murray, P. R. and Zeitinger, J. R. (1983). Evaluation of Mueller-Hinton Agar for

Disc Diffusion Susceptibility Tests. J. Clin. Microbiol. 18(5):1269.

Mwathi, J. W. (2011). Isolation, Evaluation and Molecular Characterisation of

Bacillus Thuringiensis Isolates against Prostephanus Truncatus, a Major Storage

Pest in Maize. Kenyatta University Institure Repository. 1-3.

Nathan, R. S., Ruth, E. G. and Francis, E. C. (1946). Aerobic Mesophilic

Sporeforming Bacteria. U.S. Department of Agriculture. 60-69.

National Center for Emerging and Zoonotic Infectious Diseases. (2013). CDC

Contributor Information.

Olga Martin-Belloso and Robert Soliva Fortuny. (2011). Advances in Fresh-Cut

Fruits and Vegetables Processing. Food Preservation Technology. 221-223.

Olusola Lamikanra. (2002). Fresh-Cut Fruits and Vegetables: Science, Technology,

and Market. CRC Press. 215-223.

103

Orna, R., Lars, R. and Alexander, V. G. (1990). Changes in the Stability of Specific

mRna Species in Response to Growth Stage in Bacillus subtilis. Proc. Nati. Acad.

Sci. USA. Vol. 87, pp. 8355-8359.

Patricia M. V. Azanza. (2006). Hydrogen Peroxide, Peroxyacetic Acid, Octanoic

Acid, Peroxyoctanoic Acid and 1-Hydroxyethylidene-1,1-Diphosphonic Acid as

Components of Antimicrobial Washing Solution. Chemical and Technical

Assessment. 1-7.

Patricia Shields and Laura Cathcart. (2013). Motility Test Medium Protocol.

American Society for Microbiology. 101-108.

Peggy, V. L. and Amanda, S. (2001). Safe handling of fresh fruits and vegetables.

The Texas A&M University System. E-198; 5-04.

Pirovani, M., Piagentini, A., Güemes, D. and Arkwright, S. (2004). Reduction of

Chlorine Concentration and Microbial Load During Washing-Disinfection of

Shredded Lettuce. Int. J. Food Sci. & Technol. 39: 341–347.

Pradnya, A. J. and Sonali, P. P. (2008). Microbiological analysis of fresh vegetables

& fruits and effect of anti-microbial agents on microbial load. Birla College of

Arts, Science and Commerce.

Rebecca Buxton. (2013). Nitrate and Nitrite Reduction Test Protocol. American

Society for Microbiology. 30-42.

Reuters. (2013). Guilty pleas in outbreak of listeria. Business Day The New York

Times. Available from http://www.nytimes.com/2013/10/23/business/guilty-pleas-

in-outbreak-of-listeria.html

Rosa, M. R., Jonathan, M., Robert, S. and Olga, M. (2009). Control of Pathogenic

and Spoilage Microorganisms in Fresh-cut Fruits and Fruit Juices by Traditional

and Alternative Natural Antimicrobials. Comprehensive Reviews In Food Science

And Food Safety. 8: 157-180.

Sandie Smith. (2010). Oxidase Test. BIOL 2420.

Sascha, B., Ralph, K., Walter, P. H. and Reinhold, C. (2004). Sensory and

Microbiological Quality of Shredded, Packaged Iceberg Lettuce as Affected by

104

Pre-Washing Procedures with Chlorinated and Ozonated Water. Innovative Food

Sci. Emerg. Technol. 5(1): 45-55.

Shengmin, L., Yaguang, L., Ellen, T. and Hao, F. (2007). Efficacy of Sodium

Chlorite as An Inhibitor of Enzymatic Browning in Apple Slices. Food Chemistry.

104: 824–829.

Sigfrid, L.A., Cunningham, J.M., Beeharry, N., Lortz, S., Tiedge, M., Lenzen,

S., Carlsson, C., Green, I.C. (2003). Cytokines and nitric oxide inhibit the enzyme

activity of catalase but not its protein or mRNA expression in insulin-producing

cells. J Mol Endocrinol.31(3):509-18.

Sigma. (2003). Kanamycin monosulfate from Streptomyces kanamyceticus Cell

Culture Tested. Sigma-Aldrich, Inc. (1).

Siven Benson. (2012). Botulism - Causes, Symptoms, Diagnosis and Treatment.

Online Information About Drugs. Retrieved January 29, 2014 from

http://www.tabletsmanual.com/wiki/read/botulism

Society for General Microbiology. (2011, April 11). Ozone reduces fungal spoilage

of fruits and vegetables. ScienceDaily. Retrieved January 26, 2014 from

www.sciencedaily.com//releases/2011/04/110410194709.htm

Stuart B. Levy. (2013). Alliance for the Prudent Use of Antibiotics (APUA). Boston.

Sylvia McDevitt. (2013). Methyl Red and Voges-Proskauer Test Protocol. American

Society for Microbiology. 92-98.

Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S. (2011).

MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood,

Evolutionary Distance, and Maximum Parsimony Methods. Mol. Biol. Evol. 10:

2731-9

Team: Newcastle Project. (2014). L-Forms. Retrieved May 2, 2014 from

http://2013.igem.org/Team:Newcastle/Project/L_forms

Tomoo Saga and Keizo Yamaguchi. (2009). History of Antimicrobial Agents and

Resistant Bacteria. JMAJ. 52(2): 103–108.

105

Trias, R., Bañeras, L., Montesinos, E. and Badosa, E. (2004). Lactic Acid Bacteria

from Fresh Fruit and Vegetables as Biocontrol Agents of Phytopathogenic

Bacteria and Fungi. Int Microbiol. 11(4):231-6.

UK Standards for Microbiology Investigations. (2014). Identification of Bacillus

Species. Standards Unit, Public Health England. 8-19.

Vasantha Rupasinghe and Li Juan Yu. (2013). Value-Added Fruit Processing for

Human Health. Food Industry. InTech. 7: 145-162.

Victor, R., Batia, H., Yakov, V., Genady, G., Tzipora, T. and Galit, R. (2009).

Innovations in Fresh-Cut Fruit & Vegetables Quality and Safety. Israel

Agriculture.

Wacker. (2009). Densetec®, Wacker’s High-Cell-Density Fermentation – Optimal

Space-Time Yields Combined with High Reproducibility. Biologics. 1-2.

Wareing, P., Stuart, F. and Fernandes, R. (2011). Factors Affecting the Growth Of

Micro-Organisms In Foods. Softcover. ISBN: 978-1-905224-84-5, p.437.

Wei, Z., Xiaomin, L., Tongxu, L. and Fangbai, L. (2012). Enhanced Nitrate

Reduction and Current Generation by Bacillus Sp. in the Presence of Iron Oxides.

J. Soils Sediments. 12:354–365.

Weihua Tang. (2008). Effects of Temperature, Relative Humidity, and Air Flow on

Microbial Growth on Loaded Ventilation Filters. ProQuest. 35-40.

William McGlynn. (2005). Guidelines for the Use of Chlorine Bleach as a Sanitizer

in Food Processing Operations. Oklahoma State University. 116:2.

Wizard® Genomic DNA Purification Kit. (2010). Instructions for use of Product

A1120, A1123, A1125 and A1620. Promega Corporation. 1-20.

Wizard® SV Gel and PCR Clean-Up System. (2010). Instructions for use of Product

A9280, A9281, A9282 and A9285. Promega Corporation. 1-12.

Yuk, H., Yoo, M., Yoon, J., Marshall, D. L. and Oh, D. (2006). Effect of Combined

Ozone and Organic Acid Treatment for Control of E. coli O157:H7 and Listeria.

monocytogenes on Lettuce. Journal of Food Science 71, 83–86.

106

Yuto, K., Mamoru, Y. and Yoshio, E. (1987). Screening of Bacteria with Antiviral

Activity Against Infectious Hematopoietic Necrosis Virus (IHNV) from Estuarine

and Marine Environments. Nippon Suisan Gakkaishi. 53 (12), 2179-218.

Zulfaidah, P.G., Nobukazu, N., Suharjono and Faridah, S. (2013). Toxicity Studies

for Indigenous Bacillus thuringiensis Isolates from Malang City, East Java on

Aedes Aegypti Larvae. Asian Pac J Trop Biomed. 3(2): 111–117.