THE ANTIBACTERIAL ACTIVITY OF SELECTED SMALL MOLECULE COMPOUNDS AND THEIR EFFECT ON THE MORPHOLOGY AND MECHANICAL PROPERTIES OF TAPIOCA STARCH FILMS
THESIS
Presented in Partial Fulfillment of the Requirements for The Degree Master of Science in the Graduate School of The Ohio State University.
By Zilu Wan, M.S. Graduate Program in Food Science and Technology
The Ohio State University
2016
Thesis Committee Dr. Melvin Pascall, Advisor Dr. Gireesh Rajashekara Dr. James R. Fuchs Dr. Farnaz Maleky
Copyright by Zilu Wan 2016
ABSTRACT
Microbial contamination reduces the shelf life of foods and increases the risk of foodborne illnesses. This is a major worldwide public health concern. Antimicrobial therapy is an effective way to prevent and control foodborne diseases. By incorporating antimicrobial agents into packaging material, an active food packaging system can serve as a non-thermal method of controlling foodborne pathogens. The objectives of this study were to investigate the antibacterial activity of selected small molecules, and to investigate how these compound influence the barrier and mechanical properties of starch based film when incorporated into the material.
Chapter 2 focuses on the antibacterial activity of the small molecule compounds.
The study was initiated by dissolving the small molecules (JA-144, TH-4, and TH-8) in dimethyl sulfoxide (DMSO), and incorporating them into tapioca films. These were then evaluated against E.coli K12, Listeria innocua, Campylobacter jejuni and
Campylobacter coli, respectively. The Minimum Inhibitory Concentrations (MIC) of
JA-144, TH-4 and TH-8 were determined. The bacterial growth curves for E.coli K12,
L.innocua, C.jejuni and C.coli after treatment with JA-144, TH-4 or TH-8 at the MICs were then quantitatively measured. Log reduction tests of the organisms exposed to the compounds in broth and incorporated into the films were followed by bacterial growth measurement tests to give an accurate evaluation of molecules’ efficacy when
ii incorporated into the films. Results showed that the compounds incorporated into the tapioca films and in the broth produced ≥3 log reduction in the test organisms.
Chapter 3 focuses on the effect of the small molecule compounds on the barrier and mechanical properties of the films. Specifically, thickness, moisture content, water activity, oxygen permeability coefficient (OPC), water vapor permeability
(WVP), glass transition temperature, X-ray diffraction, melting temperature and storage/loss modulus were tested as function of the compound type. Results show that the JA-144 film had the highest moisture content, OPC and WVP. This meant that it had the lowest barrier properties and mechanical property due to the plasticizing and co-plasticizing effect of hydroxyl groups within its molecules. As for TH-4 and the
TH-8 films, they had lower moisture content, OPC and WVP, which meant that they had better barrier properties, and higher mechanical strength due to the crosslinking effect of the sulfonamide group they contained. Results indicated that JA-144, TH-4 and TH-8 have great potential for antimicrobial packaging, but some modifications are still necessary for their optimal use in packaging material.
iii DEDICATION
Dedicated to my wife, my daughter, my parents and my parent-in-law
iv ACKNOWLEDGMENTS
There are so many people to thank for helping me during the last year. First, I would like to thank Dr. Melvin Pascall for his guidance, encouragement, and patience over the last two and a half years. Thank you so much for inspiring me to look at the research and my work more objectively. Your support was essential to my success here. Undoubtedly, this thesis would have not taken place had you not afforded me this opportunity.
I would like to thank Dr. Gireesh Rajashekara for offering me the opportunity to work in his lab; the experience and technique I’ve learnt were significant. I would also like to thank Dr. James R. Fuchs for his guidance, advices and help in synthesizing the small molecule compounds. Thank you Dr. Fanaz Maleky for your support and for allowing me to use the DSC instrument. The data obtained from the
DSC provided valuable information for my study. Thanks Dr. Esperanza J.
Carcache de Blanco, your toxicity data for these compounds gave me a clear path to start the experiments.
My gratitude also goes to my lab mates and friends, Dr. Anand Kumar, Janet
Antwi, Ramya Rheya, Dr. Jaesung Lee, Xu Yang, En-Huang, Hao Wang,
Musfirah Zulkurnain, Yung-hsin Chien, Yajun Wu, Bruce Ruey, Yesil Mustafa, your knowledge, experience, and most importantly, your friendship were very helpful
v towards the completion of this work.
Next, I would like to acknowledge my family. Here, I would like to thank my parents Chenggang Wan, Xueyan Gou for their love, guidance and financial support.
My appreciation goes to my parents-in-law, Deren Pei and Jinying Han for their consistent support and understanding.
Finally, thank you my love, Pei Pei, for your endless supporting and encouragement and for being here for me during the pursuit for this degree. I also want to include and acknowledge my one-year-old baby, Amy Wan, for the lack of time spent with her during the course of my study. I wish that my daughter would be healthy and bright throughout her entire life.
vi VITA
December 10, 1987 ...... Born-Xinjiang, The People’s Republic of China 2006-2010...... B.S. Department of Chemistry and Chemical Engineering Tianjin University of Techology, Tianjin, The People’s Republic of China. 2010-2012...... M.S. Department of Chemical and Biomolecular Engineering NYU Tandon School of Engineering, Brooklyn, NY, USA 2013-2015 ...... M.S. Department of Food Science and Technology The Ohio State University, Columbus, OH, USA
Publications
Teng, H. Wan, Z., Koike, Y., and Okamoto. Y. Copolymerization of pentafluorophenylhexafluoroisopropyl methacrylate (PFPHFIPMA) with trifluoroethyl methacrylate (TFEMA) and hexafluoroisopropyl methacrylate (HFIPMA). Polymers for Advanced Technologies. 2013. 24(5):520-523.
Ming, Y., Lihua, N., and Zilu, W. Cellulose converted into electricity in MFC by rumen bacteria. Journal of Shandong Chemical Industry, 2010. 39(9):8-11. (In Chinese)
Fields of Study
Major Field: Food Science and Technology
Minor Field: Biotechnology, Enology, Photography, Cinematography
vii Table of Contents
ABSTRACT ...... ii DEDICATIONS ...... iv ACKNOWLEDGEMENTS ...... v VITA ...... vii LIST OF TABLES ...... xiv LIST OF FIGURES ...... xvi LIST OF ABBREVIATIONS ...... xix
Chapters: 1. LITERATURE REVIEW ...... 1
1.1 Introduction of Edible Film ...... 1
1.2 Types of Edible Film ...... 4
1.2.1 Polysaccharide-based Edible Film/Coating ...... 6
1.2.1.1 Collagen ...... 7
1.2.1.2 Corn Zein ...... 8
1.2.1.3 Whey Protein ...... 8
1.2.2 Protein-based Edible Film/Coating ...... 9
1.2.2.1 Starch ...... 9
1.2.2.2 Carrageenan ...... 10
1.2.2.3 Cellulose Derivatives ...... 11
1.2.2.4 Chitin/Chitosan ...... 12
1.2.3 Lipid-based Edible Film Coating ...... 14
1.2.3.1 Waxes and Paraffin ...... 14
1.2.3.2 Acetylated Glycerol Monosterate ...... 15
viii 1.2.3.3 Shellac Resins ...... 16
1.2.4 Composite Edible Film Coating ...... 17
1.3 Antimicrobial Substance Incorporated in the Film ...... 19
1.3.1 Essential Oils and Plant Extracts ...... 19
1.3.1.1 Grape Seed Extract ...... 20
1.3.1.2 Oregano Oil ...... 21
1.3.1.3 Aqueous Garlic Extract ...... 21
1.3.2 Enzyme ...... 22
1.3.2.1 Proteolytic Enzymes ...... 22
1.3.2.2 Polysaccharide-degrading Enzymes ...... 24
1.3.2.3 Oxidative Enzymes ...... 25
1.3.2.4 Anti-quorum Sensing Enzymes ...... 26
1.3.3 Bacteriocin ...... 27
1.3.3.1 Bacteriocin from Eukaryotes ...... 27
1.3.3.2 Bacteriocin from Prokaryotes ...... 28
1.3.4 Inorganic Nanoparticles ...... 29
1.3.4.1 Titanium dioxide as AM agent ...... 29
1.3.4.2 Zinc Oxide as AM agent ...... 30
1.3.5 Synthesized Small Organic Molecules ...... 30
1.3.5.1 Inhibitors of Folic Acid Metabolism ...... 31
1.3.5.2 Inhibitors of Cell Wall Biosynthesis ...... 32
1.3.5.3 Inhibitors of Protein Biosynthesis ...... 32
1.3.5.4 Inhibitors of Nucleic Acid Biosynthesis ...... 33
1.4 Foodborne Diseases in Meat & Poultry Industry ...... 34
1.4.1 Main Types of Foodborne Pathogens ...... 36
1.4.1.1 Salmonella Enteritidis ...... 36
ix 1.4.1.2 Campylobacter jejuni & Campylobacter coli ...... 37
1.4.1.3 E.coli O157:H7 ...... 38
1.4.1.4 Vibrio vulnificus ...... 38
1.4.1.5 Listeria monocytogenes ...... 39
1.4.2 Antimicrobial Resistance of Foodborne Pathogens ...... 39
1.4.2.1 Salmonella ...... 40
1.4.2.2 Campylobacter ...... 41
1.4.3 Prevention and Control ...... 42
1.5 The Methods of Antimicrobial Agents Testing ...... 43
1.5.1 Commonly Used Testing Methods ...... 44
1.5.1.1 Broth Dilution Test ...... 44
1.5.1.2 Antimicrobial Gradient Method ...... 45
1.5.1.3 Disk Diffusion Test ...... 46
1.5.1.4 Automated Instrument Systems ...... 47
1.5.2 Interpretation of Susceptibility Test Results ...... 49
1.5.3 Acceptable Accuracy of Susceptibility Testing Methods ...... 49
1.6 The Methods of Characterizing Edible Film ...... 51
1.6.1 Barrier Properties ...... 51
1.6.1.1 Oxygen Transmission Rate (OTR) ...... 52
1.6.1.2 Water Vapor Transmission Rate (WVTR) ...... 53
1.6.1.3 Carbon Dioxide Transmission Rate (CO2TR) ...... 54
1.6.2 Mechanical Strength ...... 55
1.6.3 Microstructure Determination ...... 56
1.6.3.1 Fourier Transform Infrared Spectroscopy ...... 57
1.6.3.2 X-ray Diffraction ...... 57
1.6.3.3 Differential Scanning Calorimetry (DSC) ...... 59
x 1.6.3.4 Thermo-Gravimetric Analysis (TGA) ...... 60
1.6.3.5 Dynamic Mechanical Analysis (DMA) ...... 62
2. Antibacterial Activity of Selected Small Molecule Compounds against E.coli K12, Listeria innocua, Campylobacter jejuni, and Campylobacter coli bacterial species...... 63
2.1 Abstract ...... 63
2.2. Introduction ...... 65
2.3. Materials and Methods ...... 68
2.3.1 Materials ...... 68
2.3.2 Minimum Inhibitory Concentration (MIC) tests of JA-144, TH-4 and TH-8...... 68
2.3.2.1 Small molecules of JA-144, TH-4 and TH-8...... 68
2.3.2.2 Preparation of bacterial culture ...... 69
2.3.2.3 Preparation of small molecule stock solutions ...... 70
2.3.2.4 Preparation of 2,3,5-Triphenyltetrazolium Chloride solutions as color indicator.
...... 70
2.3.2.5 Preparation of working solutions...... 70
2.3.2.6 Preparation of plates...... 73
2.3.2.7 Interpreting Results...... 75
2.3.3 Bacterial Growth Curve Measurement using JA-144, TH-4, or TH-8 as inhibitors at
Minimum Inhibitory Concentration ...... 75
2.3.4 Antibacterial activity testing of JA-144, TH-4 or TH-8 in bacterial broth or incorporated into films against E.coli K12, L.innocua, C.jejuni and C.coli at each MIC value..
...... 75
2.3.4.1 Preparation of Tapioca Films with JA-144, TH-4, or TH-8...... 76
2.3.4.2 Preparation of tested films with inoculated bacterial culture ...... 78
2.3.4.3 Preparation of Plates...... 78
xi 2.3.4.4 Inoculation of Plates...... 79
2.3.5 Statistical Analysis ...... 79
2.4. Results ...... 80
2.4.1 Minimum Inhibitory Concentration (MIC) tests of JA-144, TH-4 and TH-8...... 80
2.4.2 Bacterial Growth Curve Measurement using JA-144, TH-4, or TH-8 as inhibitors at
Minimum Inhibitory Concentration...... 81
2.4.3 Antibacterial activity tests of JA-144, TH-4 or TH-8 in bacterial broth or incorporated into films against E.coli K12, L.innocua, C.jejuni and C.coli at each MIC...... 86
2.4.3.1 Antibacterial activity tests of JA-144, TH-4 or TH-8 in bacterial broth against
E.coli K12, L.innocua, C.jejuni and C.coli...... 87
2.4.3.2 Antibacterial activity tests of JA-144, TH-4 or TH-8 incorporated into tapioca films against E.coli K12, L.innocua, C.jejuni and C.coli...... 88
2.5. Discussion ...... 92
2.5.1 Minimum Inhibitory Concentration (MIC) tests of JA-144, TH-4 and TH-8 ...... 92
2.5.2 Bacterial Growth Curve Measurement using JA-144, TH-4, or TH-8 as inhibitors at
Minimum Inhibitory Concentration...... 93
2.5.3 Antibacterial activity tests of JA-144, TH-4 or TH-8 with or without films against
E.coli K12, L.innocua, C.jejuni and C.coli at each MIC...... 94
2.6. Conclusions ...... 95
3. The incorporation of Selected Small Molecule Compounds into Tapioca Films and their Effects on Thickness, Moisture Content, Oxygen and Water Barrier Properties and Mechanical Properties of the Film ...... 96
3.1 Abstract ...... 96
3.2. Introduction ...... 98
3.3. Materials and Methods ...... 101
xii 3.3.1 Materials ...... 101
3.3.2 Preparation of Tapioca Films with JA-144, TH-4, or TH-8...... 101
3.3.3 Film Moisture Content and Water Activity ...... 102
3.3.4 Oxygen Permeability Coefficient...... 103
3.3.5 Water Vapor Permeability ...... 104
3.3.6 Thermal analysis By Differential Scanning Calorimetry (DSC) ...... 105
3.3.7 X-ray Diffraction ...... 106
3.3.8 Mechanical testing by Dynamic Mechanical Analysis (DMA) ...... 107
3.3.9 Statistical Analysis ...... 107
3.4. Results ...... 108
3.4.1 Thickness, Moisture Content, and Water Activity...... 108
3.4.2 Water Vapor Permeability...... 109
3.4.3 Oxygen Permeability Coefficient ...... 110
3.4.4 Thermal Analysis by Differential Scanning Calorimetry (DSC) ...... 112
3.4.5 X-ray Diffraction Pattern ...... 113
3.4.6 Mechanical testing by Dynamic Mechanical Analysis (DMA) ...... 114
3.5. Discussion ...... 117
3.5.1 Thickness, Moisture Content, Water Activity, and barrier properties (OPC, WVP)
...... 117
3.5.2 Mechanical and Thermal Properties...... 119
3.6. Conclusions ...... 121
LIST OF REFERENCES ...... 123
APPENDIX A ...... 153
APPENDIX B ...... 154
xiii LIST OF TABLES
Table 1.1 Possible Components of Edible Films ...... 5
Table 1.2 Minimum inhibitory concentration (MIC) for chitosan against microorganisms. ... 13
Table.1.3 Antimicrobial incorporated into edible polymer film ...... 20
Table 1.4 Bacteriocins of eukaryotic organisms ...... 28
Table 1.5 Examples of bacteriocins isolated from foods ...... 29
Table 1.6 Estimated annual number of domestically acquired, foodborne illnesses, hospitalizations, and deaths due to 31 foodborne pathogens and unspecified agents in U.S. . 34
Table 1.7 Selected outbreaks in Ohio in 2013, associated with emerging foodborne pathogens and factors for their emergence...... 35
Table 2.1 Preparation of dilutions of JA-144 for use in MIC tests...... 72
Table 2.2 Preparation of dilutions of TH-4 for use in MIC tests...... 72
Table 2.3 Preparation of dilutions of TH-8 for use in MIC tests ...... 73
Table 2.4 The amount use of each constituent for MIC tests...... 74
Table 2.5 The composition of tapioca film on wet basis and dry basis ...... 76
Table 2.6 An example showed amount of bacterial culture pipetted into micro tubes based on film’s weight ...... 78
Table 2.7 Summary of Small molecules' MIC against 4 individual bacterial strains ...... 80
Table 2.8 Concentration of JA-144, TH-4 or TH-8 used for antibacterial activity tests ...... 86
Table 2.9 The summary of Log reduction tests for E.coli K12 and L.innocua ...... 88
Table 2.10 The summary of Log reduction tests for C.jejuni and C.coli ...... 90
Table 2.11 The summary of Log reduction tests for C.jejuni and C.coli ...... 90
Table 3.1 The formula of antimicrobial films based on a dry weight basis ...... 102
Table 3.2 The Summary of Thickness, Moisture Content, and Water Activity of Tapioca films with JA-144, TH-4 or TH-8 ...... 108
xiv Table 3.3 The Summary of WVP calculation of Tapioca Films with JA-144, TH-4 or TH-8….
...... 110
Table 3.4 The Summary of OPC calculation of Tapioca Films with JA-144, TH-4 or TH-8…..
...... 111
Table 3.5 The position of main X-ray diffraction peaks of Tapioca Films with JA-144, TH-4 or TH-8 ...... 114
xv LIST OF FIGURES
Figure 1.1 The structural differences of fibrous protein and globular protein...... 7
Figure 1.2 The structure of amylose and amylopectin ...... 10
Figure 1.3 The structure of kappa, iota, and lambda carrageenan...... 11
Figure 1.4 The structure of Chitin and Chitosan ...... 12
Figure 1.5 The chemical structure of Paraffin wax ...... 14
Figure 1.6. The chemical structure of Acetylated Monoglyceride ...... 16
Figure 1.7 Simplified scheme of the hydrolysis of bacterial cell walls by lysostaphin...... 23
Figure 1.8 Alginate lyase catalyzed the hydrolysis of alginate through beta-elimination ..... 25
Figure 1.9 Glucose oxidase-lactoperoxidase antimicrobial system...... 26
Figure 1.10 The biosynthesis pathway of Folates ...... 31
Figure 1.11 Campylobacteriosis Cases and Incidence Rates in Texas. Data obtained from
Texas, Department of State Health Services ...... 37
Figure 1.12 A broth micro-dilution susceptibility panel containing 96 reagent wells and a disposable tray inoculator ...... 45
Figure 1.13 S.aureus isolate tested by Antimicrobial Gradient Diffusion method with antibiotics on Mueller-Hinton agar...... 46
Figure 1.14 A disk diffusion test with an isolate of E.coli from a urine culture...... 47
Figure 1.15 The MicroScan WalkAway instrument manufactured by Siemens Healthcare
Diagnostics...... 48
Figure 1.16 A typical stress/strain curve for protein based edible film...... 56
Figure 1.17 X-ray diffraction of Methyl Cellulose, Chitosan and composite films ...... 58
Figure 1.18 DSC thermograms of amylomaize films stored at 20oC and 63.8% RH ...... 60
Figure 2.1 The chemical structure of JA-144, TH-4 and TH-8 ...... 69
Figure 2.2 The MIC tests design for JA-144, TH-4 and TH-8 ...... 74
xvi Figure 2.3 The flow chart of Tapioca/small molecules and film formation ...... 77
Figure 2.4 E.coli K12 Growth Curve measurement using JA-144 as inhibitor at 100ppm,
75ppm, 50ppm ...... 81
Figure 2.5 Listeria innocua Growth Curve measurement using JA-144 as inhibitor at 100ppm,
75ppm, 50ppm ...... 82
Figure 2.6 C.jejuni Growth Curve measurement using JA-144 as inhibitor at 6.25ppm, and
3.125ppm ...... 83
Figure 2.7 C.coli Growth Curve measurement using JA-144 as inhibitor at 6.25ppm, and
3.125ppm ...... 83
Figure 2.8 C.jejuni Growth Curve measurement using TH-4 as inhibitor at 12.5ppm, and
25ppm ...... 84
Figure 2.9 C.coli Growth Curve measurement using TH-4 as inhibitor at 12.5ppm, and 25ppm
...... 84
Figure 2.10 C.jejuni Growth Curve measurement using TH-8 as inhibitor at 25ppm, and
50ppm ...... 85
Figure 2.11 C.coli Growth Curve measurement using TH-8 as inhibitor at 25ppm, and 50ppm
...... 86
Figure 2.12a.The Log reduction tests of JA-144 against E.coli K12 and L.innocua in bacterial broth. b.The Log reduction tests of JA-144 against E.coli K12 and L.innocua incorporated into the film...... 91
Figure 2.13a.The Log reduction tests of JA-144, TH-4 or TH-8 against C.jejuni in bacterial broth. b. The Log reduction tests of JA-144, TH-4 or TH-8 against C.coli in bacterial broth.
...... 91
Figure 2.14a.The Log reduction tests of JA-144, TH-4 or TH-8 against C.jejuni incorporated into the film. b. The Log reduction tests of JA-144, TH-4 or TH-8 against C.coli incorporated into the film...... 92
xvii Figure 3.1 The summary of Moisture Content and Water Activity of Tapioca Films with
JA-144, TH-4 or TH-8...... 109
Figure 3.2 The Summary of WVP of Tapioca films with JA-144, TH-4 or TH-8 ...... 109
Figure 3.3 The Summary of OPC of Tapioca films with JA-144, TH-4 or TH-8 ...... 111
Figure 3.4 The Differential Scanning Calorimetry of Tapioca films with JA144, TH-4, or
TH-8 at 1% ...... 112
Figure 3.5 The X-ray Diffraction of Tapioca films with JA-144, TH-4 or TH-8 ...... 113
Figure 3.6 The effect of JA-144, TH-4, or TH-8 on the storage modulus of tapioca films. 116
Figure 3.7 Loss Modulus of Tapioca films with JA-144, TH-4, or TH-8...... 116
Figure 3.8 Tan Delta of Tapioca Films with JA-144, TH-4, or TH-8 ...... 117
Figure 3.9 A possible mechanism of JA-144 bind to Amylose or Glycerol...... 118
Figure A.1 The cytotoxicity test of JA-144 on Human Colon Cells (Provided by Dr.
Esperanza Carcache de Blanco’s lab) ...... 153
Figure A.2 The cytotoxicity test of TH-4 on Human Colon Cells (Provided by Dr. Esperanza
Carcache de Blanco’s lab ...... 153
Figure A.3 The cytotoxicity test of TH-8 on Human Colon Cells (Provided by Dr. Esperanza
Carcache de Blanco’s lab) ...... 154
Figure B.1 An example of 96-wells microplate showing MICs results ...... 154
xviii LIST OF ABBREVIATIONS
Abbreviation Meaning Page
HPC Hydroxy Propyl Cellulose 11
HPMC Hydroxy Propyl Methyl Cellulose 11
CMC Carboxy Methyl Cellulose 11
MC Methyl Cellulose 11
AMG Acetylated Mono-Glycerides 15
AM Antimicrobial 19
LLDPE Linear low-density polyethylene 20
MRSA Multidrug Resistant Staphylococcus Aureus 23
CDC Centers for Disease Control and Prevention 34
STEC Shiga-toxin-producing E.coli 38
Ampicillin, Chloramphenicol/florphenicol, ACSSuT 41 Streptomycin, Sulfonamides, and Tetracycline
NARMS National Antimicrobial Resistance Monitoring System 41
GMP Good Manufacturing Practices 42
HACCP Hazard Analysis Critical Control Point 42
MIC Minimal Inhibitory Concentration 44
CLSI Clinical and Laboratory Standards Institute 45
OTR Oxygen Transmission Rate 52
OPC Oxygen Permeability Coefficients 53
WVTR Water Vapor Transmission Rate 53
WVPC Water Vapor Permeability Coefficients 54
CO2TR Carbon Dioxide Transmission Rate 54 Continue
xix Continue
Abbreviation Meaning Page
CO2PC Carbon Dioxide Permeability Coefficients 55
MAP Modified Atmosphere Packaging 54
SEM Scanning Electron Microscope 56
XRD X-ray diffraction 56
DSC Differential Scanning Calorimetry 56
TGA Thermo Gravity Analysis 56
DMA Dynamic Mechanical Analysis 57
FTIR Fourier Transform Infrared Spectroscopy 57
PEI Polyetherimide 61
PC Polycarbonate 61
PET Polyethylene Terephthalate 61
PVC Polyvinyl Chloride 61
DMSO Dimethyl sulfoxide 90
TTC 2,3,5-Triphenyltetrazolium Chloride 92
TSB Tryptic Soy Broth 92
TSA Tryptic Soy Agar 92
MHB Mueller Hinton Broth 92
MHA Mueller Hinton Agar 92
SB Standard Broth 93
OD Optical Density 99
ANOVA Analysis Of Variance (one-way) 104
xx
Chapter 1
Literature review
1.1 Introduction to Edible Film
An edible film is a thin layer acting as a barrier between food and its surrounding environment. This layer can also be consumed with the food since it is edible. These coatings and films are designed to prolong the quality and shelf life of food by protecting it from physical, mechanical or biological damage (Janjarasskul & Krochta,
2010). An example of an edible film is the sausage casing, which is not removed during cooking or eating. General functions of edible coatings and films in food processing are for: enhancing quarantined treatments, improving the appearance of the food, incorporation of flavors and pigments, reduction in loss of flavors and aromas, reduction in gas diffusion and reduction in water loss (Krochta, 2002).
Edible films and coatings can either be prepared from lipids, polysaccharides or proteins, or from a combination of them (Dangaran & Krochta, 2008). Some of the polysaccharides that are suitable for use as edible films and coatings include chitosan, starches, pectin, alginates and cellulose derivatives (Mohammed, 2010). Typically they have good oxygen but poor moisture barrier properties due to their hydrophilic nature and the ability to form strong hydrogen bonding that can be used to cross-link with functional additives such as flavors, colors, and micronutrients. Animal and vegetable fats used to make films and coatings are compounds such as fatty acids,
1 acylglycerols, and waxes. These lipid compounds are quite suitable because they are excellent barriers to moisture and they add extra gloss to confectionary products.
Waxes are mainly utilized as coatings on fruits and vegetables to reduce loss of moisture and retard respiration (Valérie, 2010). Apart from the preservation factor, these films and coatings also facilitate the incorporation of food additives into the food to enhance the texture, flavor and color (Cagri, Ustunol & Ryser, 2004). Protein based films are also hydrophilic and have good mechanical strength and can be used for the individual packaging of small portions of food, such as beans, nuts and cashew nuts. Moreover, they serve as functional carriers for antimicrobial and antioxidant agents (Thawein, 2012).
The use of edible coatings and films continues to expand due to their justification by research findings in the field of active packaging. One of these active packaging applications is the incorporation of antimicrobial agents into or on edible films that can be used to successfully inhibit spoilage and pathogenic organisms from infiltrating food products. Examples of antimicrobial agents include essential oils and plants extracts, enzymes, chitosan, bacteriocins, nanoparticles and small molecule drugs (Suet-Yen, et al., 2013). However, there are still some challenges in the application of films and coatings. For example, environmental factors such as temperature and humidity that cannot be completely controlled during transportation and storage tend to make hydrophilic films more permeable
(Pascall & Lin, 2013).
The objective of this study is to develop an edible film incorporated with small
2 molecules to inhibit the growth of Campylobacter jejuni and Campylobacter coli in raw chicken & poultry products. An ideal film should have some characteristics such as good oxygen barrier property to prevent the growth of aerobic bacteria, good MIC value of small molecules when incorporated in the film, good mechanical property to wrap the poultry products as well as non-toxic, unflavored and colorless. Thus the type of edible films, type of antimicrobial agents, prevalent pathogenic bacteria, and methods of testing antimicrobial agents and films will be discussed in the following sections.
3 1.2 Types of Edible Film Material
Generally the main components of daily-consumed foods, such as proteins, carbohydrates and lipids can meet the requirements for the preparation of edible films.
As a rule of thumb, lipids are used to reduce water transmission, since lipids have good moisture barrier. However, they have low mechanical properties due to their hydrophobic structure and their inability to form cross linkages. Polysaccharides are used to control oxygen and other gas transmission, because they are hydrophilic and provide strong hydrogen bonding that can be used to cross-link with functional additives such as flavors, colors, and micronutrients. Protein films, on the other hand, have good mechanical stability so that when applied to fruits, they help to reduce injuries during transportation. These materials can be utilized individually or as mixed composite blends to form films. Table 1.1 summarizes the main types of edible films.
As shown in the table, alginate, carrageenan, cellulose and it derivatives, dextrin, pectin and starch are examples of polysaccharide films. Protein films can be made from various sources, such as corn, milk, soy, wheat and whey. Lipid based films such as waxes, glycerol esters, and resins are the oldest known edible film components, however, they are less widely used currently due to their susceptibility to oxidation and low mechanical strength.
4 Table 1.1 Possible Components of Edible Films
Types of film Examples
Protein 1) Collagen Films: Type I collagen is widely used as a biomaterial, and is the
most commercially successful edible protein films.
2) Gelatin Films: unique in forming a thermo-reversible substance with Tm
close to body temperature, which is significant in edible and
pharmaceutical applications.
3) Corn Zein Films: relatively hydrophobic thus has very good barrier
properties.
4) Wheat Gluten Films: is a water insoluble protein of wheat flour, which is a
mixture of polypeptide molecules, and considered to be globular proteins.
5) Soy Protein Films: has been extensively used as a food ingredient since it
contains high nutrition and excellent functional properties.
6) Casein Films: Transparent and flexible, but have poor water barrier
properties.
7) Mung Bean Protein Films: was found that the mechanical and water vapor
barrier properties are superior to other protein films such as casein, soy,
and gluten.
Polysaccharide 1) Starch: composed of amylose and amylopectin, is primarily derived from
cereal grains like corn.
2) Alginate: derived from seaweeds and possess good film-forming
properties.
3) Carrageenan: water-soluble polymers with a linear chain of partially
sulphated galactans.
4) Cellulose Derivatives: polysaccharides composed of linear chains of beta
glucosidic units with methyl, hydroxypropyl or carboxyl substituents.
5) Pectin: a group of plant-derived polysaccharides that appear to work well
with low moisture foods, but have poor moisture barriers. Continue
5 Table 1.1 Possible Components of Edible Films (Continue)
Polysaccharide 6) Chitin/Chitosan: an edible and biodegradable polymer derived from
chitin, the major organic skeletal substance from crustacean shells.
7) Gums: are differentiated into three groups: exudate gums (Arabic Gums);
the extractive gums (Guar Gums); and the microbial fermentation gums
(Xanthan Gums).
Lipid 1) Wax: Paraffin wax and Beeswax
2) Resins: Shellac resins, Rosin and its derivatives
3) Glycerol: acetylation of glycerol monosterate
(Thawien, 2012; Aruna and others, 2012; Subhas and others, 2014)
1.2.1 Protein-based edible film/coating.
Protein-based edible films have triggered interest in recent years because of their
advantages, including the use of edible packaging materials for individual packaging
of small portions of food, such as beans and nuts. In addition, they can be applied
between heterogeneous food at the interfaces between different layers of components
to prevent the transfer of inter-component moisture and solute migration in pizzas,
pies and candies, for example (Bryne et al., 2014). Besides, they can also function as
carriers for antimicrobial and antioxidant agents.
In its natural state, protein films can be divided into two groups, fibrous protein
films and globular protein films (Thawien, 2012). Fibrous protein chains are water
insoluble, fully extended, and associated closely with each other in parallel structures
via hydrogen bonding to form fibers (Figure.1.1a). Globular proteins are soluble in
water or acid based solutions. They fold into complicated spherical structures held
6 together by a combination of hydrogen, ionic, hydrophobic and covalent bonds
(Figure 1.1b). With regards to fibrous proteins, collagen has received the most attention as a protein-based film, while for globular proteins; examples are corn zein and whey protein.
Figure 1.1 The structural differences of fibrous protein and globular protein.
1.2.1.1 Collagen
Collagen is the main protein of connective tissue such as bone, hide, tendons cartilage and ligaments. Due to its biological properties and ready availability, which is unique among those of natural polymers, type I collagen is widely used as a biomaterial (Sisken et al., 1993). It is the most commercially successful edible protein film due to its biocompatible and non-toxic characteristics, and its structural, physical, chemical and immunological properties. It can be produced into a variety of forms,
7 and can be easily isolated and purified in large quantities (Hood, 1998).
1.2.1.2 Corn Zein
Corn Zein is the major protein in corn. Due to its high content of non-polar amino acids, it is hydrophobic and is thermoplastic in nature (Shukla & Cheryan.
2001). Corn Zein has excellent film-forming properties and can be used for the fabrication of biodegradable films. The formation of corn zein films is facilitated by the development of hydrophobic, hydrogen and limited disulfide bonds between zein chains in the film matrix (Gennadios et al., 1994), this however, results in the formation of brittle films that require the addition of plasticizers to enhance its flexibility. Its hydrophobicity characteristic enables good water vapor barrier property when compared to other edible films. It also shows the ability to reduce moisture and loss of firmness and delay color change in fresh fruit (Guilbert, 1986).
1.2.1.3 Whey Protein
Whey protein is a nutritional and highly functional protein. It is formed through the use of transglutaminase as a crosslinking agent (Mahmoud & Savello, 1993), and is shown to produce a transparent, bland, flexible, water-based edible film with excellent oxygen, aroma and oil barrier properties at low relative humidity (Miller &
Krochta, 1997). The most beneficial characteristic of whey protein edible films is their edibility and inherent biodegradability (Krochta, 2002), especially the latter feature, which is attractive to the food industry because it reduces the bio-burden on
8 the environment. However, to be of use in food packaging, the shelf life of the edible film should be longer than the shelf life of the packaged product (Krochta & De
Mulder-Johnston, 1997).
1.2.2 Polysaccharide-based edible film/coating
As shown in Table 1.1, polysaccharide films are made from starch, alginate, cellulose ethers, chitosan, carrageenan, or pectin. The great diversity of structural characteristics of polysaccharides is exhibited differences in its monosaccharide composition, linkage types and patterns, chain shapes and degree of polymerization, which influences hardness, crispiness, compactness, thickening quality, viscosity, adhesiveness, and gel forming ability. Typically, polysaccharide-based edible films have excellent gas permeability properties due to the hydrogen bonds formation between two hydrophilic subunits, thus enhancing the shelf life of the product without creating anaerobic conditions (Baldwin et al., 1995). They could also be used to extend the shelf life of muscle foods by preventing dehydration, oxidative rancidity, and surface browning (Nisperos-Carriedo, 1994). However, the hydrogen-bonding characteristic makes them poor barriers for water vapor.
1.2.2.1 Starch
Starch, is composed of amylose and amylopectin (Figure 1.2), and is primarily derived from cereal grains such as corn, wheat, potato tapioca, and rice. Starch is typically found as granules, which contain millions of amylopectin molecules
9 accompanied by even larger numbers of smaller amylose molecules (Whistler, 1985).
Amylose is responsible for the film forming capacity of starch (Claudia, 2005). Films with high amylose content are flexible; show low oxygen permeability, heat-sealable, oil resistant, but water-soluble. Starch-based films are odorless, tasteless, colorless, non-toxic, biologically absorbable, and resistant to the passage of oxygen (Krogars,
2003).
Figure 1.2 The structure of amylose and amylopectin
1.2.2.2 Carrageenan
Carrageenan is a water-soluble polymer with a linear chain of partially sulfated galactans, which plays a role in its film-forming ability. It is extracted from the cell walls of various red seaweeds (Rhodophyceae). Variations in the degree of sulfate groups present in its structure divide carrageenan into three types: kappa, iota, and
10 lambda carrageenan (Figure 1.3, 20%, 33%, and 40% w/w, respectively.).
The presence of hydroxyl and sulfate groups in the structure of carrageenan causes its hydrophilic nature. It is widely used as an agent for thickening and gelling in food and nonfood industries due to its water holding ability (Van de Velde et al.,
2002). Besides, kappa carageenans have the ability to form thermoreversible gels.
However, in the presence of a small amount of acid, this characteristic will be disrupted due to cross-link formation as a result of the presence of extra positive ions
(Park et al., 2001).
Figure 1.3 The structure of kappa, iota, and lambda carrageenan.
1.2.2.3 Cellulose Derivatives
Cellulose derivatives are polysaccharides composed of linear chains of beta-1,4 glucosidic units with methyl, hydroxypropyl or carboxyl substituents. Generally, there are four cellulose derivative forms used for edible films formation: Hydroxy Propyl
Cellulose (E463; HPC), Hydroxy Propyl Methyl Cellulose (E464; HPMC), Carboxy
11 Methyl Cellulose (E466; CMC), and Methyl Cellulose (E461; MC). The inherent hydrophilic nature of cellulose derivatives results in poor water vapor barriers and poor mechanical properties (Gennadios, 1997). Methods of enhancing the moisture barrier of these films would be by the incorporation of hydrophobic compounds, such as fatty acids into the cellulose ether matrix to develop a composite film (Morillon,
2002).
1.2.2.4 Chitin/Chitosan
Chitosan is an edible and biodegradable polymer derived from chitin by the process of deacetylation in the presence of an alkali (Figure 1.4). It is described in terms of the degree of deacetylation and average molecular weight. Chitosan has poor solubility in neutral solutions but soluble in acids such as acetic, citric and formic acids due to its cationic characteristic. It has lots of desirable properties including good oxygen and carbon dioxide permeabilities, film forming without additives, excellent mechanical properties and antimicrobial activity against bacteria, yeasts, and molds (Var Tiainen et al., 2004). The antimicrobial property of chitosan is based on
Figure 1.4 The structure of Chitin and Chitosan 12 the existence of a positive charge on the amino group and its attraction to other negatively charged polymers such as the membrane of microorganisms, cholesterol, and proteins (Muzzarelli, 1986). As an illustration, the minimum inhibitory concentration for chitosan against several microorganisms is provided in Table 1.2.
Besides its outstanding antimicrobial properties, chitosan also forms semi-permeable coatings, which can modify the internal atmosphere, thus delaying ripening and decreasing transpiration rates in fruits and vegetables (Sandford, 1989).
Table 1.2 Minimum inhibitory concentration (MIC) for chitosan against several microorganisms (Rejane et al., 2009)
13 1.2.3 Main Types of Lipid-based edible film/coating
Lipid compounds used as edible film consist of acetylated mono-glycerides, resins and natural wax. Among these compounds, paraffin wax and beeswax are most effective. The hydrophobic characteristic of lipid compounds provides excellent moisture barrier properties, however, due to their poor mechanical properties; they usually combined with other film forming agents like proteins or cellulose derivatives
(Debeaufort et al., 1993).
1.2.3.1 Waxes and Paraffin
Waxes have been used to retard desiccation of citrus fruits in China since the twelfth and thirteenth centuries. The Chinese noted that the waxes slowed water loss and caused fermentation (Hardenburg, 1967). Paraffin wax is derived from the distillate fraction of crude petroleum and consists of a mixture of solid hydrocarbon resulting from ethylene catalytic polymerization. It contains predominantly straight-chain hydrocarbons with an average chain length of 20 to 30 carbon atoms, as shown in Figure 1.5. Due to its characteristics such as non-reactive, non-toxic, good moisture barrier and colorless, it is permitted for use on raw fruits, vegetables and
Figure 1.5 The chemical structure of Paraffin wax
14 cheese since the 1930s (Kaplan, 1986). These coatings are the most efficient edible compounds blocking transport of moisture, reducing surface abrasion during handling of fruits and controlling soft scald formation in apples (Kester & Fennema, 1986).
1.2.3.2 Acetylated Glycerol Monostearate
Acetylation of glycerol monostearate by its reaction with acetic anhydride yields1-stearodiacetin. It is an emulsifier in which acetic acid is bound with monoglyceride, as shown in Figure 1.6. Acetylated monoglycerides (AMG) films display the exclusive characteristic of solidifying from the melting state to a flexible, wax-like solid. Elongation of the films can be as high as 800%, while most lipids in the solid state can be stretched to only 102%. The films are mainly used for poultry and meat cuts to retard moisture loss during storage (Bourtoom, 2008). Another application of AMG films is as an antioxidant carrier. When compared to whey protein isolate coating, an AMG coating makes some of the most effective natural antioxidants such as tocopherols migrate more freely to the surface due to the hydrophobic characteristic (Juan & John, 1997). However, AMG films are shown to have high oxygen permeability (Hoover & Nathan, 1981), therefore they did not provide protection against lipid oxidation in granulated roasted peanuts.
15 Figure 1.6. The chemical structure of Acetylated Monoglyceride
1.2.3.3 Shellac Resins
Shellac resins are secreted by the insect Laccifer lacca, and are composed of a complex mixture of aliphatic alicyclic hydroxyl acid polymers. It is not recognized as a “GRAS” substance by FDA and as such is only permitted as an indirect food additive and is mainly used in coatings for the pharmaceutical industry (Berg, et al.,
2012). Shellac resins are widely used for coating citrus and other fruits to enhance their surface glossiness thus decrease the prevalence of postharvest wilting. However, citrus with shellac resin coating typically has lower internal oxygen, higher internal
16 carbon dioxide, and higher ethanol content (an indication of off flavor) than citrus with wax coatings due to their differences in gas permeance and ability to block openings in the skin. (Bourtoom, 2008).
1.2.4 Composite edible film/coating
Edible films can be synthesized by blending polysaccharides, protein and lipids, which enables one to utilize the advantages of each class of film (Kester & Fennema,
1984). The combination could be proteins and carbohydrates, proteins and lipids, carbohydrates and lipids or synthetic or natural polymers. The aim is to improve the permeability or mechanical properties for specific purposes. The individual components of these composite films are blended in the form of an emulsion, suspension, or dispersion of the non-miscible constituents, or in successive layers, or in a solution in a common solvent. An example of a composite polymer widely used in food packaging is polyvinyl acetate. It is a nontoxic commercially available polymer prepared through emulsion polymerization and is incorporated with fungicides for protection of diverse foods or as a coating for pharmaceutical products
(Carmona-Ribeiro, 2013). For more than 50 years, techniques such as spraying and dip coating and encapsulation have been used in the pharmaceutical industries to incorporate bioactive agents with polymers. For example, an anionic copolymer based on methacrylic acid and methyl methacrylates was used for coating tablets and pills.
This coating was resistant to gastric juices but improved the protection of the tablets against moisture, light and oxygen under tropical conditions (Petereit, 2007). In
17 another example, composite polymer spheres with a sugar coating on the outside and edible polymer coating inside give them dual functionality to target and deliver drugs.
The sugar coating provides barrier to oxygen and gives taste to the tablet, while the edible polymer serves as a mechanism for delayed release of the drug. The polymer vesicles could be used to mimic a living cell or used as drug delivery vessels, and could also be used to convey drugs and biomolecules to injured or cancerous tissues in animals or humans (Schlaad, 2009).
18 1.3 Antimicrobial substance incorporated in the film
Antimicrobial (AM) agents have been found and proven to be effective against food-borne pathogens. It is reported that the effectiveness of AM packaging is greater than direct addition of AM agent onto food due to two factors. One is the lower release rate of the AM agent from the material to the food, thus enabling functionality over a longer period. The other factor is inactivation concerns (such as neutralization, hydrolysis, dilution) when directly added into the food. Functionality of AM agents in foods can be evaluated by conducting experiments with microorganisms as described by the spoilage index (Appendini, 2003). Food is considered spoil when it has a total microbial count of ≥ 107 CFU/g, and as such, studies on AM packaging have referred to a bacteria count of 107 CFU/g as a standard for shelf-life indication (Fung et al.,
1980). In this section of the literature review, five types of AM agents will be discussed; these will be essential oils, plant extracts, enzymes, bacteriocins, and synthetic small molecule compounds.
1.3.1 Essential Oils and Plant Extracts
It is well known that essential oils are rich in volatile terpenoids and phenolic particles with potential to inhibit most types of microorganisms (Solgi et al., 2009;
Burt, 2004; Tian et al., 2011; Solgi et al., 2010). The active components of plant essential oils that inhibit the growth of microorganisms do so, by causing damage to the cytoplasmic membrane, disrupting the proton motive force, electron flow, active transport and inhibition of protein synthesis (Omojate et al., 2014). Several
19 researchers have reported on the effectiveness of plant extract components in food packaging, as shown in Table.1.3.
Table.1.3 Antimicrobial incorporated into edible polymer film
Polymer Antimicrobials Target Microorganism References
Soy Edible Oregano Oil; S.aureus; Emiroglu et al., 2010 Films Thyme E.coli; E.coli O157:H7 E.coli O157:H7; Garlic Oil; S.aureus; Whey Protein Seydim & Sarikus, Oregano Oil; L.monocytogenes; Edible Films 2006 Rosemary Oil; L.plantarum; S.enteritidis; E.coli; Alginate-based S.typhimurium; Garlic Oil Yudi, et al., 2005 Edible Films S.aureus; B.cereus;
1.3.1.1 Grape Seed Extract
Grape Seed Extract received lots of interests due to its broad range of bioactive properties, including antimicrobial activity, radical-scavenging potential, and its ability to retard lipid oxidation (Ahn, et al., 2007; Mandic et al., 2008; Kulkarni, et al.,
2011). For example, linear low-density polyethylene (LLDPE) with 0.5% and 1% grape seed extract has extended the shelf life of beef stored at 3°C from 9 to 14 days
(Ha, et al., 2001). Besides, it also showed AM activities against L.monocytogenes by
1 log10 CFU/ml reduction after 1 hour incubation at 25°C when incorporated into soy protein edible film (Sivarooban, et al., 2008).
20 1.3.1.2 Oregano Oil
Cultivated in European countries, oregano plays a major role in world trade, because it is a popular herb in Mediterranean cooking (Autuono et al., 2000). The volatile oil of oregano has been used for respiratory disorders, indigestion, dental caries, rheumatoid arthritis and urinary tract disorders (Ertas, et al., 2005). Carvacrol is the major component of oregano and thus used as a food preservative (Veldhuizen et al., 2007). It is reported that polypropylene film incorporated with oregano essential oil successfully reduced the number of E.coli, Salmonella enterica, and
L.monocytogenes in salad by 1.4, 0.5 and 0.36 log10 CFU/ml, respectively after storage under 4°C for two days (Muriel-Galet et al., 2012). Carvacrol proved to be more effective against gram-negative bacteria since it affects the cell wall lipids of these classes of bacteria (Quattara et al., 1997).
1.3.1.3 Aqueous Garlic Extract
Garlic (Allium sativum) has traditional dietary and medicinal applications as an anti-infective agent (Lowson, 1998). Allicin and thiosulfinates are the main components in garlic and they have been shown to have antibiotic, anticancer, antioxidant, immune-modulatory, anti-inflammatory, and cardiovascular-protecting effects (Reuter et al., 1996). According to Iwakolun et al.’s research, the antibacterial activity of Aqueous Garlic Extract was characterized by inhibition zones of 20.2-22.7 mm for gram-positives and 19.8-24.5 mm for gram-negatives bacteria, and minimum inhibitory concentration ranges of 15.6-48.3 mg/ml and 22.9-37.2 mg/ml, respectively
21 (Iwalokun et al., 2004). The results of that study supported the use of garlic in natural products and herbal remedies.
1.3.2 Enzyme
Antimicrobial enzymes play a critical role in defending living organisms from bacterial attack. With the increasing prevalence of antibiotic resistance, these enzymes are under intense investigation. An ideal antimicrobial enzyme should be able to directly attack the microorganism, interfere with biofilm formation, destroy the biofilm, and catalyze reactions which result in the production of antimicrobial compounds (Barbara, et al., 2013). Typically, antimicrobial enzymes function by degrading microbial DNA, polysaccharides, proteins of microorganisms, and the prevention of biofilm formation. Advances in synthetic biology, enzyme engineering and whole DNA-Sequencing technologies have shown great potential for use in the development of effective antimicrobial and anti-biofilm enzymes.
1.3.2.1 Proteolytic enzymes
Proteases are protein-hydrolyzing enzymes, which are classified into two major groups, exopeptidases and endopeptidases.
Subtilisins and Lysostaphin are representatives of exopeptidases and endopeptidases enzymes, respectively. Produced by Bacillus spp., subtilisins are serine proteases that cleave proteins in which serine serves as the nucleophilic amino acid (Molobela et al., 2010). They hydrolyze adhesins, which are a type of virulence
22 factor that facilitate adhension between cells or cell to surfaces, hence preventing co-aggregation of microorganisms, which could potentially form biofilm (Leroy et al.,
2008). Lysostaphin, first isolated by Schindler and Schuhardt, exhibits three different catalytic activities: glycyl-glycine endopeptidase, endo-beta-N-acetyl gluycosamidase and N-acetyl muramyl-L-alanine amidase activity (Recsei et al., 1987). It cleaves
Staphylococci spp.’s cell walls on the third and fourth glycine residues of the pentaglycine cross-bridge, as shown in Figure 1.7 (Kumar, 2008; Kumar et al., 2008).
This enzyme is attracting significant interest for the control of Multidrug Resistant
Staphylococcus Aureus (MRSA). In the dairy industry, lysostaphin prevents mastitis
(a serious food-borne disease leading to $4 billion loss per year in U.S. and Europe) by inserting lysostaphin related genes into cows’ cells. This causes the expression of
Figure 1.7 Simplified scheme of the hydrolysis of bacterial cell wall by lysostaphin
23 bacterial derived lysostaphin in the milk, thus inhibiting the growth of mastitis causing Staphylococcus spp. (Hermoso et al., 2007).
1.3.2.2 Polysaccharide-degrading enzymes
Among the polysaccharide hydrolyzing enzymes, amylases, alginate lysases and lysozymes are the most commonly developed.
Lysozyme is derived from chicken egg white, and attacks gram-positive bacteria and some gram-negatives by hydrolyzing 1,4-beta-linkages in the cell wall (Proctor et al., 1988). It is used to increase the shelf life of many fresh foods such as fruits, vegetables, meat, and cheese (Cunningham et al., 1991). When immobilized in polyvinyl alcohol films, it can inhibit food-spoiling or pathogenic microorganisms such as Kelbsiella pneumonia, Bacillus anthracis, Bacillus subtilis, and
Staphylococcus aureus (Bang et al., 2011).
Alginate lyase is another antimicrobial enzyme, which cleaves beta-glycosidic bonds of bacterial alginate polymer (Alkawash et al., 2006). It is found in algae, invertebrates and microorganisms. The antimicrobial mechanism consists of three steps: 1) removing the negative charge on the carboxylate anion; 2) abstraction of the
proton on the C5 carbon; 3) beta-elimination of the 4-O-glycosidic bond, as shown in
Figure 1.8. It has been used to control Pseudomonas aeruginosa, an organism known to colonize the respiratory tracts of patients with cystic fibriosis (Gacesa et al., 1987).
Amylase inhibits the biofilm formation of S.aureus (Oulahal-Lagsir et al., 2003).
It can also be used in combination treatments with proteases to enhance the removal
24 of biofilms and prevent bacterial growth, especially for Pseudomonas fluorescens
(Leroy et al., 2008).
Figure 1.8 Alginate lyase catalyzes the hydrolysis of alginate through beta-elimination.
1.3.2.3 Oxidative enzymes
Superoxide anions are considered “primary” reactive oxygen species. By dismutating into hydrogen peroxide, the peroxidases can be used to destroy invading pathogens by oxidizing halides and isocyanate to produce more potent antimicrobial compounds (Leskovac et al., 2005). Furthermore, hydrogen peroxide itself, which is dismutated from superoxide anions, can directly react with metals to generate hydroxyl free radicals, which are stronger antimicrobial compounds. The procedure is shown in Figure 1.9. Glucose oxidase uses glucose as an electron donor substrate and
reduces oxygen, resulting in the production of H2O2, which in turn is used by lacto-peroxidase to oxidize halides to more potent antimicrobial compounds.
25 Figure 1.9 Glucose oxidase-lactoperoxidase antimicrobial system.
1.3.2.4 Anti-quorum sensing enzymes.
Bacteria use quorum sensing molecules to regulate various physiological activities, including virulence, competence, conjugation, antibiotic and bacteriocin production, and spore and biofilm formation (Pearson et al., 1999). Among the quorum-sensing molecules, acyl homoserine lactones (AHLs) are the most commonly used molecules in more than 50 bacterial species (Fernandes et al., 2006).
Anti-quorum sensing enzymes such as lactonases can hydrolyze the ester bond of the homoserine lactone ring of acylated homoserine lactones, thus preventing AHLs from binding to the target regulators. By inhibiting the virulence of pathogenic microorganisms, these enzymes play a significant role in biotechnology and agriculture industries.
26 1.3.3 Bacteriocins
Bacteriocins generated by bacteria are gaining in popularity due to their heat-stability and acidic tolerance. Bacteriocins are metabolic by-products produced by the bacterial defense system and the objective is to inhibit the growth of other strains of bacteria in the environment. Bacteriocins protect the host cell by permeabilizing the target cell membrane, resulting in an irreversible leakage of cellular material and consequently leading to cell death (Nissen-Meyer et al., 1997).
The activity spectrum of a bacteriocin can be narrow and confined to inhibition of closely related species, or it can be relatively broad and include many different bacterial species (Klaenhammer, 1993). Highly acceptable bacteriocins are those from lactic acid bacteria, which serve a key role in food fermentation.
1.3.3.1 Bacteriocin from Eukaryotes
Bacteriocins from eukaryotes show varying degrees of toxicity. Table 1.4 provides examples of many bacteriocins produced by eukaryotic organisms. As shown in Table 1.4, some bacteriocins act against a specific group of competing organisms, while others have broad-spectrum antimicrobial efficacy and serve as a general defense mechanism.
27 Table 1.4 Bacteriocins of eukaryotic organisms
(Oren et al., 1996; Marri et al., 1996; Helmerhorst et al., 1997; Goulard et al., 1995)
1.3.3.2 Bacteriocin from Prokaryotes
Bacteriocins were first characterized in gram-negative bacteria. The colicins produced by E.coli attracted intense attention in that colicins constitute a diverse group of antibacterial proteins, which kill closely related bacteria not only by inhibiting cell wall synthesis, but also inhibiting RNase or DNase activity (Lazdunski,
1988). As for gram-positive bacteria, the lactic acid bacteria have been studied for production of bacteriocins with food applications, as shown in Table 1.5.
28 Table 1.5 Examples of bacteriocins isolated from foods
(Gomez et al., 1997; Miteva et al., 1998; Bennik et al., 1998; Yildirim et al., 1998, Franz et al., 1998; Herbin et al., 1997; Cai et al., 1997; Ennahar et al., 1996; Jack et al., 1996; Marsnier-Patin et al., 1996)
1.3.4 Inorganic nanoparticles
Since inorganic nanoparticles have good stability to withstand harsh process conditions such as high pressure or temperature during the process, the utilization of inorganic nanoparticles as AM agents has recently gain popularity. The most
extensively studied particles for AM purposes have been titanium dioxide (TiO2) and zinc oxide (ZnO).
1.3.4.1 Titanium dioxide as an AM agent
Titanium dioxide is non-toxic and approved by FDA for the use in foods, drugs,
and food contact materials. To inactivate the growth of microorganisms, TiO2 generates hydroxyl radicals and reactive oxygen species via light reaction, which can
29 inactivate microorganisms by oxidizing the polyunsaturated phospholipids component of the cell membrane (Chawengkijwanich et al., 2008). When incorporated in
polyethylene film, TiO2 films exhibited inhibition percentage of 89.3% for E.coli and
95.2% for S.aureus, respectively (Xing et al., 2012).
1.3.4.2 Zinc Oxide as an AM agent
Applications of ZnO nanoparticle coating systems are of recent interest because of their AM activity towards both the gram-negative and gram-positive bacteria. For example, ZnO-coated PVC films showed significant AM effect against E.coli and
S.aureus compared with a control blank film (Li et al., 2010). Results also indicated that the ZnO nanoparticles adhered well to the PVC film, and particles showed little effect on the tensile strength and elongation at break of the PVC material. Therefore, the PVC film coated with nano-ZnO particles has a good potential to be used as an active coating system for food packaging.
1.3.5 Synthesized small organic molecules
Small organic molecules have always been of interest to chemists and biochemists due to their capability of exerting powerful effects on the functions of macromolecules that comprise living systems (Marians et al., 1997). As one of the most important therapeutic agents, small organic molecules have benefits, such as improved stability over peptides in oral administration, synthetic accessibility, and optimization convenience for compound bioactivity when comparing with
30 macromolecules (Pathania et al., 2009). Synthesized small molecules are generally
used to affect the growth of bacteria in two ways: by killing the bacteria, or inhibiting
the growth of the bacteria. On the basis of the mechanism of their action, small
molecules are typically classified as four types: 1) those that block specific steps in
folic acid metabolism; 2) those that affect bacterial cell-wall biosynthesis; 3) those
that interfere with protein biosynthesis; 4) those that affect nucleic acid biosynthesis
and transcription (Williams et al., 2002).
1.3.5.1 Inhibitors of Folic Acid Metabolism
Folic acid is a key cofactor required for the biosynthesis of many cellular
components in all living organisms. It is an intermediate in the transfer of methyl,
Figure 1.10 The biosynthesis pathway of Folates
31 formyl, and other single-carbon fragments in the biosynthesis of purine nucleotides
(Finch et al., 2003). As for microorganisms, they must synthesize folates through folate synthetic pathway showed in Figure 1.10, while for mammals, folates are not necessary, thus folate synthetic pathway is attractive for antimicrobial drug design
(Bermingham et al., 2002). Inhibition of folate biosynthesis results in the inability of the bacteria to grow since folate acid is indispensable for further nucleic acid biosynthesis.
1.3.5.2 Inhibitors of Cell Wall Biosynthesis
The bacterial cell wall serves several functions, these are: 1) to maintain the cell’s integrity; 2) to provide a semi-permeable barrier; 3) to counteract changes in the osmotic pressure within the cell environment; 4) to prevent digestion by host enzymes
(Williams et al., 2002). When comparing with mammalian cells, all bacterial cell walls differ dramatically in structure and function, and enzymes found in bacterial cell walls are not found in mammalian cells. Consequently, it provides the possibility for antimicrobial drugs design. Currently, there are two main types of drugs that target bacterial cell wall. These are: beta-lactams (penicillins, cephalosporins, carbapenems, and monobactams), and glycopeptides (vancomycin) (Williams et al., 2002). Though their target sites and mechanism of action are different, they all kill bacteria by disrupting the normal function of the cell wall, leading to cell lysis.
32 1.3.5.3 Inhibitors of Protein Biosynthesis
This class of molecules exerts their effects by inhibiting ribosomally mediated protein biosynthesis. The ribosomes of bacteria consist of two subunits (30S and 50S), which contain suitable structural differences from eukaryotic ribosomes, thus they allow molecules to be targeted for AM purposes. The molecules do not bind to nor interfere with the function of human 80S ribosomal subunits at normal doses.
However, the molecules can interfere with bacterial protein biosynthesis by preventing repair, cellular growth, and the reproduction process (William et al., 2002).
The major classes of protein synthesis inhibitors are aminoglycosides, tetracyclines, and macrolides, respectively.
1.3.5.4 Inhibitors of Nucleic Acid Biosynthesis
As for inhibitors of nucleic acid biosynthesis, there are two main targets associated with them; these are DNA gyrase and topoisomerase IV. DNA gyrase is responsible for introducing negative supercoils into DNA, hence allowing superhelical tension ahead of the polymerase to be released to continue the replication process (Yoshida et al., 1993). As for topoisomerase IV, its function is similar to
DNA gyrase and also allows chromosome separation during cell division in gram-positive bacteria (Marians et al., 1997). As for inhibitors, fluoroquinolones, bind to DNA gyrase and topoisomerase IV interferes with cell growth and division, leading to cell death. However, these compounds are non-toxic to mammalian cells since they cannot bind to topoisomerase II found in eukaryotes (Drlica et al., 1997).
33 1.4 Foodborne diseases in Meat & Poultry Industry
Foodborne illnesses can result in major public health implications in the U.S. and around the world. According to recently published CDC (Centers for Disease Control and Prevention) data (Table 1.6), foodborne diseases account for approximately 8 million illnesses, and 9,000 deaths each year in the U.S. alone (CDC, 2011). The epidemiology of foodborne diseases is rapidly changing as newly recognized pathogens emerge and well-studied pathogens increase in prevalence or associate with new food vehicles, as shown in Table 1.7 (CDC, 2013). Apart from acute gastroenteritis, some foodborne diseases may cause chronic illness or disability.
Listeriosis, for instance, can cause miscarriages or meningitis in patients with pre-existing chronic diseases (Schuchat et al., 1991). As meat and meat products are the major source of foodborne infection and the most important link between food-producing animal and humans, the study of foodborne pathogens isolated from meat and poultry is indispensable.
Table 1.6 Estimated annual number of domestically acquired, foodborne illnesses, hospitalizations, and deaths due to 31 foodborne pathogens and unspecified agents in U.S.* Estimated Estimated Estimated annual Foodborne annual number annual % number of % % Agents of illnesses (in number of hospitalizations million) deaths 31 known 9.4 55,961 1,351 20 44 44 pathogens (6.6–12.7) (39,534–75,741) (712–2,268) Unspecified 71,878 1,686 38.4 (19.8–61.2) 80 56 56 agents (9,924–157,340) (369–3,338) 47.8 127,839 3,037 Total 100 100 100 (28.7–71.1) (62,529–215,562) (1,492–4,983) * Data obtained from CDC: http://www.cdc.gov/foodborneburden/2011-foodborne-estimat es.html#annual
34 Table 1.7 Selected outbreaks in Ohio in 2013, associated with emerging foodborne pathogens and factors for their emergence.* Genus Species Serotype or Location of Total Total Food Genotype Consumption illness Death Vehicle C.jejuni Private Home 21 0 Unknown Salmonella Enteritidis Restaurant 6 0 enterica Sit-down dining Campylobacter Picnic 6 0 spp. Salmonella Typhimurium Private Home; 32 0 Prime rib and enterica Restaurant aus jus Sit-down dining Salmonella Braenderup Picnic; private 5 0 enterica home Cryptosporidium Private home 8 0 Apple cider, unpasteurized Listeria Private Home; 6 1 Cheese monocytogenes Restaurant Sit-down dining E.coli, Shiga O157:H7 5 0 toxin-producing E.coli, Shiga O26 26 0 Lettuce toxin-producing Clostridium Workplace 7 0 Sandwich; perfringens unspecified Bacillus cereus Other 5 0 Landjaeger Salmonella Lomalinda Restaurant 2 0 Egg, ground enterica Sit-down dining beef Salmonella Heidelberg Private Home; 2 0 Chicken and enterica Restaurant noodles. Sit-down dining Staphylococcus Church, temple, 33 0 Chicken and spp. religious location barley Clostridium Private Home; 2 0 Chicken, perfringens; Restaurant buffalo wings Norovirus Sit-down dining C.jejuni Private home 2 0 Milk, whole milk, unpasteurized Clostridium Caterer 17 0 Roast beef perfringens *Data obtained from CDC website: http://www.cdc.gov/foodborneoutbreaks/Default.aspx
35 1.4.1 Main Types of Foodborne Pathogens
According to the statistical data from CDC, five pathogens account for over 90% of estimated food-related deaths: Samonella, Listeria, Campylobacter, E.coli
O157:H7 and Vibrio (CDC, 2011). Although many of these diseases result in a self-limiting diarrheal illness in humans, severe invasive diseases or prolonged illnesses in immune-compromised individuals can occur and may require antimicrobial therapy. Below are the details of above-mentioned foodborne bacterial pathogens.
1.4.1.1 Salmonella Enterica
The genus Salmonella currently includes more than 2400 different serotypes.
Salmonella species are unique in the environment and can colonize and cause disease in a variety of animals. Salmonellosis, caused by non-typhoidal Salmonella strains, typically results in a self-limiting diarrhea that do not need antimicrobial therapy, while in some rare cases, the infections of Salmonella Enterica can lead to life-threatening systematic syndrome which require effective chemotherapy (Lee et al.,
1994). For Salmonella spp., in some cases, a rapid spread through the animal production systems seems to have occurred at a global level in 1980s (Rodrigue et al.,
1990). It is reported that S.Enteritidis appeared simultaneously around most of
European countries and U.S., and also spread into the poultry production systems of developing countries later in the 1990s (Matope et al., 1998)
36 1.4.1.2 Campylobacter jejuni & Camylobacter coli
Campylobacters are thin, curved, motile gram-negative rods. They are generally micro-aerophilic, though some strains are aerobic and anaerobic. Currently, campylobacters are recognized as the leading cause of foodborne gastroenteritis in the
U.S. and one of the most frequent causes of acute bacterial enteritis worldwide (Mead et al., 1999). Gastroenteritis caused by Campylobacter is an acute diarrheal disease that typically causes high fever, abdominal cramping, and diarrhea that last from several days to more than one week. It is to be noted that C.jejuni, and C.coli
(clinically indistinguishable) are the most common species associated with diarrheal illness, causing more than 95% of Campylobacter enteritis (Harris et al., 1986). The reports of campylobacteriosis cases have been continuously increasing in many parts of the world，as proved by statistical data from Texas Department of State Health
Services shown in Figure 1.11. Most infections are sporadic single cases resulting from the consumption of contaminated food, milk or uncooked and mishandled poultry (Friedman et al., 2000).
Campylobacteriosis Cases and Incidence Rates in Texas, 2001-2014 15 3000
Incidence Rates Reported Cases
10 2000
5 1000 100,000 Reported Cases 0 0 Incidence Rates per 2002 2004 2006 2008 2010 2012 2014 Year
Figure 1.11 Campylobacteriosis Cases and Incidence Rates in Texas. Data obtained
from Texas, Department of State Health Services
37 1.4.1.3 E.coli O157:H7
Shiga-toxin-producing E.coli (STEC) was first recognized as an emerging human pathogen in 1982 when E.coli O157:H7 was implicated in two outbreaks of hemorrhagic colitis associated with consumption of uncooked beef (Wells et al.,
1983). The CDC estimates that E.coli O157:H7 causes approximately 73,000 illness and 60 deaths each year in the U.S., in which 85% of these cases are attributed to foodborne transmission. Human infection with STEC can lead to non-bloody diarrhea or bloody diarrhea, or more serious and fatal syndrome such as hemorrhagic colitis and hemolytic uremic syndrome. It is proved that the most important virulence factors associated with STEC infection are Shiga toxins (stx1, stx2 or variants) (Schmidt et al., 1999).
1.4.1.4 Vibrio vulnificus
Vibrio vulnificus is a gram-negative bacterium commonly found in estuarine and coastal habitats throughout the northern Gulf of Mexico. This species is an opportunistic human pathogen that can cause primary septicemia, wound infection and gastroenteritis (Strom et al., 2000). Comparing with gastroenteritis, primary septicemia is the most common and severe syndrome caused by V.vulnificus, with mortality rate of more than 50% (Blake et al., 1979). Most reported cases revealed that consumption of raw shellfish and eastern oyster is the main cause of infections
(Strom et al., 2000). Besides, V.vulnificus can produce severe skin and soft tissue infections in patients with pre-existing wounds who come in contact with the
38 bacterium via seawater or by handling seafood (Howard et al., 1988).
1.4.1.5 Listeria monocytogenes
Many studies indicated that Listeria monocytogenes grows well at refrigeration temperatures and with minimal nutrients, and is able to survive and even grow in plants, soil and water (Schuchat et al., 1991). The foodborne transmission has been recognized as a major source of human listeriosis since 1982, though the first reported human listeriosis was in 1929 (Rocourt et al., 1997). The widespread nature of
L.monocytogenes allows easy access to food products during various phases of production, processing, manufacturing, and distribution, thus it has been found in lots of food products, including fresh vegetables, raw milk, raw meats, and eggs. Many illnesses are associated with refrigerated processed foods (ready-to-eat) consumed without prior cooking or reheating. The incidence of listeriosis has increased over the past two decades throughout the world. It is estimated by CDC that in the U.S., there are 2500 cases with 500 deaths attributed to listeriosis annually, mostly involving pregnant women, newborn babies, the elderly, and immune-compromised people. The spectrum of listeriosis is broad, ranging from asymptomatic infection and flu-like symptoms, to miscarriage, stillbirth, and meningitis (Robert R., 2003).
1.4.2 Antimicrobial resistance of foodborne pathogens
Antimicrobials are used for the control and treatment of bacterial associated infectious diseases as well as for growth purposes. However, it is inevitable that the
39 use of antimicrobials in animals and humans can select for resistant bacterial populations. As a consequence, zoonotic foodborne bacterial pathogens can become resistant to antimicrobials and transmit to humans as food contaminants. Among the antimicrobial resistant pathogens, Salmonella enterica serovar Typhimurium DT104 and Campylobacter jejuni are the most representatives, and they are becoming increasingly prominent in recent years (CDC, 2011). Compared to patients with susceptible infections, patients with antimicrobial-resistant infections are more likely to require hospitalization and to be hospitalized for longer periods.
1.4.2.1 Salmonella
The multidrug-resistant phenotypes have been increasingly described among
Salmonella species. For example, the ampicillin resistance in Salmonella species increased from 8 to 44%, tetracycline resistance from 1 to 42%, chloramphenicol resistance from 1.7 to 26%, and nalidixic acid resistance from 0.1 to 11%, respectively, according to a 7-year study in Spain (Prats et al., 2000). In the case of life-threatening salmonellosis, fluoroquinolones and expanded-spectrum cephalosporins are the choice against multidrug-resistant strains but increasing reports describing decreased efficacy of these AM agents among Salmonella species (Fey et al., 2000). The emergence and spread of multidrug-resistant Salmonella enterica serovar Typhimurium DT104 strain is the main cause of decreased efficacy of many antimicrobial agents (Threlfall et al., 2000). Threlfall et al.’s research revealed that
Salmonella enterica serovar Typhimurium DT104 isolates showed a unique
40 chromosomal gene cluster that encodes for the complete spectrum of the ACSSuT
(Ampicillin, Chloramphenicol/florphenicol, Streptomycin, Sulfonamides, and
Tetracycline) resistance phenotype. It is not known whether the spread of DT104 is as a result of the use of antimicrobial in the animal production environment or via clonal dissemination of pathogen, or both, thus it still remains challenging and limiting their negative effects in veterinary and human safety is imperative.
1.4.2.2 Campylobacter
Campylobacter jejuni/coli are generally susceptible to a variety of antimicrobial agents, however, increasing reports have shown that some species are resistant to drugs. A Canadian study indicated that resistance to tetracycline increased in C.jejuni from 19.1% to 55.7% from 1985 to 1995, respectively (Gaudreau et al., 1998).
Among the C.jejuni isolates of human origin tested through the U.S. National
Antimicrobial Resistance Monitoring System (NARMS), 54% were resistant to one or more antimicrobial agents, 20% were resistant to two or more agents. While among the C.coli isolates, 50% were resistant to one or more antimicrobials, and 35% were resistant to two or more agents, respectively. The most common resistance phenotypes observed were to tetracycline (46%), nalidixic acid (20%), and ciprofloxacin (18%). ((http://www.cdc.gov/narms/annuals.htm). The high level of fluoroquinolone-resistant Campylobacter spp. in humans and poultry has prompted the Center for Veterinary Medicine at the FDA to announce a proposal to withdraw approval of the new animal drug application for use of the fluoroquinolones in poultry
41 (Ruiz et al., 1998).
1.4.3 Prevention and Control
The prevention and control of foodborne disease depends on Good
Manufacturing Practices (GMP) of food production, including the handling of raw ingredients and the preparation of finished products. If not, hazards can be introduced at any point from the farm to the table. The introduction of the Hazard Analysis
Critical Control Point (HACCP) system greatly improved hygiene control in processing plants. Such programs require food industries to identify points in food production where contamination may occur and target resources toward processes that reduce or eliminate foodborne hazards (Goodfellow, 1995). However, infections cannot be fully eliminated since young animals or poultry are highly susceptible to pathogens. Milner and Shaffer indicated that the infective dose of S.Typhimurium for one-day-old birds with an oral administration was as low as 10 CFUs (Milner et al.,
1952). This deficiency could be overcome by oral administration of a saline suspension with mature micro-flora from adult birds. In this way, adult-type microflora would be established in young birds and thus prevent them from pathogenic infections by the phenomenon known as “competitive exclusion” (Rantala et al., 1973).
42 1.5 The Methods of Antimicrobial Agents Testing
The performance of antimicrobial susceptibility tested by a microbiology lab is crucial for confirmation of the efficacy of the compound, or to detect resistance in individual isolated microbial strains. The most widely used testing methods include broth micro-dilution or rapid automated instrument methods (Recio et al., 1989). Disk diffusion and gradient diffusion methods provide flexibility at reasonable cost compared with automated methods. Each method has strengths and weaknesses, including organisms that may be accurately tested or not. The test system should ideally be simple, rapid, reproducible, and inexpensive and maximize high sample throughput in order to cope with a varied number of extracts and fractions (Hadacek et al., 2000).
Currently, methods for testing antimicrobial activity of AM agents fall into three groups, including bioautographic, diffusion, and dilution methods, in which the first two methods are known as qualitative techniques since these methods can only give an idea of the presence or absence of antimicrobial agent with activity. However, dilution methods are considered quantitative assays since they determine the minimal inhibitory concentration of the antimicrobial compound (Vanden Berghe et al., 1991).
In general current testing methods provide accurate detection of common antimicrobial resistance mechanisms.
43 1.5.1 Commonly Used Testing Methods
1.5.1.1 Broth Dilution Test
The broth dilution test is one of the earliest antimicrobial susceptibility testing methods (Ericsson et al., 1971). The test involves preparing two-fold dilutions of the
AM agent, (e.g. 1000ppm, 500ppm, 250ppm, 125ppm) in a liquid growth medium dispensed in test tubes, which are inoculated with a standardized bacterial suspension of 103 to 106 CFU/ml. Following overnight incubation at a desired temperature, the tubes are examined for visible bacterial growth as proved by turbidity (Jorgensen et al., 2007). The lowest concentration of the AM agent that prevented growth represented the minimal inhibitory concentration (MIC). Precision of this method is considered to be plus or minus one two-fold concentration, based on numerous practices of manually preparing serial dilutions of the AM agent (Balows, 1972).
The use of small, disposable, plastic “micro-dilution trays” has made broth dilution test practical and popular (Figure 1.12). Micro-dilution panels are typically prepared using dispensing instruments that aliquot precise volumes of pre-weighed and diluted AM agents in broth into the individual wells of the trays. Inoculation of bacterial strains is accomplished by a disposable device that transfers 0.01 to 0.05 mL of standardized bacterial suspension into each well of the micro-dilution tray.
Following incubation, MICs are determined using a manual or automated viewing device for inspection of each well. Broth Dilution Test can be used to generate quantitative results, and is reproducible and convenient by having pre-prepared panels.
However, due to the fact that most broth micro dilution antimicrobial test panels are
44 prepared commercially, this methodology is less flexible than agar dilution or disk diffusion in adjusting to the changing needs of the surveillance/monitoring program
(Clinical and Laboratory Standards Institute, 2009).
Figure 1.12 A broth micro-dilution susceptibility panel containing 96 reagent wells and a disposable tray inoculator
1.5.1.2 Antimicrobial Gradient Method
The antimicrobial gradient diffusion method uses the principle of establishing an antimicrobial concentration gradient in an agar medium as a mean of determining efficacy. Typically a plastic test strip with dried AM agent concentration gradient underside and marked concentration reading on the surface is placed on the surface of agar plate inoculated with a standardized organism suspension. After overnight incubation at a desired temperature, the test is read by viewing the strip from the top of the plate. The MIC is determined by the intersection of the organism growth with the strip as measure using the scale inscribed on the strip, as shown in Figure 1.13.
45 This method is greatly applied to situations in which an MIC for only 1 or 2 AM agents is needed or when a fastidious organism requires enriched medium (Huang et al., 1992). Generally, these results have correlated well with MICs generated by broth dilution method. However, systematic biases toward higher or lower MICs exist when testing certain organism-antimicrobial agent combinations (Jorgensen et al., 1994).
Figure 1.13 S.aureus isolate tested by Antimicrobial Gradient Diffusion method with antibiotics on Mueller-Hinton agar.
1.5.1.3 Disk Diffusion Test
Disk diffusion test is simple and practical thus has been well standardized. The test is performed by applying a bacterial inoculum of approximately 1.0-2.0×108
CFU/ml to the surface of agar plate. The commercially prepared, fixed concentration, paper antimicrobial disks are placed on the inoculated agar surface, as shown in
Figure 1.14. The plates are incubated for 24 hours at a desired temperature. The
46 zones of growth inhibition around each of the antimicrobial disk are measured to the nearest millimeter. The susceptibility of the isolated strains and diffusion rate of the antimicrobial molecule are interpreted using the criteria published by the Clinical and
Laboratory Standards Institute (CLSI) or those included in the FDA approved product inserts for the disks (Clinical and Laboratory Standards Institute, 2009). Results of disk diffusion test are qualitative, in that the susceptibility is derived from the test rather than MIC. The advantages of the disk diffusion method are the convenience, which does not need any special equipment, the flexibility in selection of disks for testing, and economy. The downsides of this method are lack of mechanization or automation of the test (Korgenski et al., 1998).
Figure 1.14 A disk diffusion test with an isolate of E.coli from a urine culture.
1.5.1.4 Automated Instrument Systems
The usage of instruments can standardize the reading of end points and often
47 produce susceptibility test results in a shorter period than manual readings. This is so because sensitive optical systems allow detection of subtle changes in bacterial growth. The MicroScan WalkAway instrument (Siemens Healthcare Diagnostics) is a large self-contained incubator/reader device that can incubate and analyze 40-96 micro-dilution trays in one cycle (Figure 1.15). The instrument utilizes standard size micro-dilution trays that could be inoculated manually prior to placing them in the incubator slots in the instrument. The instrument incubates the trays for the appropriate period, and then examines them periodically with either a photometer or fluorometer to measure the bacterial growth curve (Richter et al., 2007). These automated Instrument systems can result in substantial direct cost savings, and are more flexible than manual test methods and produce results in a shorter time (Doern et al., 1994).
Figure 1.15 The MicroScan WalkAway instrument manufactured by Siemens Healthcare Diagnostics.
48 1.5.2 Interpretation of Susceptibility Test Results.
The results of susceptibility tests must be properly interpreted before being used as part of a report to someone else. Optimal interpretation of MICs requires knowledge of the pharmacokinetics of the AM agent in humans, and information on the likely success of the AM agent in inhibiting target bacteria at various body sites
(Clinical and Laboratory Standards Institute, 2008). Both MIC values and disk diffusion zone diameters must be interpreted using a table of values that relate to proven clinical efficacies of AM agents and for various bacterial species (Clinical and
Laboratory Standards Institute, 2008).
The CLSI zone size and MIC interpretive criteria are established by analysis of three kinds of data. These are: 1) Microbiologic data, which include a comparison of
MICs and zone sizes on a large number of bacterial strains, including those with known mechanisms of resistance that have been defined either phenotypically or genotypically; 2) Pharmacokinetic and pharmacodynamics data; 3) Clinical studies results (including comparisons of MIC and zone diameter with microbiological inhibition and clinical efficacy) obtained during studies before FDA approval and marketing of an AM agent (Clinical and Laboratory Standards Institute, 2009).
1.5.3 Acceptable Accuracy of Susceptibility Testing Method
When evaluating the accuracy of various susceptibility testing methods as compared to standard reference methods, the terms “very major” and “major errors” have been used to describe false-susceptible or false-resistant results. It is
49 indispensable to examine a representative number of strains that are resistant to various drugs for the evaluation of new susceptibility testing methods (Jorgensen,
1993). The FDA requires that very major errors of a test device should be <1.5% for individual species/drug comparisons, major errors should be within 3%, and an overall essential MIC agreement of >90% of device MICs of a CLSI reference MIC
(U.S. Food and Drug and Administration, 2003). The emergence of new antimicrobial resistance mechanisms, including some that may be difficult to detect requires that the performance of susceptibility devices be frequently assessed and updated. In some cases, it is necessary to introduce special ancillary testing methods to supplement routine testing by a commercial instrument system (Clinical and Laboratory Standards
Institute, 2009).
50 1.6 The Methods of Characterizing Edible Film
Knowledge of an edible film’s characteristics is indispensable for its application in the food industry. Packaging material, plasticizers, as well as incorporated substance determine the functions and compatibility with the intended product.
Besides, edible films also exert their influence on packed product in the form of mouth feel, appearance, and flavor. An ideal edible film should have the following characteristics (Milda et al., 2009):
1) Contain no toxic, allergic and non-digestible components.
2) Provide structural stability and prevent mechanical damage during
transportation, handling and display.
3) Have good adhesion to the surface of the food to be protected providing
uniform coverage.
4) Control water migration both in and out of protected food to maintain
desired moisture content.
5) Provide semi-permeability to maintain internal equilibrium of gases
involved in aerobic and anaerobic respiration, thus retarding senescence.
6) Prevent loss or uptake of components that stabilize aroma, flavor, nutritional,
and organoleptic characteristics necessary for consumer acceptance while
not adversely altering the taste or appearance.
7) Provide biochemical properties while protecting against contamination, pest
infestation, microbe proliferation, and other types of decay.
8) Maintain or enhance aesthetics and sensory attributes of product.
51 9) Serve as carrier for desirable additives such as flavor, fragrance, coloring,
nutrients, and vitamins.
10) Be easily manufactured and economically viable.
Typically the characteristics of edible film include desirable barrier property, mechanical strength, optical, and thermal properties, to the intended food.
1.6.1 Barrier Properties
Determination of the barrier properties of an edible film is crucial for estimation and prediction of the product-package shelf life. An edible film with good oxygen barrier properties can significantly prolong the shelf life of product, as it reduces lipid oxidation and nutrition loss if applied to certain foods (Han et al., 2008). Generally, edible films are relatively permeable to small molecules such as gases, water vapor, organic vapors and liquids and they provide a broad range of mass transfer characteristics, ranging from excellent to low barrier values, respectively. Water vapor and oxygen are two of the main permeate studies in food packaging, since they can cause changes to a product’s quality and shelf life (Germain, 1997). Carbon dioxide is important in influencing the shelf life of fresh fruits, vegetables, meat, and bakery products (Preeti et al., 2012).
1.6.1.1 Oxygen Transmission Rate (OTR)
The oxygen transmission rate of a food packaging material serves as an important role in shelf life, especially for those oxygen sensitive products such as
52 fresh produce and high-fat products (Preeti et al., 2012). The literature reports that
OTR is highly relative to its crystallinity. Films with higher crystallinity values typically have good oxygen barrier properties (Lacroix, 2009). According to ASTM
D3985 (2010) standard method, the oxygen barrier property is quantified by the oxygen permeability coefficients (OPC), which describes the amount of oxygen that permeates per unit area and time through a packaging material with units given as cm3×mil/(m2×s×Pa). The OTR value indicates the quantity of oxygen gas passing through a unit area of the parallel surfaces of a film per unit time under the conditions of the test (cm3×m/(m2×s)). The OPC is correlated to the OTR by the following equation:
��� = ���× ! (1.1) △!
where l is the thickness of the film (m), △P is the oxygen partial pressure of oxygen (Pa), which is the mol fraction of oxygen multiplied by the total pressure
(nominally, one atmosphere). In the test gas side of the diffusion cell, the partial
pressure of O2 on the carrier gas side is considered zero. (Siracusa et al., 2008)
1.6.1.2 Water Vapor Transmission Rate (WVTR)
The water vapor barrier properties of packaging material play a key role in maintaining or extending product shelf life. This is so for products whose physical or chemical deterioration are related to its equilibrium moisture content. The experiment is typically done by using a Water Vapor Transmission (WVT) cup, with salt solutions or any substance with a know relative humidity inside and sealed by the test
53 film. The cups are then placed into a humidity-controlled chamber and weight changes are recorded until reaching equilibrium (Prommakool et al., 2011). Thus, the water vapor transmission rate (WVTR) of the package can be determined by the change in weight over time (Equation 1.2). According to the ASTM E96-92 method, water vapor barrier is quantified by the water vapor permeability coefficients
(WVPC), which indicate the amount of water vapor that permeates per units of area and time through a packaging material g*mil/(m2*s*Pa), the WVPC is correlated to the WVTR as described in Equation 1.3 as shown below:
���� = ! (1.2) !
���� = ����× ! (1.3) !×△!
Where: A is the surface area (m2) of the sample exposed to moisture;
△P is the driving force, describing the humidity difference between two sides of film (mmHg);
T is thickness of the film (m);
Q is weight change of WVT cups (g)
t is period of time (day)
1.6.1.3 Carbon Dioxide Transmission Rate (CO2TR)
Films with good carbon dioxide barrier property are usually used in Modified
Atmosphere Packaging (MAP) applications. The literature reports that carbon dioxide gas serves as a natural antimicrobial agent and is effective against the growth of some
psychotropic microorganisms (Hotchkiss et al., 1996). MAP with CO2 has led the
54 evolution of fresh and minimally processed foods for the past two decades and has helped these foods to maintain the same quality for a longer time (Jayas et al., 2002).
Similar to oxygen and water vapor barrier properties, carbon dioxide barrier is
quantified by the carbon dioxide permeability coefficients (CO2PC), which indicates the amount of carbon dioxide that permeates per unit of area and time through a packaging material (cm3×m/(m2×s×Pa)). As is the case with the OTR, there is also an
2 equation describing CO2TR (expressed in cc/(m ×s) that is correlated to the CO2PC.
1.6.2 Mechanical Strength
It is well known that adequate mechanical strength of an edible film is necessary to protect the integrity of the packaging throughout its distribution. The most commonly used test for mechanical strength is the tensile test, in which four properties, tensile strength, elongation, yield strength, and modulus of elasticity can be obtained from a single test. An example of a tensile test curve for a film is shown in Figure 1.16 (Hongda, 1995). Tensile strength is the maximum tensile stress that a film can sustain and still return to its original dimension if released from the stress.
The term elongation refers to as the point of break and is expressed as the percentage change in original gauge length of the specimen. Yield strength is the stress at which a predetermined amount of permanent deformation occurs. The modulus of elasticity is the ratio of stress to strain over the linear range and it measures the intrinsic stiffness of the film. Typically, the tensile strength and elongation are reported most frequently for the simplicity of data processing (Hongda, 1995). As a standard testing
55 method, ASTM D882 (2010) is used for experiments. It requires that the test sample must be conditioned at 23oC, 50% humidity for at least 48 hours and then cleanly cut to a rectangular shape and mounted on two separate grips on the equipment. When tension is applied at a constant rate, the force versus extension displacement is recorded thereby tensile strength and elongation at break point could be easily determined from the stress-strain curve.
Figure 1.16 A typical stress/strain curve for protein based edible film.
1.6.3 Microstructure determination
The properties of polymeric films and coatings depend on several factors such as the ratio of crystalline to amorphous zones, polymeric chain mobility, and specific interactions between functional groups of polymers and incorporated substances within the amorphous zones. Common tests used to determine film structure and properties include Scanning Electron Microscopy (SEM), X-ray diffraction (XRD),
Differential Scanning Calorimetry (DSC), Thermo Gravity Analysis (TGA), Dynamic
56 Mechanical Analysis (DMA), and Fourier Transform Infrared Spectroscopy.
1.6.3.1 Fourier Transform Infrared Spectroscopy (FTIR)
Fourier Transform Infrared Spectroscopy has been used to determine the functional groups of materials (Tanaka et al., 2001) on the basis of vibrational responses from the functional groups due to the absorption of infrared energy (Diem,
1993). It is fast, simple, non destructive, no sample preparation is required and no waste material is produced during the test. The FTIR region of the electromagnetic spectrum spans from 14,000 to 50 cm-1 and is divided into three areas: near IR
(14,000 to 4000 cm-1), mid IR (4,000 to 400 cm-1), and far IR (400 to 50 cm-1), respectively. Within the fingerprint region (1500 to 400 cm-1), it can be used to provide information on the biochemical composition and chemical shifts in the sample. As for edible films, the presence of plasticizers and emulsifiers can be easily determined. The main application of FTIR is to develop a fast authentication system for the testing of ingredients in a product. Examples of its application could be for adulteration and fraudulent additives in certain foods such as purees adulterants
(Rodriguez-Saona et al., 2011). By using Partial Least-Squares Near IR models,
Contal et al. showed that adulteration of strawberry or raspberry juice with apple juice could be detected at levels >10% (Contal et al., 2002).
1.6.3.2 X-ray diffraction (XRD)
X-ray diffraction patterns of edible films, either of a single component or
57 composite materials, will show an amorphous-crystalline structure characterized by sharp peaks associated with the crystalline diffraction and an amorphous zone. The crystalline fraction can be estimated by the relative area of the upper regions above the smooth curve, while the amorphous regions can be estimated by the area between the smooth curve drawn following the scattering hum and the baseline joining the background within the low and high angle points. (Snyder & Bish, 1989; Köksel et al.,
1983). X-ray diffraction patterns of composite films generally represent a mixture of component features in which the characteristic peaks of individual component can be identified. In the case of starch-based films, peak width decreased slightly and peak intensities increased, suggesting a growth in crystallite size along with a slow recrystallization process. For example, the X-ray diffraction pattern for composite
Methyl Cellulose/Chitosan films are similar to those obtained for Methyl Cellulose alone, although peak intensity at 2θ=8° (distinct to Methyl Cellulose films) decreased with an increasing Chitosan concentration. This suggested that Chitosan interfered
Figure 1.17 X-ray diffraction of Methyl Cellulose, Chitosan and composite films
58 with the extent of the crystallinity in Methyl Cellulose, as shown in Figure 1.17
(Chen et al., 2003). Also, the broad base of the peak is an indication of an increase in the size distribution of the crystals.
1.6.3.3 Differential Scanning Calorimetry (DSC)
Differential Scanning Calorimetry is a highly sensitive technique that could be used to study the thermo-tropic properties of many different biological macromolecules and extracts such as lipids or proteins (McElhaney, 1982). By measuring the temperature dependence of the partial heat capacity, DSC provides immediate access to the thermodynamic mechanism that determines a conformational equilibrium, for instance, the folded and the unfolded protein, or single and double stranded DNA, or the percent crystallinity in a plastic material (Ilian et al., 1999). The machine is typically equipped with twin cells (sample cell and reference cell) that operate in a differential mode. When heated at constant rate, the heat capacities of the sample cell and reference cell are different, thus a certain amount of electrical power is required to zero the temperature difference between two cells. The power difference
(Js-1) is a direct measure of the heat capacity difference between the material in the sample cell and nitrogen in reference cell.
In the case of an edible film, DSC test is commonly used to estimate crystallization and melting point temperatures. These are important since they impact mechanical and barrier properties of the film under specific conditions of application and storage. In the study of evolution of the film matrix crystalline structure during
59 storage performed by García et al., starch films obtained by alkaline treatment showed an endothermic transition with a peak at temperature around 50oC during 90 days storage. The peak became narrower and its melting temperature and the corresponding enthalpy increased with increasing storage time (Figure 1.18). The
DSC transition could be associated with several processes, such as the crystal growth of short chains and recrystallization of amylose or other long lateral amylopectin chains (García et al, 2000). Thus DSC can be used to determine how an additive
influences Tg and Tm thus the performance of a material exposed to a certain application.
Figure 1.18 DSC thermograms of amylomaize films stored at 20oC and 63.8% RH.
1.6.3.4 Thermo-Gravimetric Analysis (TGA)
Thermo-Gravimetric analysis (TGA) is finding increasing utility in
60 investigations of the pyrolysis and combustion behavior of solids (Broido, 1969).
Typically it is carried out by observing the onset temperature, in a given atmosphere, of the decomposition of a polymer. In this way an accurate ranking of relative stability of different polymers can be obtained at given temperature ranges.
Polyetherimide (PEI), polycarbonate (PC), polyethylene terephthalate (PET) and polyvinyl chloride (PVC) differ in their thermal stabilities due to the chemical make-up of their backbones (in the order PEI>PC>PET>PVC) (Paul, 2008). One of the applications is to assess relative thermal stability of materials that are used under adiabatic conditions. Skachkova et al. investigated the thermo-oxidative stability of the commercially important polypropylene-EPDM thermoplastic rubbers contained a hydrocarbon oil plasticizer as did control samples of polypropylene and EPDM rubber.
Results indicated that the oil degraded and became integrated into the oxidized polymer surface at high temperatures forming a multi-layer, which inhibited the ingress of oxygen and hence produced protection of the rubbers (Skachkova et al.,
2003). Though TGA is used in a number of degradation studies to determine the kinetics of decomposition, in some cases, problems exist in identifying the crucial weight loss mechanism because some polymers have more than one weight loss region (Paul, 2008). The combination of TGA and FTIR can be used to characterize the most abundant volatile products of certain polymers, including poly blends and co-polymers and the mechanism of their formation (Pielichowski et al., 2005). TGA can also be used in conjunction with DSC to discover if thermal events are associated with mass loss.
61 1.6.3.5 Dynamic Mechanical Analysis (DMA)
Dynamic Mechanical Analysis can be simply described as applying an oscillating force to a sample and analyzing the material’s response to the force over a given temperature range. During a typical test, the slope of the line obtained from the stress-strain curve provides the relationship of stress to strain and is a measurement of the material’s stiffness, the modulus, which is dependent on the temperature and the applied stress. It is excellent for detecting thermal events that involve subtle changes in the mechanical properties of the sample and is usually associated with the measurement of glass transitions and other related phenomena (Duncan et al., 1995).
DMA tests have been widely used in the characterization of viscoelastic materials, particularly in polymer science and the pharmaceutical industry. Typically, the following information concerning polymeric systems may be obtained from a dynamic mechanical analysis:
1) Quantitative modulus, i.e. storage and loss modulus;
2) Glass transitions (primary, secondary);
3) Rate and extent of polymeric curing;
4) Quantification of gelation, e.g. sol-gel transitions;
5) Damping properties, i.e. characterization of the ratio of loss to storage modulus at
defined temperatures.
6) Polymer morphology/compatibility
7) Interactions between polymeric components or between drug molecules and
polymeric constituents of pharmaceutical systems. (David, 1999)
62
Chapter 2
Antibacterial Activity of Selected Small Molecule Compounds
against E.coli K12, Listeria innocua, Campylobacter jejuni, and
Campylobacter coli bacterial species.
2.1 Abstract
The antibacterial activity of synthesized small molecule compounds (JA-144,
TH-4, and TH-8) dissolved in Dimethylsulfoxide and incorporated into tapioca films, were evaluated against E.coli K12, Listeria innocua, Campylobacter jejuni and
Campylobacter coli. Minimum Inhibitory Concentrations (MIC) for JA-144, TH-4 and TH-8 against E.coli K12, L.innocua, C.jejuni and C.coli in Mueller-Hinton broth at 37oC or 42oC were determined. The MICs for JA-144 were 100ppm against E.coli
K12, and 50 to 100ppm against L.innocua at 37oC. For C.jejuni and C.coli, the MICs for JA-144 at 42oC were 3.125 to 6.25ppm, respectively. The TH-4 and TH-8 compounds were not active against E.coli K12 and L.innocua, thus only C.jejuni and
C.coli were tested. The MICs for TH-4 were 12.5 to 25ppm against both C.jejuni and
C.coli at 42oC, respectively. For TH-8, the MICs were 25 to 50ppm against both
C.jejuni and C.coli at 42oC, respectively. The bacterial growth curves for E.coli K12,
L.innocua, C.jejuni under the treatment of JA-144, TH-4 and TH-8 at MICs showed that JA-144 completely inhibited the growth of E.coli K12 and L.innocua at 100ppm,
C.jejuni at 6.25ppm, respectively. TH-4 completely inhibited the growth of C.jejuni at
63 25ppm. TH-8 completely inhibited the growth of C.jejuni at 50ppm, respectively. The log reduction results showed that, the MICs for JA-144 in the broth against E.coli
K12, L.innocua, C.jejuni and C.coli caused reductions of 5.86, 5.52, 6.35, 6.27 log10
CFU/ml, respectively after 24 hours. The same concentration of JA-144 incorporated into the tapioca film caused E.coli K12, L.innocua C.jejuni and C.coli reductions of
3.31, 3.51, 6.36 and 6.14 log10 CFU/ml, respectively after 24 hours. Thus, the efficacy losses for JA-144 due to film incorporation were 43.48%, 36.42%, -0.25% and 2.03%, respectively. The MICs of TH-4 in the broth for C.jejuni and C.coli caused reductions
of 6.37 and 6.16 log10 CFU/ml, respectively after 24 hours. The same concentration of
TH-4 incorporated into the tapioca film caused C.jejuni and C.coli reductions of 5.99
and 4.37 log10 CFU/ml, respectively after 24 hours. Thus, the efficacy losses of TH-4 due to film incorporation were 6.09% and 29.02%, respectively. The MICs of TH-8 in
the broth for C.jejuni and C.coli caused reductions of 5.39 and 5.51 log10 CFU/ml, respectively after 24 hours. The same concentration of TH-8 incorporated into the
tapioca film caused C.jejuni and C.coli reductions of 4.01 and 2.87 log10 CFU/ml, respectively after 24 hours. Thus, the efficacy losses of TH-8 due to film incorporation were 25.22% and 48.01%, respectively. This study demonstrated that
JA-144 was effective against E.coli K12, L.innocua, C.jejuni and C.coli in broth and in the film. TH-4 and TH-8 were effective only against C.jejuni and C.coli in broth and in the film.
64 2.2 Introduction
Microbial contamination is capable of reducing the shelf life of foods and increases the risk of foodborne illnesses. This is a major worldwide public health concern. It is reported that over 48 million people get sick every year in the United
States (CDC 2014). According to statistical data from the CDC, five pathogens account for over 90% of the estimated food-related deaths. These microorganisms are
Samonella, Listeria, Campylobacter, E.coli O157:H7 and Vibrio (CDC, 2014).
Among these five pathogens, illness associated with Campylobacter, termed
“Campylobacteriosis”, is one of the most common forms of foodborne gastroenteritis in developed countries (Blaser, M.J., et al, 2008). It is reported that cases of diarrhea and bacteremia caused by Campylobacter spp. are seen in HIV patients in developing countries (Coker, A.O., et al, 2002). Apart from Campylobacter spp., the CDC estimates that E.coli O157:H7 causes approximately 73,000 illnesses and 60 deaths each year in the U.S., in which 85% of these cases are attributed to foodborne transmission. Human infection with E.coli O157:H7 can lead to non-bloody or bloody diarrhea, or more serious and fatal syndromes such as hemorrhagic colitis and hemolytic uremic syndrome. Listeriosis, another serious disease usually caused by eating food contaminated with the bacterium Listeria monocytogenes, is also an important public health problem. It is estimated that in the U.S., there are about 2500 cases with 500 deaths annually attributed to listeriosis. The spectrum of listeriosis is broad, ranging from asymptomatic infection and flu-like symptoms, to miscarriage, stillbirth, and meningitis (CDC, 2014).
65 Antimicrobial therapy is one of the most effective ways to prevent and control bacterial diseases. Currently, the most commonly used antimicrobials are macrolides
(erythromycin) and fluoroquinolones (ciprofloxacin) with tetracycline used as an alternative choice (Moore, J.E., et al., 2006). However, as the use of antimicrobials for therapy and prophylaxis increase in both human and animal medicine, increasing numbers of bacteria have developed drug resistance. As a solution to this antimicrobial-resistance problem, antimicrobial compounds could be developed to completely kill all pathogens. However, since this is not practical, a workable approach is needed. The target selection for drug design should be substrate-specificity, therefore, it is possible for drugs to inhibit or kill pathogenic bacteria without affecting the beneficial ones (Jongbloed et al., 2000).
An active food packaging system can serve as a non-thermal method to reduce foodborne pathogen contamination in food. By definition, active packaging is defined as packaging in which subsidiary constituents have been deliberately included in or either on the packaging material or the packaging headspace to enhance the performance of the packaging system (Robertson, 2006). One application of active packaging is to incorporate antimicrobial substances that can control microbial contamination by reducing the growth rate and maximum population by extending the lag-phase of the target microorganism (Han, J.H., 2000).
The objectives of this study were to investigate the antibacterial activity of synthesized small molecules (JA-144, TH-4 and TH-8) dissolved into dimethyl sulfoxide (DMSO), and also incorporated into tapioca edible films. These will be
66 tested against E.coli K12 and Listeria innocua, Campylobacter jejuni and
Campylobacter coli. Among these strains, E.coli K12 and L.innocua were used as bacteria surrogates of E.coli O157:H7 and L.monocytogenes, respectively.
67 2.3 Materials and Methods.
2.3.1 Materials.
Tapioca starch powder was purchased from a local supermarket in Columbus,
OH. It was irradiated at the OSU Nuclear Reactor Lab in order to achieve sterilization.
This was used as the main ingredient in the films. Distilled water was used to make a suspension of the starch powder. Glycerol ≥99.0% (Sigma-Aldrich®) was obtained from Fisher Scientific (Fisher Scientific, Fair Lawn, NJ) and used as a plasticizer.
Acetic Acid (ACS Reagent ≥99.7%), Dimethyl Sulfoxide (ACS Reagent ≥99.9%) and
2,3,5-Triphenyltetrazolium Chloride (≥95.0%) were purchased from Sigma-Aldrich® and used as a solvent and bacterial dye agent, respectively. E.coli K12 and Listeria innocua were purchased from the American Type Culture Collection (Manassas, VA).
Campylobacter jejuni and Campylobacter coli were obtained from Dr. Rajashekara’s lab, Wooster, OH. Tryptic Soy Broth (TSB) and Tryptic Soy Agar were purchased from Difco (Sparks, MD). Mueller Hinton Agar, Mueller Hinton Broth and Fisher
BioReagents Peptone (Granulated) were purchased from Thermo Scientific®. 1.5ml
Micro Centrifuge Tubes, 96-Well Microplates, 100mm Petri Dishes were purchased from Thermo Scientific®, respectively.
2.3.2 Minimum Inhibitory Concentration (MIC) tests for JA-144, TH-4
and TH-8.
2.3.2.1 Small molecules of JA-144, TH-4 and TH-8.
The synthesized small molecules referred to as JA-144, TH-4 and TH-8 were
68 obtained from Dr. James Fuch’s lab at the College of Pharmacy, OSU. The chemical structures of these molecules are shown in Figure 2.1
Figure 2.1 The chemical structure of JA-144, TH-4 and TH-8.
2.3.2.2 Preparation of bacterial culture.
E.coli K12, L.innocua，C.jejuni and C.coli were used in the assay. The bacteria were maintained on nutrient agar slants at 4oC. Before use, a single colony of each bacterium was transferred into 50ml tubes containing MHB using aseptic techniques.
The tubes were capped and placed in an incubator overnight at 37oC, aerobically (for
E.coli and L.innocua), and at 42oC (for C.jejuni and C.coli), microaerobically. After
24 hours incubation, the broths were centrifuged at 4000 rpm for 5 min using appropriate aseptic precautions. The supernatants were discarded and the pellets resuspended using 20 ml of sterile peptone water and centrifuged again at 4,000 rpm for 5 min. This step was repeated until the supernatants were clear. The pellets were then suspended in 20 ml of sterile peptone water, and were labeled as Standard Broth
(SB). As for E. coli K12, and L. innocua, six consecutive 10-fold dilutions were made
69 to reach the initial inoculum of 103 CFU/ml. As for C.jejuni and C.coli, the initial inoculum was obtained by measuring the optical density of the SB at 595nm, and serial dilutions carried out until the optical density reached 0.05, which was equal to
106 CFU/ml.
2.3.2.3 Preparation of small molecule stock solutions.
The small molecule solutions were prepared by dissolving 100 mg of each compound at room temperature in 1 ml DMSO in separate test tubes to reach a concentration of 100,000 ppm. A vortex mixer was used to ensure that each compound was well dissolved into a homogenous solution. The stock solutions were then stored at -20oC.
2.3.2.4 Preparation of 2,3,5-Triphenyltetrazolium Chloride solutions as color indicator.
The TTC stock solution was prepared by dissolving 20 mg in 1 ml distilled water at room temperature to reach a concentration of 20,000 ppm. A vortex mixer was used to ensure that it was well dissolved. The stock solution was stored at -20oC for future use.
2.3.2.5 Preparation of working solutions.
The range of concentrations for the small molecules in the films depended on previous cytotoxicity assay conducted by Dr. Esperanza Carcache de Blanco’s
70 laboratory in the College of Pharmacy at OSU. This test determined the highest concentrations of the compounds to cause lysis of human colon cells. The results showed that the values for JA-144, TH-4 and TH-8 were 200 µM. When converted to ppm values, they were 66.9 ppm, 61.1 ppm, 60.4 ppm, respectively (Appendix
Figure A.1, A.2, A.3). The survival rates of colon cells for JA-144, TH-4 and TH-8 at
200 µM were 99%, 60%, and 79%, according to Appendix Figure A.1, A.2, A.3.
Since JA-144 showed less toxicity on human colon cells, the starting concentration for this study was 100ppm; while for TH-4 and TH-8, the starting concentration were
50ppm, respectively.
Consequently, according to previous studies, the DMSO in MHB was 1%, TTC in MHB was 100 ppm, and the upper limits for JA-144, TH-4, and TH-8 in MHB were 100 ppm, 50 ppm, and 50 ppm, respectively. Two-fold series dilutions started from the upper limit of each compound were made and shown in Table 2.1, 2.2, 2.3, respectively. A total of 10 dilutions were made in order to obtain a sufficient working range for the MIC assay.
71 Table 2.1 Preparation of dilutions of JA-144 for use in MIC tests.
JA-144 conc. Vol.of Final conc. in Vol. stock JA-144 conc. (mg/L) in stock DMSO medium after solution (ml) (mg/L) solution (ml) addition of MHB 100,000 1 9 10,000 100
10,000 1 1 5,000 50
5,000 1 1 2,500 25
2,500 1 1 1,250 12.5
1,250 1 1 625 6.25
625 1 1 313 3.13
313 1 1 157 1.57
157 1 1 78 0.78
78 1 1 34 0.34
34 1 1 17 0.17
Table 2.2 Preparation of dilutions of TH-4 for use in MIC tests.
Vol. of Final conc. in TH-4 conc. (mg/L) Vol. stock TH-4 conc. DMSO medium after in stock solution solution (ml) (mg/L) (ml) addition of MHB
100,000 1 19 5,000 50
5,000 1 1 2,500 25
2,500 1 1 1,250 12.5
1,250 1 1 625 6.25
625 1 1 313 3.13
313 1 1 157 1.57
157 1 1 78 0.78
78 1 1 34 0.34
34 1 1 17 0.17
17 1 1 9 0.09
72 Table 2.3 Preparation of dilutions of TH-8 for use in MIC tests.
Vol. of Final conc. in TH-8 conc. (mg/L) Vol. stock TH-8 conc. DMSO medium after in stock solution solution (ml) (mg/L) (ml) addition of MHB 100,000 1 19 5,000 50
5,000 1 1 2,500 25
2,500 1 1 1,250 12.5
1,250 1 1 625 6.25
625 1 1 313 3.13
313 1 1 157 1.57
157 1 1 78 0.78
78 1 1 34 0.34
34 1 1 17 0.17
17 1 1 9 0.09
2.3.2.6 Preparation of plates.
A sterile 96 well plate was used for all MIC tests. A positive control was used in
this study. This positive control was made of 198.6µl of MHB with E.coli K12,
L.innocua, C.jejuni, and C.coli at a concentration of 106 CFU/ml, 0.4µl of
Chloramphenicol (2µl/ml in broth), and 1µl TTC stock solution (100ppm in broth).
This was pipetted into the first column of the plate. The MIC test groups were then
placed in row 2 to row 11, plus 197µl of MHB inoculated with E.coli K12, L.innocua,
C.jejuni, or C.coli at 106 CFU/ml, 2µl of DMSO with diluted JA-144, TH-4 or TH-8,
and 1ul TTC stock solution. A negative control group was prepared by repeating the
above but without JA-144, TH-4 or TH-8. This negative control group was pipetted
into the last column of the plate. Triplicate tests were done for each bacterial strain.
73 Figure 2.2 and Table 2.4 show the MIC tests design. The 96-well micro plates with
E.coli K12 and L.innocua were incubated at 37oC aerobically for 24 hours; while the plates with C.jejuni and C.coli were incubated at 42oC microaerobically for 24 hours, respectively.
Figure 2.2 The MIC tests design for JA-144, TH-4 and TH-8.
Table 2.4 The amount of each constituent used for MIC tests.
Positive Control Group Experimental Group Negative Control Group
Constituents Vol. (µl) Constituents Vol. (µl) Constituents Vol. (µl)
MHB with tested MHB with MHB with 198.6µl 197µl 197µl bacteria tested bacteria tested bacteria
DMSO with Chloramphenicol 0.4µl 2µl DMSO only 2µl Small molecules
TTC 1µl TTC 1µl TTC 1µl
74 2.3.2.7 Interpreting Results.
The MIC is the lowest concentration of the agent that completely inhibits visible growth of the test microorganisms as judged by the naked eye. In this study the MIC values depended on a color change. No color change was observed when the growth of the bacteria was inhibited.
2.3.3 Bacterial Growth Curve Measurement for all test compounds as inhibitors at Minimum Inhibitory Concentration.
The bacterial growth curve measurement was a follow up experiment for the
MIC tests. As for E.coli K12 and L.innocua, the bacterial incubations were followed by measurement of the OD (Optical Density) values every 15 minutes in an automated SunriseTM Tecan Spectrophotometer (Tecan Co.) during the incubation period of 24 hours at 37oC. For C.jejuni, the incubation experimental design was similar, but the measurement period occurred every 4 hours. From the data collected, comparisons were made with the control samples and statistical analyses used to determine the significance between the means for all observations
2.3.4. Antibacterial activity of all test compounds in bacterial broth or incorporated into films against all test microorganisms at each MIC value.
The antibacterial activity tests using tapioca starch films incorporated with the
JA-144, TH-4 or TH-8 were followed by MIC and bacterial growth curve tests.
75 Preparation of the bacterial cultures was the same as for the MIC tests.
2.3.4.1 Preparation of Tapioca Films with JA-144, TH-4, or TH-8.
The film forming solutions were prepared using blends of tapioca starch and
small molecule compounds (JA-144, TH-4 and TH-8) dissolved in acetic acid with
glycerol as plasticizer. Aliquots of 100 mg of each small molecule compound (JA-144,
TH-4 or TH-8) were first dissolved in 1 ml acetic acid. Tapioca starch (5% w/w) and
acetic acid solutions (0.7% w/w) with the small molecules and 1.8% w/w glycerol
were dissolved into 100 ml distilled water, as shown in Table 2.5a. All dispersions
were heated in a water bath (70oC) for 15 min with glass rod stirring until completely
gelatinized. An Ultrasonic Sonicator (Graymills Co., Chicago, IL) was used to
remove tiny bubbles from the gelatinized solutions, which was done by immersing the
beakers containing the film-forming solutions into sonic water at 70oC for 1 hour. The
edible films were prepared by casting the solution mixtures (107g) into 10-inch radius
Teflon plates. These plates were oven dried at 45±2oC for 12 hours, then the dried
films peeled off from the plate surfaces. The final concentration of each small
Table 2.5 The composition of tapioca film on wet basis and dry basis. a. Wet basis b. Dry basis
Composition Weight % Composition Weight %
Tapioca starch 5％ Tapioca starch 72.78%
Glycerol 1.8％ Glycerol 26.20%
Acetic acid 0.7％ Small molecules 1.02%
Small molecules 0.07%
Distilled water 93.23%
Total 100％ Total 100%
76 molecule in the dry film was approximately 1% w/w, as shown in Table 2.5b.
Figure 2.3 summarizes all the steps for the films’ preparation. The method of achieving the percent small molecules in the films was adapted from a study of fast dissolving oral films reported by Arun et al, (2010). In that study, the percent drugs in the films ranged from 1% to 25%. Films with the same ingredients but without small molecule compounds were also prepared as controls. The film thicknesses were measured using Magna-Mike 8500 Thickness Gage (Olympus, Japan), with resolution of 0.001mm. A total of 5 measurements per film at various locations were taken to determine the film thickness.
Figure 2.3 The flow chart of Tapioca/small molecules and film formation.
77 2.3.4.2 Preparation of test films with inoculated bacterial culture
The films prepared in 2.3.4.1 were cut into small pieces that were capable of fitting into 1.5 ml micro-tubes. The weight of each film sample ranged from 5 to 15 mg. The micro-tubes with the films were then exposed to UV light for 18 hours to eliminate possible contamination that may have occurred during the cutting and transferring process. The amount of bacterial culture in each tube depended on two factors: the amount of small molecules in the film, and the MIC value that completely inhibited the bacterial growth. Table 2.6 shows an example of the amount of bacterial culture used in a micro-tube. After adding the proper amount of bacterial culture, the micro-tubes were placed in an incubator aerobically at 37oC (for E.coli and
L.innocua), or microaerobically at 42oC (for C.jejuni and C.coli), for 24 hours, respectively.
Table 2.6 An example of amount of bacterial culture pipetted into micro tubes based on film’s weight. Volume or Weight
Films incorporated with small molecules 15mg
Small molecules in the film 0.15mg
Amount of bacterial culture pipetted into Micro Tubes* 1.5ml
*Estimated MIC of small molecule was 100ppm.
2.3.4.3 Preparation of plates
Mueller Hinton Agar (MHA) was used as a growth medium in this test. A 4% w/w solution was prepared by dissolving 40g MHA powder in 1,000 ml distilled
78 water and then sterilizing it at 121oC for 25 min. It was then transferred to a 50oC water-bath and allowed to cool. Afterwards, it was poured into pre-labeled sterile
Petri dishes on a level surface at room temperature (23oC) and dried so that no drops of moisture remained on surface of the agar.
2.3.4.4 Inoculation of Plates
After 24 hours incubation, the broth was centrifuged at 4,000 rpm for 5 min at
23oC with appropriate aseptic precautions. The supernatant was discarded and the pellet resuspended using 1 ml of sterile peptone water and centrifuged again at 4000 rpm for 5 min. This step was repeated until the supernatant was clear. The pellets were then suspended in 1 ml sterile peptone water, and seven consecutive 10-fold dilutions were made to obtain inoculums ready for the plate counts. For the experimental groups with the small molecule compounds, 0.1 ml of diluted broth
(dilution of 10-2; 10-4; 10-6) were inoculated on to the plates. For the control groups,
0.1 ml of diluted broth (dilution of 10-6 and 10-7) was added to the plates. They were then incubated at 37oC aerobically (for E.coli and L.innocua), or 42oC microaerobically (for C.jejuni and C.coli) for 24 hours, and then colony numbers on each plate counted using a Darkfield Colony Counter manufactured by American
Optical (Buffalo, NY).
2.3.5 Statistical Analysis
All experiments were repeated 3 times and differences in the means analyzed by
79 a SAS statistical program. One-way analysis of variance (ANOVA) was carried out to evaluate significance in differences (p<0.05) between the influences of the small molecules on the microbial growth.
2.4 Results
2.4.1 Minimum Inhibitory Concentration tests of JA-144, TH-4 and
TH-8.
The MICs of JA-144, TH-4 or TH-8 against E.coli K12, L.innocua，C.jejuni and
C.coli are shown in Table 2.7. The MIC of JA-144 against E.coli K12 was 100ppm, while the result for L.innocua, ranged from 50ppm to 100ppm. The MIC of JA-144 against C.jejuni and C.coli ranged from 3.125ppm to 6.25ppm, respectively. The results showed that TH-4 had no antibacterial activity against E.coli K12 and
L.innocua. However, the MIC of TH-4 against C.jejuni and C.coli ranged from
12.5ppm to 25ppm, respectively. TH-8 also did not show antibacterial activity against
E.coli K12 and L.innocua, but for C.jejuni and C.coli, the MIC ranged from 25ppm to
50ppm, respectively. Appendix Figure B.1 is a photograph of the 96-wells
Table 2.7 Summary of Small molecules' MIC against 4 individual bacterial strains.
MIC (ppm) Strain JA-144 TH-4 TH-8 E.coli K12 100 / /
L.innocua 50 to 100 / /
C.jejuni 3.125 to 6.25 12.5 to 25 25 to 50
C.coli 3.125 to 6.25 12.5 to 25 25 to 50
80 micro-plate showing the color changes that occurred in the samples and from which the microbial data were obtained.
2.4.2 Bacterial Growth Curve Measurement using JA-144, TH-4, or
TH-8 as inhibitors at Minimum Inhibitory Concentration.
The bacterial growth curve measurements revealed how the bacterial populations were affected by JA-144, TH-4 or TH-8 under their MICs. As for JA-144, 50ppm,
75ppm, 100ppm concentrations were tested for E.coli K12 and L.innocua strains as shown in Figures 2.4 and 2.5. Concentrations of 3.125ppm, 6.25ppm were tested for
C.jejuni and C.coli strains as shown in Figures 2.6 and 2.7, respectively. Regarding
TH-4, concentrations of 12.5ppm and 25ppm were tested against C.jejuni and C.coli strains as shown in Figures 2.8 and 2.9. For TH-8, 25ppm and 50ppm were tested against C.jejuni and C.coli strains as shown in Figure 2.10 and 2.11 respectively.
E.coli K12 Growth Curve using JA-144 as inhibitor at 100ppm, 75ppm, 50ppm 0.98 P CNTL 0.88 100ppm ave 0.78 75ppm ave 0.68 50ppm ave 0.58 N CNTL 0.48 0.38 0.28 OD value at 595nm 0.18 0.08 0 5 10 15 20 Time in hours Figure 2.4 E.coli K12 Growth Curve using JA-144 as inhibitor at 100ppm, 75ppm, 50ppm.
81 Listeria innocua Growth Curve using JA-144 as inhibitor at 100ppm,75ppm, and 50ppm 0.28 P CNTL 0.26 100ppm ave 0.24 75ppm ave 0.22 50ppm ave 0.2 N CNTL 0.18 0.16 0.14
OD value at 595nm 0.12 0.1 0.08 0 5 10 15 20 Time in hours Figure 2.5 L.innocua Growth Curve using JA-144 as inhibitor at 100ppm, 75ppm, 50ppm.
As shown in Figure 2.4, the results indicated that the growth of E.coli K12 exposed to JA-144 at 50ppm was lower than the negative control. For 75ppm, the growth of E.coli K12 was almost zero, however, the curve showed a growing trend after 15-hour incubation, suggesting that the bacteria was not completely inhibited. At
100ppm concentration level, the growth of E.coli K12 was not detected. Results for
L.innocua shown in Figure 2.5 were similar to E.coli K12, the concentration level for
JA-144 that completely inhibited the growth of L.innocua was 100ppm.
As for C.jejuni in Figure 2.6, the growth was not completely inhibited by
JA-144 at the 3.125ppm concentration after 12 hours incubation. For the 6.25ppm treatment, its efficacy was greater than the positive control since the growth curve was below the positive control at all times. In Figure 2.7, it was shown that C.coli growth was inhibited by JA-144 at both 3.125ppm and 6.25ppm concentrations.
82 C.jejuni Growth Curve using JA-144 as inhibitor
0.465 P CNTL 0.415 N CNTL
0.365 JA-144 6.25ppm 0.315 JA-144 3.125ppm 0.265 OD value at 595nm 0.215
0.165
0.115 0 5 10 15 20 25 Time in hours Figure 2.6 C.jejuni Growth Curve using JA-144 as inhibitor at 6.25ppm, and 3.125ppm.
C.coli Growth Curve using JA-144 as inhibitor
1.800 P CNTL 1.600 JA-144 6.25ppm 1.400 JA-144 3.125ppm
1.200 N CNTL 1.000
0.800
0.600
OD value at 595nm 0.400
0.200
0.000 0 4 8 12 16 20 24 Time in hours Figure 2.7 C.coli Growth Curve using JA-144 as inhibitor at 6.25ppm, and 3.125ppm.
83
C.jejuni Growth Curve using TH-4 as inhibitor
0.465 P CNTL
0.415 N CNTL
TH-4 25ppm 0.365 TH-4 12.5ppm
0.315
0.265 OD value at 595nm 0.215
0.165
0.115 0 5 10 15 20 25 Time in hours Figure 2.8 C.jejuni Growth Curve using TH-4 as inhibitor at 12.5ppm, and 25ppm.
C.coli Growth Curve using TH-4 as inhibitor
1.800 P CNTL 1.600 TH-4 25ppm 1.400 TH-4 12.5ppm
1.200 N CNTL 1.000
0.800
0.600 OD value at 595nm 0.400
0.200
0.000 0 4 8 12 16 20 24 Time in hours Figure 2.9 C.coli Growth Curve using TH-4 as inhibitor at 12.5ppm, and 25ppm.
84 Results for the TH-4 tests shown in Figures 2.8 and 2.9 are similar to those shown in Figures 2.6 and 2.7. C.jejuni was inhibited by TH-4 at 12.5ppm when compared with the negative control. At the 25ppm concentration, TH-4 had a greater impact on inhibiting the growth of C.jejuni. As for C.coli in Figure 2.9, it was more sensitive than C.jejuni because the growth curve was inhibited by TH-4 at 12.5ppm.
For the TH-8 tests, as shown in Figures 2.10 and 2.11, results show that the growth of both C.jejuni and C.coli was completely inhibited by the concentration at
50ppm. In Figure 2.10, C.jejuni appear to be more resistant to TH-8 at 25ppm concentration when compared to C.coli in Figure 2.11.
C.jejuni Growth Curve using TH-8 as inhibitor
0.465 P CNTL 0.415 N CNTL
0.365 TH-8 50ppm 0.315 TH-8 25ppm 0.265 OD value at 595nm 0.215
0.165
0.115 0 5 10 15 20 25 Time in hours Figure 2.10 C.jejuni Growth Curve using TH-8 as inhibitor at 25ppm, and 50ppm.
85 C.coli Growth Curve using TH-8 as inhibitor
1.800 P CNTL 1.600 TH-8 50ppm 1.400 TH-8 25ppm 1.200 N CNTL 1.000
0.800
0.600
OD value at 595nm 0.400
0.200
0.000 0 4 8 12 16 20 24 Time in hours Figure 2.11 C.coli Growth Curve using TH-8 as inhibitor at 25ppm, and 50ppm.
2.4.3. Antibacterial activity tests of all compounds in bacterial broth or incorporated into films against all tested organisms at each MIC.
The antibacterial efficacies of JA-144, TH-4 and TH-8 in the broth and incorporated into the film against E.coli K12, L.innocua, C.jejuni and C.coli were tested. The results are summarized in Table 2.8.
Table 2.8 Concentration of JA-144, TH-4 and TH-8 tested for antibacterial activities.
E.coli K12 L.innocua C.jejuni C.coli
JA-144 100ppm 100ppm 6.25ppm 3.125ppm
TH-4 / / 25ppm 12.5ppm
TH-8 / / 50ppm 50ppm
86 2.4.3.1 Antibacterial activity tests of all compounds in bacterial broth against all tested organisms.
For the broth inoculated with E.coli K12 and L.innocua, those treated with
JA-144 at 100ppm resulted in significant reductions (p<0.05) of 5.86 and 5.52 log10
CFU/ml, respectively after 24 hours incubation (Figure 2.12a,b), when compared with the control.
For C.jejuni and C.coli in the broth, JA-144 at 6.25ppm and 3.125ppm
concentration resulted in significant reductions (p<0.05) of 6.35 and 6.27 log10
CFU/ml, respectively after 24 hours incubation (Figure 2.13a,b), when compared with the control.
In regards to TH-4 at 25ppm and 12.5ppm, concentrations against C.jejuni and
C.coli in the broth resulted in significant reductions (p<0.05) of 6.37 and 6.16 log10
CFU/ml, respectively after 24 hours incubation (Figure 2.13a,b), when compared with the control.
When C.jejuni and C.coli in the broth were treated by TH-8 at 50ppm, the results
showed significant differences (p<0.05) of 5.39 and 5.51 log10 CFU/ml, respectively after 24 hours incubation (Figure 2.13a,b), when compared with control. All log reduction-testing results are summarized in Table 2.9.
87 Table 2.9 The summary of Log reduction tests for E.coli K12 and L.innocua.
E.coli K12 L.innocua
a b In JA-144 2.96±0.30 3.14±0.11 Broth CNTL 8.81±0.15a 8.66±0.09b
Log10 CFU/ml reduction 5.86 5.52
JA-144 5.58±0.08a 5.22±0.07b In CNTL 8.90±0.13a 8.73±0.29b Film
Log10 CFU/ml reduction 3.31 3.51
% Of efficacy loss due to incorporation 43.48% 36.42%
Values within the same row with the same letters are not significantly different (p>0.05).
2.4.3.2 Antibacterial activity tests of all compounds incorporated into tapioca films against all tested organisms.
The antibacterial activity testing of JA-144, TH-4 and TH-8 incorporated into tapioca films were investigated against E.coli K12, L.innocua, C.jejuni and C.coli.
The antibacterial activity of JA-144, TH-4 or TH-8 was enumerated by the viable cell count method to give a quantitative estimation of the efficacy. Samples were enumerated for bacteria viability after 24 hours. As a comparison, the control films with no added small molecules were also tested. Results obtained in this section were compared with those obtained from the broth studies, and the percentages of antibacterial activities lost due to film incorporation were compared.
For E.coli K12 and L.innocua, 1% w/w of JA-144 film resulted in significant
reductions (p<0.05) of 3.31 and 3.51 log10 CFU/ml, respectively after 24 hours incubation (Figure 2.12b), when compared with control. The percentages of
88 antibacterial activity lost due to film incorporation were 43.48% and 36.42%, respectively.
For C.jejuni and C.coli, JA-144 at 6.25ppm and 3.125ppm resulted in significant
reductions (p<0.05) of 6.36 and 6.14 log10 CFU/ml, respectively after 24 hours incubation (Figure 2.14a,b), when compared with control. The percentages of antibacterial activities lost due to film incorporation were 0.25% and 2.03%, respectively.
In regards to TH-4 at 25ppm and 12.5ppm, as for C.jejuni and C.coli, the results
were 5.99 and 4.37 log10 CFU/ml (p<0.05), respectively after 24 hours incubation
(Figure 2.14a,b), when compared with control. The percentages of antibacterial activity lost due to film incorporation were 6.09% and 29.02%, respectively.
When C.jejuni and C.coli were treated by TH-8 at 50ppm, the results were 4.01
and 2.87 log10 CFU/ml (p<0.05), respectively after 24 hours incubation (Figure
2.14a,b), when compared with control. The percentages of antibacterial activity lost due to film incorporation were 25.52% and 48.01%, respectively. All log reduction-testing results were summarized in Table 2.10 and Table 2.11.
89 Table 2.10 The summary of Log reduction tests for C.jejuni and C.coli.
C.jejuni
C.jejuni Control Log10 CFU/ml reduction
JA-144 2.90±0.09a 9.25±0.01ab 6.35
TH-4 2.78±0.09a 9.15±0.06ab 6.37
In TH-8 3.64±0.05a 9.03±0.08ab 5.39
Broth C.coli
C.coli Control Log10 CFU/ml reduction
JA-144 2.93±0.26a 9.20±0.02ab 6.27
TH-4 3.21±0.12a 9.38±0.04ab 6.16
TH-8 4.11±0.05a 9.62±0.04ab 5.51
Values within the same row with the same letters are not significantly different (p>0.05).
Table 2.11 The summary of Log reduction tests for C.jejuni and C.coli.
C.jejuni
Log10 CFU/ml % Efficacy loss due C.jejuni Control. reduction to incorporation JA-144 2.86±0.13a 9.22±0.05ab 6.36 0.25
TH-4 3.16±0.14a 9.15±0.07ab 5.99 6.09
In TH-8 5.10±0.07a 9.11±0.12ab 4.01 25.52
Film C.coli
Log10 CFU/ml % Efficacy loss due C.coli Control. reduction to incorporation JA-144 2.93±0.16a 9.07±0.05ab 6.14 2.03
TH-4 5.03±0.07a 9.40±0.04ab 4.37 29.02
TH-8 6.83±0.06a 9.70±0.07ab 2.87 48.01 Values within the same row with the same letters are not significantly different (p>0.05).
90 JA-144 against E.coli K12and Tapioca ilm with JA-144 against JA-144 E.coli K12 and L.innocua at MIC L.innocua at MIC (100ppm) JA-144 (100ppm) Control CNTL 10.00 10.00
9.00 9.00 8.00 8.00
7.00 6.00 7.00 5.00 6.00
4.00 Log CFU/ml Log CFU/ml 5.00 3.00 4.00 2.00 3.00 1.00 0.00 2.00 E.coli Listeria E.coli Listeria
Figure 2.12 a.The Log reduction tests of JA-144 against E.coli K12 and L.innocua in bacterial broth. b.The Log reduction tests of JA-144 against E.coli K12 and L.innocua incorporated into the film.
JA-144(6.25ppm), JA-144(3.125ppm), TH-4(25ppm) and TH-4(12.5ppm) and C.coli TH-8(50ppm) in broth against C.jejuni TH-8(50ppm) in broth against C.jejuni C.coli Control Control 10.00 10.00 9.00 9.00 8.00 8.00 7.00 7.00
6.00 6.00 5.00 5.00 4.00
Log CFU/ml 4.00 Log CFU/ml 3.00 3.00 2.00 2.00 1.00 1.00 0.00 0.00 JA-144 TH-4 TH-8 JA-144 TH-4 TH-8
Figure 2.13 a.The Log reduction tests of JA-144, TH-4 or TH-8 against C.jejuni in bacterial broth. b. The Log reduction tests of JA-144, TH-4 or TH-8 against C.coli in bacterial broth.
91 JA-144(6.25ppm), TH-4(25ppm) JA-144(3.125ppm), and TH-8(50ppm) in tapioca C.jejuni TH-4(12.5ppm) and C.coli ilm against C.jejuni TH-8(50ppm) in tapioca ilm Control Control against C.coli 10.00 10.00 9.00 9.00
8.00 8.00 7.00 7.00 6.00 6.00
Log CFU/ml 5.00 Log CFU/ml 5.00 4.00 4.00 3.00 3.00 2.00 2.00 1.00 1.00 0.00 0.00 JA-144 TH-4 TH-8 JA-144 TH-4 TH-8
Figure 2.14 a.The Log reduction tests of JA-144, TH-4 or TH-8 against C.jejuni incorporated into the film. b. The Log reduction tests of JA-144, TH-4 or TH-8 against C.coli incorporated into the film.
2.5 Discussion
2.5.1 Minimum Inhibitory Concentration (MIC) tests for JA-144, TH-4
and TH-8.
In the present study, MIC values for JA-144, TH-4 and TH-8 against E.coli K12,
L.innocua, C.jejuni, and C.coli were determined. It is to be noted that MIC values for
JA-144 against C.jejuni and C.coli were significantly lower than E.coli K12 and
L.innocua, suggesting that Campylobacter was more sensitive to JA-144. This can be
ascribed to the fact that aerobic bacteria grow 30 to 50 times faster than microaerobic
bacteria (Mosen, A., 2006). This faster growth rate of aerobic bacteria is indicative of
their greater resistance to drugs, when compared with microaerobes (Diana et al.,
1991). The results also revealed that JA-144 inhibited all tested bacteria, while TH-4
92 and TH-8 were only effective against the Campylobacter species. This could be attributed to the polar hydroxyl group in the JA-144 molecule. Rotunda et al., (2010) reported that hydroxyl groups could disrupt the integrity of biological membranes by binding to the phospholipid bilayer hydrophobic core, solubilizing membrane-associated proteins, and finally causing membrane breakdown and lysis of the bacterial cell membrane.
2.5.2 Bacterial Growth Curve Measurement using JA-144, TH-4, or
TH-8 as inhibitors at Minimum Inhibitory Concentration.
The bacterial growth curve measurement revealed how the bacterial populations were affected by JA-144, TH-4 and TH-8 under their MICs. The OD values provided quantitative information and thus narrowed down the MICs of JA-144, TH-4 and
TH-8 to specific values. However, the OD method is a rough estimate, and the bacterial growth is not observed with OD value until the cell concentration reaches
107 CFU/ml approximately (Christina et al., 2014). Thus it can be concluded that even a small increase observed in the OD values in these experiments could mean that significant changes in microbial populations occurred. Therefore statistical analysis of these OD measurements cannot objectively reflect differences in the microbial populations.
93 2.5.3 Antibacterial activity tests of JA-144, TH-4 or TH-8 with or
without films against E.coli K12, L.innocua, C.jejuni and C.coli at each
MIC.
The viable cell count method used in the antibacterial activity testing provided
quantitative estimations for the efficacies of JA-144, TH-4 and TH-8 against E.coli
K12, L.innocua, C.jejuni and C.coli at their MICs. In this present study, besides
investigating the antibacterial activities of JA-144, TH-4 or TH-8 in aqueous solutions,
the small molecules were also tested to see if they would retain the antibacterial
activities when incorporated into the tapioca films. Typically, when an antimicrobial
agent is incorporated into a film or bonded to the surface as a coating, it can lose some
of its efficacy. If the functional groups of the antimicrobial agent bind too tightly to
the polymer, the releasing rate will be highly restricted when it contacts a
contaminated surface (Han, J.H., 2000), as a consequence, the microorganism can
grow instantly before the antimicrobial agent is released. On the other hand, when the
releasing rate of antimicrobial agent is faster than the growth rate of the target
microorganism, the antimicrobial agent will be depleted and lost its efficacy quickly
since packaged food has an almost infinite volume compared to the amount of
antimicrobial agent (Raija Ahvenainen, 2003). In addition, the processing method of
the film could act to interfere with the antimicrobial function of the active agent
(Wen-Xian et al., 2015).
Reduction in the activities of the compounds when incorporated into the films
could be attributed to their functional groups if they interact negatively with the
94 polymers. For example, the hydroxyl group in JA-144 was more likely to interact with the tapioca starch granules and glycerol since they are hydrophilic. However, as for
C.jejuni and C.coli, the losses in efficacies for JA-144 were only 0.25% and 2.03%.
These were significantly lower than the reductions obtained when exposed to E.coli
K12 or L.innocua. This could be attributed to the microaerobic nature of the
Campylobacter species and the fact that this lead to their slow growth rate. Based on this premise, it can be concluded that the release rate of JA-144 is optimal when considering the Campylobacter’s growth rate.
2.6 Conclusion
In this study, the antimicrobial effect of the synthesized small molecules (JA-144,
TH-4 and TH-8) against E.coli K12, L.innouca, C.jejuni and C.coli were evaluated qualitatively and quantitatively. The MICs of these molecules were investigated and log reduction numbers at the MIC values were determined. The study also investigated the effects of incorporation of the compounds into the films on the antibacterial activity of these molecules. It could be concluded that tapioca films incorporated with JA-144 have great potential to be used in packaging, and reduce bacterial growth on ready-to-eat meats, fresh produce, and other foods. However, additional researches on the release rate of the compounds from the films are needed since this is indispensable to understanding of how the molecules will behave within the film. Besides, the mechanical properties of the films should be investigated to ascertain if the functionality is affected by the addition of small molecule compounds.
95
Chapter 3
The Incorporation of Selected Small Molecule Compounds into
Tapioca Films and their Effects on Thickness, Moisture Content,
Oxygen and Water Barrier Properties and Mechanical Properties
of the films
3.1 Abstract
The synthesized small molecules (JA-144, TH-4 and TH-8) in this study were incorporated into the tapioca starch film, and their effects on the film’s thickness, moisture content, water activity, water vapor permeability (WVP), oxygen permeability coefficient (OPC), melting temperature, glass transition temperature,
X-ray diffraction, as well as storage/loss modulus as function of temperature were investigated. The thickness, moisture content, and water activity of JA-144 incorporated tapioca film were 0.198±0.004mm, 16.021±0.664%, 0.270, which were significantly different (p<0.05) from control film 0.173±0.005mm, 12.900±0.210%,
0.266, respectively. For TH-4 and TH-8 films, they were 0.170±0.06mm,
10.183±0.107, 0.252, and 0.172±0.002mm, 9.824±0.100, 0.259, respectively, which were not significantly different (p>0.05) from control film. The WVP of JA-144 film was 8.183×10-11±2.752×10-13 g×m-1×s-1×Pa-1, which was significantly different
(p<0.05) from control film 6.546×10-11±9.688×10-13g×m-1×s-1×Pa-1. As for TH-4 and
TH-8 films, their WVPs were 5.186×10-11±1.386×10-12g×m-1×s-1×Pa-1,
96 5.389×10-11±4.636×10-13g×m-1×s-1×Pa-1, respectively, which were also significantly different (p<0.05) from control film. The OPC of JA-144 and TH-4 films were
1.084×10-13±2.708×10-15 cc×m-1×s-1×Pa-1, 7.406×10-14±4.206×10-15 cc×m-1×s-1×Pa-1, which were significantly different (p<0.05) from control film 8.564×10-14±3.489×10-15 cc×m-1×s-1×Pa-1. For TH-8 film, the OPC was 8.310×10-14±1.247×10-15 cc×m-1×s-1×Pa-1, which was not significantly different (p>0.05) from control film. The
Differential Scanning Calorimetry (DSC) tests showed that the melting temperature of
JA-144 was 119.84oC. When compared with control film (122.63oC), it decreased by
2.28%. For TH-4 and TH-8 films, the melting temperatures were 126.49oC and
130.68oC, respectively. When compared with control film, they increased by 3.15% and 6.56%, respectively. X-ray diffraction tests showed JA-144 did not significantly increase the film’s crystallinity, as no extra peak was observed in X-ray pattern,
However, for TH-4 and TH-8, an extra sharp peak was observed in X-ray patterns indicating that these two molecules were highly crystallized and formed crosslinking effect with amylose/amylopectin or glycerol molecules. The Dynamic Mechanic
o Analysis (DMA) tests showed that Tg of the control and JA-144 films were -67.249 C
o o and -67.249 C, respectively. As for TH-4 and TH-8 film, Tg values were -66.508 C and -66.508oC, respectively. The DMA tests also showed the films’ storage modulus change as function of temperature. The addition of JA-144 decreased storage modulus at temperature above -57oC, while the addition of TH-4 or TH-8 significantly increased storage modulus at temperatures above -14oC. Overall, our results show that
JA-144 film has high moisture content, high water activity, high OPC and WVP,
97 which means low barrier properties, but good mechanical property. As for TH-4 and
TH-8 films, the less moisture content, low water activity, low OPC and WVP, which means good barrier properties, but low mechanical property. These results indicate that JA-144, TH-4 and TH-8 have great potential for antimicrobial packaging compounds, while some modifications are still necessary for their optimal use in packaging.
3.2 Introduction
Increasing demands for environmental-friendly packaging material have triggered sustained researches in the development of starch-based bio-composite films.
Starch is one of the most preferred green packaging materials due to its rapid biodegradable nature, renewable sources and availability at relatively low cost (Liu et al., 2011). Films made from starch could be used to cover food surfaces, separate incompatible zones and ingredients in complex food mixes, form a barrier against oxygen, aroma, oil and moisture, and used to make pouches or wraps for food packaging. Besides, edible films can be used as carriers of functional agents, such as antioxidants or antimicrobials, and can be used to improve the appearance of selected foods (Kester and Fennema, 1986).
Tapioca is obtained from the cassava plant, which is a significant crop in South
America, Africa, Latin America and Asia, and is an economical source of starch (FAO,
2004). Films made from tapioca starch exhibit appropriate physical characteristics, since they are odorless, tasteless, colorless, and good barriers to oxygen. However,
98 tapioca starch-based films could show brittleness with inadequate mechanical properties if not properly made. To overcome this weakness, a plasticizer is generally required because it could reduce the intermolecular forces and increase the mobility of polymer chains, therefore improving the flexibility and extensibility of the film.
Glycerol is one example of a plasticizer used in filmmaking. It shows stability and compatibility with hydrophilic bio-polymeric materials used for packaging (Fernández
Cervera et al., 2004). However, high water solubility and poor water vapor barrier limit the application of hydrophilic materials such as starch-based films. To solve these problems, the blending of starch with different biopolymers or the addition of hydrophobic materials such as oils or waxes have been proposed (Xu et al., 2005;
García et al., 2000; Anker et al., 2001).
Antimicrobial (AM) agents have been found to be effective against food-borne pathogens. It is reported that the effectiveness of AM packaging is greater than direct addition of AM agents into foods due to two factors. One is the lower release rate of the AM agent from the material to the food, thus enabling functionality over a longer period. The other factor is inactivation concerns (such as neutralization, hydrolysis, or dilution) when the AMs are directly added into the food. Various types of AM agents have been incorporated into edible films. Examples of these include benzoic, sorbic, propionic, and lactic acids, nisin, and lysozyme, to retard surface growth of bacteria, yeasts, and molds on a wide range of products, including meats and cheeses (Islam et al., 2002; Lahellec, et al., 1981; Lueck, E., 1976; Moir, et al., 1992; Reddy, et al.,
1982; Sofos, et al., 1981). Small organic molecules have always been of interest to
99 chemists and biochemists due to their capability of exerting powerful effects on the functions of macromolecules that comprise living systems (Marian et al., 1997). As one of the most important therapeutic agents, small organic molecules have benefits such as improved stability over peptides in oral administration, synthetic accessibility, and optimization convenience for compound bioactivity when compared with macromolecules (Pathania et al., 2009). Synthesized small molecules are generally used to affect the growth of bacteria in two ways: by killing the bacteria, or inhibiting the growth of the bacteria. However, the incorporation of additives into the matrix of a polymer may alter its mechanical and barrier properties, which are two important factors known to affect the performance of edible films.
The objectives of this study were to investigate how the addition of synthesized small molecules JA-144, TH-4 and TH-8 could affect a tapioca starch-based film’s thickness, morphology, moisture content, oxygen and water vapor permeabilities, glass transition and melting temperatures and mechanical properties.
100 3.3 Materials and Methods
3.3.1 Materials
Tapioca starch powder was purchased from a local supermarket in Columbus,
OH and was irradiated at the OSU Nuclear Reactor Lab, in order to sterilize it. This was used as the main ingredient in the films. Distilled water was used to make a suspension of the starch powder. Glycerol ≥99.0% (Sigma-Aldrich®) was obtained from Fisher Scientific (Fisher Scientific, Fair Lawn, NJ) and used as a plasticizer.
Acetic Acid (ACS Reagent ≥99.7%) was purchased from Sigma-Aldrich® and used as a solvent. Synthesized small molecules referred to as JA-144, TH-4 and TH-8 were obtained from Dr. James Fuch’s lab at the College of Pharmacy, OSU.
3.3.2 Preparation of Tapioca Films with JA-144, TH-4, or TH-8.
Film forming solutions were prepared using blends of tapioca starch and the small molecule compounds (JA-144, TH-4 and TH-8) dissolved in acetic acid with glycerol as a plasticizer. Aliquots of 100 mg of each small molecule compound
(JA-144, TH-4 or TH-8) were first dissolved in 1 ml acetic acid. Tapioca starch 5% w/w and acetic acid solutions (ranging from 0.7% w/w to 5.6% w/w) with small molecules and 1.8% w/w glycerol were dissolved into 100 ml distilled water, as shown in Table 3.1. All dispersions were heated in a water bath (70oC) for 15 min with a glass rod stirring until completely gelatinized. An ultrasonic sonicator
(Graymills Co., Chicago, IL) was used to remove tiny bubbles in the gelatinized solution. The edible films were prepared by casting the solution mixture (107g) into
101 10-inch radius Teflon plates. These plates were oven dried at 45±2oC for 12 hours, then the dried films peeled off from the plate surfaces. The final concentration of each small molecule in the dry film is shown in Table 3.1. The method of achieving the percent small molecules in the films was adapted from a study of fast dissolving oral films reported by Arun et al, 2010. In that study, the percent drugs in the films ranged from 1% to 25%. Films with the same ingredients but without small molecules were also prepared and used as controls. The film thicknesses were measured using a
Magna-Mike 8500 Thickness Gage (Olympus, Japan), with a resolution of 0.001mm.
A total of 5 measurements per film at various locations were taken to determine the film thickness.
Table 3.1 The formula of antimicrobial films based on a dry weight basis.
Amount (g) Amount of Antimicrobial Tapioca Small distilled film Glycerol Acetic Acid starch Molecules water (mL) Control 5 1.8 0.7 0 100
JA-144 5 1.8 0.7 0.07 100
TH-4 5 1.8 0.7 0.07 100
TH-8 5 1.8 0.7 0.07 100
3.3.3 Film Moisture Content and Water Activity.
The moisture content of the prepared films was determined by a gravimetric method. To accomplish this, the samples were dried at 105±2oC in a laboratory oven
(UNE PA, Memmert, Germany) until constant weight was achieved (Jiang et al.,
102 2010). The tests were done by using approximately 1.0g film samples that were previously conditioned for 24 hours at 23oC and 50% relative humidity. These were placed in previously dried and cooled glass petri dishes and kept in the oven for 8 hours. Weights of the samples were taken before and after drying using a 5 decimal point XSE Analytical Balance (±0.01 mg) (Mettler Toledo Co. Toledo, OH). All tests were conducted in triplicates and the average values were recorded.
As for water activity measurement, each sample (2.0g) was placed into weighting scale, and their initial water activity was determined using a water activity meter
(AquaLab ®, Decagon Devices Inc., Pullman, Washington, USA).
3.3.4 Oxygen Permeability Coefficient.
Oxygen (O2) permeability of the films was tested using an OX-TRAN® Model
2/21 Series OTR instrument (Mocon Inc., Minneapoils, MN). The method of oxygen permeability testing was done according to the ASTM D3985 method with some modifications. The oxygen transmission rates of the film samples were measured at
23oC and 0% relative humidity. An Aluminum mask manufactured by Modern
Controls Inc. (Minneapolis, MN) was placed on each film to make a test area of 5 cm2.
Tests were performed after 12 hours conditioning. Nitrogen carrier gas was used to purge the chamber of the diffusion cell while oxygen gas flowed over one side of the sample. The flow rate of the nitrogen carrier gas was 10 cm3/min. Oxygen gas that permeated through the film into the nitrogen carrier gas was transported to the detector at the flow rate of 10 cm3/min. An oxygen-sensitive coulometric sensor was
103 used to measure the quantity of oxygen that permeated the material (ASTM, 2004).
The results were obtained using a Model 34401A multimeter manufactured by
Hewlett-Packard Company (Loveland, CO). Frequent calibration of the instrument was performed with a standard PET film sample obtained from Modern Control Inc.
(Minneapolis, MN). Duplicate measurements on two pouches for each condition were obtained.
3.3.5 Water vapor permeability (WVP)
The WVP tests were conducted using the ASTM E96 (1996) Method with some modifications. Test cups with 50cm2 open area were filled with 10g anhydrous calcium sulfate to produce a relative humidity of 0% inside the cup. The film samples were placed on top of the cup and sealed with an O-ring. A high vacuum silicone sealant was applied between the O-ring and the film samples, between the sealing lip of the cup and the sample, before clamping them with 4 screws. The cups containing the desiccants were weighed to give the initial weight and then placed in a humidity chamber at room temperature (23±2oC) and relative humidity of 55±2%. At an hour intervals the cups were weighed until a steady state was reached. The water vapor transmission rate (WVTR) through the film was estimated from the linear portion of the plot of weight gained versus time and the slope divided by the film exposure area according to Equation 3.2. Three replicates per film were tested. The WVP of the film was calculated by multiplying the WVTR by the film’s thickness and dividing saturated by the vapor pressure difference across the film and surface area of the
104 sample exposed to the storage environment. (Equation 3.3).
���� = ! (3.2) !
���� = ����× ! (3.3) !×△!
Where: A is the surface area (m2) of the sample exposed to moisture;
ΔP is the driving force, describing the humidity difference between two sides of film (Pa). In this test, Relative Humidity (RH) was determined by measuring dry bulb and wet bulb temperature during the tests and RH value was obtained by using Psychometric Chart. The driving force ΔP was based on Saturation Pressure of
Water Vapor, which was corresponding to dry bulb temperature during the test. In this case, water vapor saturation pressure was 7.30×103 Pa according to Saturation
Pressure & Temperature Chart.
T is thickness of the film (m);
Q is weight change of WVT cups (g)
t is period of time (day)
All tests were conducted in triplicate and the units for WVPC were g×m-1×s-1×Pa-1.
3.3.6 Thermal analysis By Differential Scanning Calorimetry (DSC)
A TA instrument (Q2000, USA) differential scanning calorimeter equipped with a data collection station was used to scan the thermal transitions of the tested films.
The samples were weighed (ranging from 5 to 7 mg) using a 5 decimal point XSE
Analytical Balance (±0.01 mg) (Mettler Toledo Co. Toledo, OH) in aluminum pans
105 followed by sealing with inverted lids, for optimum thermal conductivity. The reference was an empty aluminum pan sealed in the same manner. Both pans were then equilibrated at 20oC for 30 sec to stabilize the baseline followed by scanning until 200oC at a heating rate of 10oC min-1. Thermograms were recorded and analyzed
by the TA Instrument software (Universal Analysis 2000, Version 4.1D). The Tm
(Melting temperature) was identified as the inflexion point of the baseline. Three replicates per film were tested.
3.3.7 X-ray Diffraction
X-ray diffraction was measured using a Rigaku Miniflex 600 diffractometer with vertical goniometer was used (Cu Kα radiation λ= 1.542Å). Operation was performed at 40kV and 20mA. Samples were mounted on a glass and were attached to the equipment holder and X-ray intensity was recorded with a scintillation counter in a scattering angel (2θ) range of 3-50o with a scanning speed of 1o/min. Distances between the planes of the crystals d(Å) where calculated from the diffraction angels (o) obtained in the X-ray pattern, according to Bragg’s law:
nλ = 2dsin(θ) (3.4)
Where λ is the wavelength of the X-ray bean and n is the order of reflection.
From the scattering spectrum, the effective percent crystallinity of the films was determined, according to Hermans and Weidinger (1961), as the ratio of the integrated crystalline intensity to the total intensity. Crystalline area was evaluated on the basis of the area of the main peaks. Due to the complexity of the system, the calculated
106 crystallinities were not taken as absolute value, but were rather used for comparative purposes.
3.3.8 Mechanical testing by Dynamic Mechanical Analysis (DMA)
The mechanical properties of the films were determined using a stress-controlled
Dynamic-Mechanical Analyzer (DMA 2980, TA Instruments, Surrey, England) at a frequency of 1Hz from -80 to 100oC with a heating rate of 5oC min-1. Film samples
(25 mm in length, 5 mm in width, and 6-8 mil in thickness) were equilibrated at room temperature (23±2oC) and relative humidity of 55±2% for 48 hours prior to the analysis. The storage (‘E) and loss modulus (“E) as well as tan δ of the film samples were monitored as a function of temperature. Three replicates per film were tested.
3.3.9 Statistical Analysis
All data were analyzed using the analysis of variance (ANOVA), and Tukey’s multiple comparisons test was used to determine the significant differences between the means at a level of p<0.05. The statistical analysis was used to compare and determine the significant effect of the addition of JA-144, TH-4, TH-8 on the thickness, moisture content, and the mechanical and barrier properties of the tapioca films. SPSS Version 10.0 software (SPSS Inc., Chicago, IL) was used for this purpose.
Data are presented as mean and standard deviation of duplicate or triplicate analysis.
107 3.4 Results
3.4.1 Thickness, Moisture Content, and Water Activity
The thickness of the films was average from 5 readings taken from 5 random places on the films. Results are shown in Table 3.2. The film thicknesses ranged from
0.171±0.006mm for the TH-4 films to 0.198±0.004mm for JA-144 films. The thickness of the films did not significantly (p>0.05) increased after the addition of
TH-4 and TH-8. However, it significantly (p<0.05) increased after the addition of
JA-144 when compared with the control film.
The moisture content and water activity of the Tapioca films with and without
JA-144, TH-4, TH-8 were measured and are also shown in Table 3.2, and Figure 3.1.
From the results obtained, the JA-144 film had the highest moisture content value and water activity of 16.021±0.664%, 0.270.
Table 3.2 A Summary of Thicknesses, Moisture Contents and Water Activity of Tapioca films with JA-144, TH-4 or TH-8. Moisture Sample Compound Thickness1 Standard Standard Water Content2 ID conc. % (mm) Deviation Deviation Activity (%) Control 0 0.173a 0.005 12.900a 0.210 0.266
JA-144 1 0.198b 0.004 16.021b 0.664 0.270
TH-4 1 0.170a 0.006 10.183c 0.107 0.252
TH-8 1 0.172a 0.005 9.824c 0.100 0.259 1 Each data point is expressed as the mean of five measurements 2 Each data point is expressed as the mean of three measurements a-c In a given column, values with same letters are not significantly different (p>0.05), while different letters are significantly different (p<0.05).
108 Moisture Contents Water activity
18.000 0.275
16.000 0.27 14.000 0.265
12.000 w a 0.26 10.000
8.000 0.255
Moisture Content in the Films % 6.000 0.25 Ja-144 Th-4 Th-8 Control Ja-144 Th-4 Th-8 Control Figure 3.1 The summary of Moisture Content and Water Activity of Tapioca Films with JA-144, TH-4 or TH-8.
3.4.2 Water Vapor Permeability.
The water vapor permeabilities of the films are shown in Table 3.3 and Figure
3.2. As can be seen from the results, the WVP of the tapioca film without compound was 6.546×10-11±9.688×10-13 g×m-1×s-1×Pa-1. The JA-144, TH-4, and TH-8 films produced WVP values of 8.183×10-11±2.752×10-13, 5.186×10-11±1.386×10-12,
5.389×10-11±4.636×10-13 g×m-1×s-1×Pa-1, respectively. The WVP of the films was
Water Vapor Permeabilities for Tapioca Films with JA-144, TH-4 or TH-8 9E-11
-1 8E-11 × Pa -1 7E-11 × s -1 6E-11
5E-11
WVP in g× m 4E-11 Ja-144 Th-4 Th-8 Control Figure 3.2 The Summary of WVP of Tapioca films with JA-144, TH-4 or TH-8.
109 significantly decreased (p<0.05) after the addition of TH-4 and TH-8. However, it was significantly increase (p<0.05) after the addition of JA-144 compared with the control group.
Table 3.3 The Summary of WVP calculations for Tapioca Films with JA-144, TH-4 or TH-8.
Sample WVTR Relative WVP1 Standard ΔP (Pa) ID (g/s) Humidity1 (%) (g×m-1×s-1×Pa-1) Deviation
JA-144 8.344×10-6 56.9 4.15×103 8.183×10-11a 2.752×10-13
TH-4 5.962×10-6 56.9 4.15×103 5.186×10-11b 1.386×10-12
TH-8 6.323×10-6 56.9 4.15×103 5.389×10-11b 4.636×10-13
Control 7.708×10-6 56.9 4.15×103 6.546×10-11c 9.688×10-13 1 Each data point is expressed as the mean of three measurements. a-c In a given column, values with same letters are not significantly different (p>0.05), while different letters are significantly different (p<0.05).
3.4.3 Oxygen Permeability Coefficient.
The Oxygen Permeability Coefficients (OPC) of the films is shown in Table 3.4 and Figure 3.3. As can be seen from the results, the OPC of the tapioca film without compound was 8.564×10-14±3.489×10-15 cc×m-1×s-1×Pa-1. The JA-144, TH-4, and
TH-8 films produced OPC values of 1.084×10-13±2.708×10-15,
7.406×10-14±4.206×10-15, 8.310×10-14±1.247×10-15 cc×m-1×s-1×Pa-1, respectively. The
OPC of the films did not significantly decreased (p<0.05) after the addition of TH-8.
However, it was significantly increased (p<0.05) after the addition of JA-144 or decreased on the addition of TH-4 and TH-8 when compared with control group.
110 Oxygen Permeabilities Coef icient for Tapioca Starch with JA-144, TH-4, or TH-8 × -1 1.2E-13
1.1E-13
1.0E-13
9.0E-14 -1 8.0E-14 × Pa -1 s 7.0E-14
6.0E-14
5.0E-14
4.0E-14
Oxygen Permeability Coef icienct cc× m Ja-144 Th-4 Th-8 Control
Figure 3.3 The Summary of OPC of Tapioca films with JA-144, TH-4 or TH-8.
Table 3.4 The Summary of OPC calculations for Tapioca Films with JA-144, TH-4 or TH-8.
Sample OTR Permeant OPC1 Standard ID (cc×m-2×s-1) Conc. (%) (cc×m-1×s-1×Pa-1) Deviation
Control 1.519×10-5 21 8.564×10-14a 3.489×10-15
JA-144 1.470×10-5 21 1.084×10-13b 2.708×10-15
TH-4 1.241×10-5 21 7.406×10-14c 4.206×10-15
TH-8 1.195×10-5 21 8.310×10-14a 1.247×10-15 1 Each data was expressed as the mean of three measurements. a-c In a given column, values with same letters are not significantly different (p>0.05), while different letters are significantly different (p<0.05).
111 3.4.4 Thermal Analysis by Differential Scanning Calorimetry
The differential Scanning Calorimetric curves in Figure 3.4 display the thermally induced endothermic transitions for JA-144, TH-4 and TH-8 tapioca films from 20oC
o to 200 C. As shown in Figure 3.4, the melting temperature (Tm) of the control,
JA-144, TH-4 and TH-8 films were 122.63oC, 119.84oC, 130.68oC, and 126.49oC, respectively. Both the control and JA-144 films exhibited a single endothermic peak, which indicated homogeneity of the films. This is an indication that JA-144 blended well within the molecular structure of the starch. For the TH-4 or TH-8 films, an extra small peak was observed at 87.14oC or 90.95oC, respectively, suggested that TH-4 or
TH-8 was not completely incorporated into the molecular structure of the starch film.
The results also reflected that the addition of JA-144, TH-4, and TH-8 caused a shift
Figure 3.4 The Differential Scanning Calorimetry of Tapioca films with JA144, TH-4, or TH-8 at 1%.
112 in the Tm, indicated the film’s crystallinity was affected by the compounds. In addition, a broadening of the peaks when the compounds were added to the starch is an indication of increased variability of the crystals within the molecular structure of the starch.
3.4.5 X-ray diffraction pattern
The X-ray patterns of Tapioca film only, and with 1% w/w JA-144, TH-4, or
TH-8 films are presented in Figure 3.5. The main peak positions of each sample are summarized in Table 3.5.
Control (A) JA-144 (B) 2500 4000 3500
2000 -1 -1 3000 1500 2500 2000 1000 1500 Intensity s Intensity s 1000 500 500 0 0 0 10 20 30 40 50 0 10 20 30 40 50 2-theta 2-theta
TH-4 (C) TH-8 (D) 6.0E+05 1.6E+05 5.0E+05 1.4E+05
-1 1.2E+05 s-1 4.0E+05 1.0E+05 3.0E+05 8.0E+04 2.0E+05 6.0E+04 Intensity Intensity s 4.0E+04 1.0E+05 2.0E+04 0.0E+00 0.0E+00 0 10 20 30 40 50 0 10 20 30 40 50 2-theta 2-theta
Figure 3.5 The X-ray Diffraction of Tapioca films with JA-144, TH-4 or TH-8.
113 Table 3.5 The position of main X-ray diffraction peaks of Tapioca Films with JA-144, TH-4 or TH-8.
Sample Peak Position (o) ID Control 3.8 7.2 7.6 19.4 21.2 28.5 31.2 38.7
JA-144 3.8 7.6 19.4 21.2 28.5 31.2 38.7
TH-4 3.8 7.6 11.4 19.4 31.2 34.8 38.7 42.5
TH-8 7.6 9.8 19.4 21.2 28.5
As shown in Figure 3.5, all samples show different diffraction patterns. The
Tapioca film was mostly amorphous in nature with one diffuse peak located at 21.20o
and other small crystalline fraction imbedded in the amorphous matrix as summarized
in Table 3.5. By adding JA-144 into the film, it showed increase intensity towards
crystallization in the 21.2o region. By adding TH-4 into the film, the X-ray pattern
showed a strong peak at 3.8o and small distinct peaks at 7.6o, 11.4o, 19.4o and smaller
peaks between 30o and 40o, as shown in Table 3.5. When TH-8 was added to the
tapioca, the X-ray patterns showed a strong peak at 7.6o, smaller peaks at 9.8o, 19.4o,
21.2o and 28.5o, respectively.
3.4.6 Mechanical testing by Dynamic Mechanical Analysis (DMA)
In Dynamic Mechanical Analysis (DMA) experiments, information on the
storage modulus, loss modulus and tan δ of the tapioca films with JA-144, TH-4, and
TH-8 were obtained. The data were used to determine glass transition temperature Tg
as well as the stress-strain curve as a function of time. By definition, temperature
114 corresponding to a sharp decrease in storage modulus, or a maximum value of loss modulus and tan δ during a temperature sweep is the glass transition temperature
(Sperling, 2001). Storage modulus provides important information of film’s ability to store energy in response to an applied force at given temperatures. It is also called elastic modulus and relates to the inherent stiffness of the sample. Figure 3.5 shows the effects of JA-144, TH-4, TH-8 addition on the storage modulus over the entire temperature range of the DMA. The data show that the addition of JA-144, TH-4, and
TH-8 had significant effects (p<0.05) on the modulus of the test films. Particularly, the addition of JA-144 decreased the storage modulus at temperature above -57oC, while the addition of TH-4 or TH-8 significantly increased storage modulus at temperatures above -14oC, as shown in Figure 3.6.
The results shown in Figures 3.7 and 3.8 were used to determine the effect of
JA-144, TH-4, or TH-8 on the loss modulus and tan δ of the films. From this
information the Tg for the films were determined, which was estimated to be
o o -67.249 C and -67.249 C, respectively. As for TH-4 and TH-8 film, Tg values were the highest, which were -66.508oC and -66.508oC, respectively.
115 Figure 3.6 The effect of JA-144, TH-4, or TH-8 on the storage modulus of tapioca films.
Figure 3.7 Loss Modulus of Tapioca films with JA-144, TH-4, or TH-8.
116 Dynamic Mechanic Analysis of Tapioca Films with JA-144, TH-4, or TH-8 1 Tan Control 0.99 Tan TH-4 0.98 Tan JA-144 0.97 Tan TH-8
0.96
0.95
Tan Delta 0.94
0.93
0.92
0.91
0.9 -71.266 -55.966 -40.972 -25.927 -11.009 4.015 19.015 34.005 48.994 64.007 79.020 93.995 Temperature oC Figure 3.8 Tan Delta of Tapioca Films with JA-144, TH-4, or TH-8.
3.5 Discussion
3.5.1 Thickness, Moisture Content, Water Activity, and barrier properties
(OPC, WVP)
The results showed that thicknesses of the films did not significantly increase after the addition of TH-4 and TH-8. However, it was significantly increased after the addition of JA-144 compared with control group. The thickness increase caused by
JA-144 was possibly due to the hydroxyl groups in the compound and its hydrogen bonding to the polymeric chains (Lagos et al., 2015). The existence of hydroxyl group in JA-144 also played a key role in film’s moisture content and water activity due to its hygroscopic character. This interaction with the film’s matrix increased the spaces between the chains, thus facilitating water migration into the film, and consequently,
117 increasing its moisture content and water activity, as shown in Figure 3.9 (da Matta, et al., 2011). These structural changes within the JA-144 films further affected their barrier properties, which are shown in the OPC and WVP tests. As shown in Table
3.3 and Table 3.4, the WVP and OPC of JA-144 are 8.183×10-11 g×m-1×s-1×Pa-1,
1.084×10-13 cc×m-1×s-1×Pa-1, respectively, which are 25.00% and 26.58% higher than the control film. (Chien-Hsien, et al., 2007).
As for TH-4 and TH-8 films, the results indicate that there was no significant change in thickness, and they had lower moisture content and water activity, but good water and oxygen barrier properties. These could be attributed to the sulfonamide functional group in TH-4 and TH-8. The sulfonamide functional group is rigid, hydrophobic, and typically has a tendency toward crystallization (Seong et al., 2001).
These characters of sulfonamide functional group in TH-4 and TH-8 could probably enhance the crosslinking effect within the film matrix, and consequently lead to less moisture content, less water activity, but good oxygen and water barrier properties.
Figure 3.9 A possible mechanism of JA-144 bind to Amylose or Glycerol.
118 3.5.2 Mechanical and Thermal Properties
As discussed previously, JA-144 film showed hydrophilic characteristics, while
TH-4 and TH-8 films were hydrophobic, rigid, and had a tendency toward crystallization due to the sulfonamide functional group. Therefore, it is expected that
JA-144 film should have lower Tg, lower Tm, and less storage modulus due to its low crystallinity, higher amorphous, and high flexibility characteristics compared to the control film. As for TH-4 and TH-8 films, it is expected that they should show a
higher Tg, higher Tm, and more storage modulus due to the presence of halogens in their structure. These have a tendency to increase crosslinking and subsequently increase the crystallinity of the polymer (Jianwei et al., 2005).
These expectations were proved by the DSC, X-ray diffraction and DMA tests.
o The Tm of JA-144 was 119.84 C, which is 2.28% lower than that of the control; while for TH-4 and TH-8 films, they are 3.15% and 6.56% higher than the control, respectively. The storage modulus reflected the stiffness of film, showed a value
-57oC for JA-144 film, and this was lower than the control film. For the TH-4 and
TH-8 films, higher storage modulus values were expected and the results show that these values were significantly higher than the control film (above -11oC), as shown in
Figure 3.6.
X-ray diffraction tests further detect these minor changes. As shown in Figure
3.5, the addition of JA-144 resulted in a slightly increase of crystallinity. This could be due to the intra-molecular interactions of hydroxyl groups between amylose and
JA-144 that led to the formation of intramolecular hydrogen bonding. The increase in
119 peak intensity is in the amorphous regions of the control sample, which indicates good blending of JA-144 with the starch molecules and increase in its crystallinity.
However, the addition of TH-4 and TH-8 resulted in a dramatic increase of crystallinity. X-ray patterns for these two were significantly changed, which could be ascribed to two possibilities. First, it is possible that crosslinking effect between
TH-4/TH-8 and starch amylose were formed when adding TH-4/TH-8 into the film forming solution. Crosslinking restricts molecular mobility, tying the polymer backbones together thus resulting in crystallinity increase (Shulamit et al., 2008).
Second, it is possible that TH-4/TH-8 molecules are not well blended into the film as compared to JA-144. Previous research demonstrated that sulfonamide groups are rigid, hydrophobic, and typically has a tendency toward crystallization (Seong et al.,
2001). Since the film forming solution is hydrophilic, it is possible that these
TH-4/TH-8 molecules were self-assembled or aggregated during the blending process
(Kazunari et al., 1992). As a result, a high intensity peak shown in the X-ray diffraction was observed.
An ideal edible film with incorporated compounds for antimicrobial application should have less moisture content, good barrier properties, and good mechanical properties (Bhanu et al., 2015). Based on these criteria, JA-144 film showed high moisture content, high OPC and WVP, which means low barrier properties, but not strong mechanical properties due to the plasticizing and co-plasticizing effect of hydroxyl group with water molecules. This means that the JA-144 films will dissolve faster in the mouth and needs less chewing since it showed a lower mechanical
120 strength and higher moisture holding capacity. As for TH-4 and TH-8 films, when compared with JA-144, they showed less moisture content, low OPC and WVP, which mean good barrier properties, but higher mechanical strength due to the crosslinking effect of sulfonamide group. This means the TH-4 and TH-8 films show better characteristics for applications such as packaging and wrapping, which require more mechanical strength and less gas permeability. Also, these compounds were not completely incorporated into the film as both DSC curves showed an extra peak at
87.14oC and 90.95oC. Consequently, future work should be focused on how to optimize the structure of these molecules, or to introduce another chelating agent to offset drawbacks of each compound. By doing so, it is to be expected that these compounds could be better incorporated into the film.
3.6 Conclusion
In this study, the synthesized small molecules (JA-144, TH-4 and TH-8) were incorporated into the tapioca starch film, and their effects on film’s thickness, moisture content, water vapor and oxygen permeabilities, melting and glass transition temperatures, X-ray diffraction, as well as storage/loss modulus were investigated. It is concluded that the JA-144 films had high moisture content, high OPC and WVP, which meant a low barrier properties, but good mechanical property due to the plasticizing and co-plasticizing effect of hydroxyl group with water molecules, which could make the JA-144 films good for chewing. As for TH-4 and TH-8 films, the lower moisture content, OPC and WVP mean that they have good barrier properties,
121 but stronger mechanical strength due to the crosslinking effect of sulfonamide groups.
As a conclusion, JA-144, TH-4 and TH-8 have great potential for antimicrobial packaging compounds, while some modifications are still necessary for their optimization.
122
LIST OF REFERENCES
1. Ahn J., Grün I.U., Mustapha A., 2004, Antimicrobial and antioxidant activities
of natural extracts in vitro and in ground beef. Journal of Food Protection,
Vol.67, pp.148-155.
2. Alkawash, M.A., Soothill, J.S., Schiller, N.L., 2006, Alginate lyase enhances
antibiotic killing of mucoid Pseudomonas aeruginosa in biofilms. APMIS.
Vol.114, pp.131-138.
3. Anker, M., Berntsen, J., Hermansson, A.M., Stading, M., 2001. Improved water
vapour barrier of whey protein films by addition of an acetylated monoglyceride.
Innovative Food Science and Emerging Technologies. Vol.3, pp.81–92.
4. Antuono L.F., Gulletti G.C., Bocchini P., 2000, Variability of essential oil
content and composition of Origanum vulgare L. populations from a north
Mediterranean area. Annuals of Botany, Vol.86, pp.471-478.
5. Appendini, P., Hotchkiss, J.H. 2003, Review of antimicrobial food packaging.
Innovative Food Science and Emerging Technologies. Vol.3, 113-126.
6. Arun A., Amrish C., Vijay S., Kamla P., 2010, Fast Dissolving Oral Films: An
Innovative Drug Delivery System and Dosage Form. International Journal of
ChemTech Research, Vol.2, No.1, pp.576-583.
123 7. Aruna D., Sasikala P., Lavanya R., Kavitha V., Yazhini G., M.Shakila B., 2012,
Edible Films from Polysaccharides. Food Science and Quality Management,
Vol.3 ISSN 2224-6088.
8. Arunya P., Tanaboon S., Theeranun J., John M., 2011, Krochtac.Whey protein–
okra polysaccharide fraction blend edible films: tensile properties, water vapor
permeability and oxygen permeability. J Sci Food Agric. Vol.91, pp.362-369.
9. Attila E. Pavlath and William Orts, 2009. Edible Films and Coatings: Why,
What, and How? In:Ambuscade ME, Huber KC. Edible Films and coatings for
food applications. Springer Science Business Media, LLC, New York, NY.
pp.18-19.
10. Baldwin E.A., Nisperos-Carriedo M.O., Baker R.A., 1995, Use of Edible
Coatings to Preserve Quality of Lightly Processed Products. Crit. Rev. Food Sci,
Vol.35, Issue.6, pp.509-524.
11. Balows A., 1972, Current techniques for antibiotic susceptibility testing.
Springfield, IL: Charles C. Thomas.
12. Bang, S.H., Jang, A., Yoon, J., Kim, P. et. al., 2011, Evaluation of whole
lysosomal enzymes directly immobilized on titanium (IV) oxide used in the
development of antimicrobial agents. Enzyme Microb. Technol. Vol.49,
pp.260-265.
13. Bennik, M.H., Vanloo, B., Brasseur, R., Gorris, L.G., Smid, E.J., 1998. A novel
bacteriocin with a YGNGV motif from vegetable-associated Enterococcus
124 mundtii: full characterization and interaction with target organisms. Biochim.
Biophys. Acta VOl.1373, pp.47-58.
14. Berg S., Bretz M., Hubbermann E.M., Schwarz K., 2012, Influence of different
pectins on powder characteristics of microencapsulated antho-cyanins and their
impact on drug retention of shellac coated granulate. Journal of Food
Engineering, Vol.108, No.1, pp.158-165.
15. Bermingham, A.; Derrick, J. P., 2002, The folic acid biosynthesis pathway in
bacteria: evaluation of potential for antibacterial drug discovery. Bioessays.
Vol.24, Issue.7, pp.637-48.
16. Bhanu M., Anu K., Harsha K. 2015. Antimicrobial food packaging: potential
and pitfalls. Frontiers in Microbiology, Vol.6, pp.611-619.Chien-Hsien C.,
Lih-Shiuh L., 2008. Mechanical and water vapor barrier propertiesof tapioca
starch/decolorized hsian-tsao leaf gum films in the presence of plasticizer. Food
Hydrocolloids. Vol.22, pp.1584-1595.
17. Blake P.A., 1979, Disease caused by a marine vibrio—clinical characteristics
and epidemiology. N. Engl. J. Med. Vol.300, pp.1-5.
18. Blaser, M. J., J. Engberg, I. Nachamkin, and C. Szymanski. 2008. Clinical
aspects of Campylobacter jejuni and Campylobacter coli infections.
Campylobacter: 99-121.
19. Bochner, B.R., Savageau, M.A., 1977. Generalized indicator plate for genetic,
metabolic, and taxonomic studies with microorganisms. Applied and
Environmental Microbiology 33, 434-444.
125 20. Bourtoom T., 2008. Edible films and coatings: characteristics and properties.
International Food Research Journal, Vol.15, No.3, pp. 237-248.
21. Briodo A., 1969, A Simple, Sensitive Graphical Method of Treating
Thermogravimetric Analysis Data, Journal of Polymer Science: Part A-2 Vol.7,
pp.1761-1773.
22. Bryne Mubururu, Dinga N. Moyo, Perkins Muredzi. Production of Artificial
Sausage Casings from Whey Proteins. International Journal of Nutrition and
Food Sciences. Special Issue: Optimizing Quality and Food Process Assessment.
Vol. 3, No. 6-1, 2014, pp. 30-38.
23. Burke M.D., Schreiber S.L., 2004, A Planning Strategy for Diversity-Oriented
Synthesis. Angew. Chem., Int. Ed., Vol.43, pp.4658.
24. Burt, S., 2004, Essential oils: their antibacterial properties and potential
applications in foods-a review. International Journal of Food Microbiology,
Vol.94, pp.223-253.
25. Cagri A., Ustunol, Z., Ryser, E., 2004.Antimicrobial edible films and coatings.
Journal of Food Protection Vol.67. pp.833-848.
26. Cai, Y., Ng, L.K., Farber, J.M., 1997. Isolation and characterization of
nisin-producing Lactococcus lactis subsp. lactis from bean-sprouts. J. Appl.
Microbiol. Vol.83, pp.499-507.
27. Carmona-Ribeiro A.M., Carrasco L.D., 2013, Cationic antimicrobial polymers
and their assemblies. International Journal of Molecular Sciences, Vol.14, No.5,
pp.9906–9946.
126 28. CDC. 2014. Incidence and Trends of Infection with Pathogens Transmitted
Commonly Through Food — Foodborne Diseases Active Surveillance Network,
10 U.S. Sites, 2006–2013. MMWR Morb Mortal Wkly Rep. Vol.63, Issue.15.
pp.328-332.
29. Chawengkijwanich, C., Hayata, Y. 2008. Development of TiO2 powder-coated
food packaging film and its ability to inactivate Escherichia coli in vitro and in
actual tests. International Journal of Food Microbiology, Vol.123, 288-292.
30.
31. Chen M., Deng J., Yang F., Gong Y., Zhao N., Zhang X., 2003, Study on
physical properties and nerve cell affinity of composite films from chitosan and
gelatin solutions. Biomaterials Vol.24, pp.2871-2880.
32. Christina L.L., Caelli C.C., Andre G.S. 2014. Mass and Density Measurements
of Live and Dead Gram-Negative and Gram-Positive Bacterial Populations.
Appl. Environ. Microbiol., Vol.80, Issue.12, pp.3622-3631.
33. Claudia A.R.B., Bello-Perez L.A., Gacia M.A., Martino M.N., Solorza-Feria J.,
Zaritzky N.E., 2005, Carbohyd. Polym. Vol.3, Issue.2, pp.156-161.
34. Clinical and Laboratory Standards Institute. 2008, Development of in vitro
susceptibility testing criteria and quality control parameters; approved guideline.
Third ed. CLSI document: M23–A3. Wayne, PA: Clinical and Laboratory
Standards Institute.
127 35. Clinical and Laboratory Standards Institute. 2009, Methods for dilution
antimicrobial susceptibility testing for bacteria that grew aerobically. Approved
Standard M7–A10. Wayne, PA: Clinical and Laboratory Standards Institute.
36. Clinical and Laboratory Standards Institute. 2009, Performance standards for
antimicrobial susceptibility testing. Nineteenth informational supplement
M100–S19. Wayne, PA: Clinical and Laboratory Standards Institute.
37. Coker, A. O., R. D. Isokpehi, B. N. Thomas, K. O. Amisu, and C. L. Obi. 2002.
Human campylobacteriosis in developing countries. Emerging infectious
diseases 8:237-244.
38. Contal L., Leon V., Downey G. 2002. Detection and quantification of apple
adulteration in strawberry and raspberry purees using visible and near infrared
spectroscopy. J. Near Infrared Spectrosc. Vol.10, Issue.4, pp.289-99.
39. Cunningham, F.E., Proctor, V.A., Goetsch, S.J., 1991, Egg-white lysozyme as a
food preservative: an overview. World’s Poult. Sci. J. Vol.47, 141.
40. da Matta, M. D., Jr., Sarmento, S. B. S., Sarantopoulos, C. I. G. L., Zocchi, S. S.
2011. Barrier properties of films of pea starch associated with xanthan gum and
glycerol. Polimeros-Ciencia e Tecnologia, Vol.21, Issue.1, pp.67-72.
41. Dangaran, K. L. and J. M. Krochta. 2008. Whey protein films and
coatings. In Whey Processing, Functionality and Health Benefits. C.
Onwulata and P. Huth (eds.) Blackwell Publishing, Ames, IA.
128 42. David S.J., 1999, Dynamic mechanical analysis of polymeric systems of
pharmaceutical and biomedical significance Vol.179, Issue.2, pp.167-178.
43. Debeaufort, F., Voilley, A., Meares, P., 1994. Water vapor permeability and
diffusivity through methylcellulose edible films. J. Membr. Sci. No.91,
pp.125-133.
44. Diane J.W.B., Speece R.E. 1991. A database of Chemical Toxicity to
Environmental Bacteria and Its Use in Interspecies Comparisons and
Correlations. Research Journal of the Water Pollution Control Federation.
Vol.63, No.3, pp.198-207.
45. Diem M., 1993, Introduction to Modern Vibrational Spectroscopy.New York:
Wiley-Interscience.
46. Drlica, K.; Zhao, X., 1997, DNA gyrase, topoisomerase IV, and the
4-quinolones. Microbiol Mol Biol Rev. Vol.61, Issue.3, pp.377-92.
47. Duncan Q.M., Fiona A.J., 1995, Pharmaceutical applications of dynamic
mechanical thermal analysis. Thermochimica Acta, Vol.97, pp.97-115.
48. Eidus, L., Diena, B.B., Greenberg, L., 1959. Observations on the use of
tetrazolium salts in the vital staining of bacteria. Canadian Journal of
Microbiology 5, 245-250.
49. Emiro_glu, Z. K., Yemis¸, G. P., Cos¸kun, B. K., & Cando_gan, K. 2010.
Antimicrobial activity of soy edible films incorporated with thyme and oregano
essential oils on fresh ground beef patties. Meat Science, Vol.86, 283-288.
129 50. Ennahar, S., Aoude-Werner, D., Sorokine, O., Van Dorsselaer, A., Bringel, F.,
Hubert, J.C., Hasselmann, C., 1996. Production of pediocin AcH by
Lactobacillus plantarum WHE 92 isolated from cheese. Appl. Environ.
Microbiol. Vol.62, pp.4381-4387.
51. Ericsson J.M., Sherris J.C. 1971, Antibiotic sensitivity testing: report of an
international collaborative study. Acta Pathol Microbiol Scand Vol.217, Suppl.
pp.1–90.
52. Ertas O.N., Guler T., Ciftci M., Darlkilic B., Simsek U.G., 2005, The effect of
an essential oil mix drive from oregano, clove and anise on broiler performance.
International Journal of Poultry Science, Vol.4, Issue.11, pp.879-884.
53. FAO, Food and Agriculture Organization, 2004. Proceedings of the Validation
Forum on the Global Cassava Development Strategy. In: Global Cassava
Market Study Business Opportunities for the Use of Cassava, Vol. 6.
International Fund for Agricultural Development, Roma, Italia.
54. Fernández Cervera, M., Karjalainen, M., Airaksinen, S., Rantanen, J., Krogars,
K., Heinämäki, J., Iraizoz Colarte, A., Yliruusi, J., 2004. Physical stability and
moisture sorption of aqueous chitosan–amylose starch films plasticized with
polyols. European Journal of Pharma- ceutics and Biopharmaceutics Vol.58,
pp.69–76.
55. Fernandes, P., 2006, Antibacterial discovery and development: the failure of
success? Nat. Biotechnol. Vol.24, pp.1497-1503.
130 56. Fey P.D., Safranek T.J., Rupp M.E., Dunne E.F., Ribot E., Iwen P.C., et al.,
2000, Ceftriaxone-resistant Salmonella infection acquired by a child from
cattle, N. Engl. J. Med. Vol.342, pp.1242-1249.
57. Finch, R. G.; Greenwood, D.; Norrby, S. R.; Whitley, R. J., 2003, Antibiotic and
Chemotherapy: Anti-infective agents and theri use in therapy. 8th ed.; Churchill
Livingstone, An imprint of Elsevier Science Limited: United Kingdom.
58. Franz, C.M., Du, T.M., Olasupo, N.A., Schillinger, U., Holzapfel, W.H., 1998.
Plantaricin D, a bacteriocin produced by Lactobacillus plantarum BFE 905
ready-to-eat salad. Lett. Appl. Microbiol. Vol.26, pp.231-235.
59. Friedman C.R., Neimann J., Wegener H.C., Tauxe R.V., 2000, In: I. Nachamkin,
M.J. Blaser (Eds.), Campylobacter, 2nd ed., ASM Press, Washington DC, pp.
130-130.
60. Fung, D. Y. C., Kastner, C. L., Lee, C. Y., Hunt, M. L., Dikeman, M. E., &
Kropf, D. 1980. Mesophilic and psychrotrophic populations on hot boned and
conventionally processed beef. Journal of Food Protection, Vol.43, 547-550.
61. Gacesa, P., 1987, Alginate-modifying enzymes: A proposed unified mechanism
of action for the lyases and epimerases. FEBS Lett. Vol.212, pp.199-202.
62. García, M.A., Martino, M.N., Zaritzky, N.E. 2000. Microstructural
characterization of plasticized starch-based films. Starch/ Stärke Vol.52, Issue.4,
pp.118–124.
131 63. Gaudreau C., Gilbert H., 1998, Antimicrobial resistance of clinical strains of
Campylobacter jejuni subsp. jejuni isolated from 1985 to 1997 in Quebec,
Canada, Antimicrob. Agents Chemother. Vol.42, pp.2106-2108.
64. Gennadios A., Hanna M.A., Kurth L.B., 1997, Applications of edible coatings
on meats, poultry and seafoods: a review. Food Science and Technology, Vol.30,
Issue.4, pp.337-350.
65. Germain Y., 1997, Conception de films polymer à perm[abilité contrôlée pour
l'embalage alimentaire. Industriée Alimentaire et Agricules, pp.137–140.
66. Gomez, S., Cosson, C., Deschamps, A.M., 1997. Evidence for a bacteriocin-like
substance produced by a new strain of Streptococcus sp., inhibitory to
gram-positive food-borne pathogens. Res. Microbiol. Vol.148, pp.757-766.
67. Goodfellow, S. J. 1995. Implementation of the HACCP program by meat and
poultry slaughterers. In HACCP in Meat, Poultry and Fish Processing, eds. A.
M. Pearson & T. R. Dutson, pp. 58–71. Glasgow, UK, Blackie Academic &
Professional.
68. Goulard, C., Hlimi, S., Rebuffat, S., Bodo, B., 1995. Trichorzins HA and MA,
antibiotic peptides from Trichoderma harzianum: I. Fermentation, isolation and
biological properties. J. Antibiot. Tokyo Vol.48, pp.1248–1253.
69. Goy Rejane C., Britto Douglas de, Assis Odilio B.G., 2009, A Review of the
Antimicrobial Activity of Chitosan Polímeros. Ciência e Tecnologia, Vol.19,
No.3, pp.1-7.
132 70. Guilbert S., Gontard N., Cup B., 1986, Technology and application of edible
protective films. Packaging Technology and Science. Vol.8, Issue.6,
pp.339-346.
71. Ha, J. U., Kim, Y. M., & Lee, D. S. 2001, Multilayered antimicrobial
polyethylene films applied to the packaging of ground beef. Packaging
Technology and Science, Vol.14, 55-62.
72. Hadacek, F., Greger, H. 2000. Testing of antifungal natural products:
methodologies, comparability of results and assay choice. Phytochem Anal.,
Vol.11, pp.137-147.
73. Han, J.H., 2000. Antimicrobial food packaging. Food Technology 54:56-65.
74. Han J.H., Hwang H.M., Min S., Krochta J.M., 2008, Coating of peanuts with
edible whey protein film containing alpha-tocoperol and ascorbic palmitate. J
Food Sci. Vol.73, pp.349-355.
75. Hardenburg R.E. 1967. “Wax and Related Coatings for Horticultural Products.
A Bibliography.” Agr. Res. Bull. 15-51, Washington, D.C.: U.S. Dept. of
Agric.,
76. Harris N.V., Weiss N.S., Nolan C.M., 1986, The role of poultry and meats in the
etiology of Campylobacter jejuni/coli enteritis, Am. J. Public Health Vol.76,
pp.407-411.
77. Helmerhorst, E.J., van’t Hof, W., Veerman, E.C., Simoons-Smit, I., Nieuw
Amerongen, A.V., 1997. Synthetic histatin analogues with broad-spectrum
antimicrobial activity. Biochem. J. Vol.326, Pt.1, pp.39–45.
133 78. Hermans, P. H., Weidinger, A., 1961. On the determination of the crystalline
fraction of poly-ethylenes from X-ray diffraction. Makromolecular Chemistry,
Vol.44-46, pp.24-36.
79. Herbin, S., Mathieu, F., Brule, F., Branlant, C., Lefebvre, G., Lebrihi, A., 1997.
Characteristics and genetic determinants of bacteriocin activities produced by
Carnobacterium piscicola CP5 isolated from cheese. Curr. Microbiol. Vol.35,
pp.319-326.
80. Hermoso, J.A., García, J.L., García, P., 2007, Taking aim on bacterial pathogens:
from phage therapy to enzybiotics. Curr. Opin. Microbiol. VOl.10, pp.461-472.
81. Hongda C., 1995, Functional Properties and Applications of Edible Films made
of Milk Proteins. Journal of Dairy Science, Vol.78, No.11.
82. Hood E.E., Shen Q.X., Varner J.E., 1988, A developmentally regulated
hydroxyproline-rich glycoprotein in maize pericarp cell walls. Plant Physiology,
Vol.1, pp.138-142.
83. Hoover M.W., Nathan, P. J., 1981. Influence of tertiary butylhydroquinone and
certain other surface coatings on the formation of carbonyl compounds in
granulated roasted peanuts. J. Food Sci. Vol.47, pp.246-248.
84. Hotchkiss J.H., Chen J.H., 1996, Microbiological effects of the direct
acidification of CO2 to pasteurized milk. Journal of Dairy Science Vol.79,
Suppl.87–92.
85. Howard R.J., Lieb S., 1988, Soft-tissue infections caused by halophilic marine
vibrios. Arch. Surg. Vo.l.123, pp.245-249.
134 86. Huang M.B., Baker C.N., Banerjee S., Tenover F.C. 1992, Accuracy of the E
test for determining antimicrobial susceptibilities of Staphylococci, Enterococci,
Campylobacter jejuni, and gram-negative bacteria resistant to antimicrobial
agents. J Clin Microbiol. Vol.30, pp.3243-8.
87. Ilian J., Hans R.B., 1999, Isothermal titratio calorimetry
anddifferential scanning calorimetry as complementary tools to investigatethe
energetics of biomolecular recognition. J. Mol. Recognit. Vol.12, pp.3-18.
88. Ilian J., Hans R.B., 1999, Isothermal titratio calorimetry
anddifferential scanning calorimetry as complementary tools to investigatethe
energetics of biomolecular recognition. J. Mol. Recognit. Vol.12, pp.3-18.
89. Islam, M., J. Chen, M. P. Doyle, and M. Chinnan. 2002. Control of Listeria
monocytogenes on turkey frankfurters by generally-recognized-as-safe
preservatives. J. Food Prot. Vol.65, pp.1411–1416.
90. Iwakokun B. A., Ogunledun A., Ogbolu D.O., Bamiro S.B., Jimi-Omojola J.,
2004, In Vitro Antimicrobial Properties of Aqueous Garlic Extract Against
Multidrug-Resistnat Bacteria and Candida Species from Nigeria. J. Med. Food,
Vol.7, Issue.3, pp.327-333.
91. Janjarasskul T., Krochta J.M. 2010, Edible Packaging materials. Annu Rev Food
Sci Technol. Vol.1, pp.415-448.
92. Jack, R.W., Wan, J., Gordon, J., Harmark, K., Davidson, B.E., Hillier, A.J.,
Wettenhall, R.E., Hickey, M.W., Coventry, M.J., 1996. Characterization of the
chemical and antimicrobial properties of piscicolin 126, a bacteriocin produced
135 by Carnobacterium piscicola JG126. Appl. Environ. Microbiol. Vol.62,
2897-2903.
93. Jayas D.S., Jeyamkondan S., 2002, Modified atmosphere storage of grains,
meats, fruits and vegetables. Biosystems Engineering Vol.82, pp.235-251.
94. Jeannine, B.L., N´ıvea M.V., Rodolfo M.C., Dos, S., Ana-Monica Q.B., Paulo
J.A., 2015. Mechanical properties of cassava starch films as affected by
different plasticizers and different relative humidity conditions. International
Journal of Food Studies. Vol.4, pp.116–125.
95. Jianwei X., Xueming L., Tingting L., Junchao H., Chaobin H. 2005. Synthesis
and Self-Assembly of Di-functional Halogen-Bonding Molecules: A New
Family of Supramolecular Liquid-Crystalline Polymers. Macromolecules,
Vol.38, pp.3554-3557.
96. Jongbloed, J.D., Grieger, U., Antelmann, H., Hecker, M., Nijland, R., Bron, S.,
and van Dijl, J.M. (2004) Two minimal Tat translocases in Bacillus. Mol
Microbiol 54: 1319–1325.
97. Jorgensen J.H. 1993, Selection criteria for an antimicrobial susceptibility testing
system. J Clin Microbiol Vol.31, pp.2841-4.
98. Jorgensen J.H., Ferraro M.J., McElmeel M.L., Spargo J., Swenson J.M.,
Tenover F.C. 1994, Detection of penicillin and extended-spectrum
cephalosporin resistance among Streptococcus pneumoniae clinical isolates by
use of the E test. J Clin Microbiol. Vol.32, pp.159-63.
136 99. Jorgensen J.H., Turnidge J.D. 2007, Antibacterial susceptibility tests: dilution
and disk diffusion methods. In: Murray PR, Baron EJ, Jorgensen JH, Landry
ML, Pfaller MA, eds. Manual of clinical microbiology. 9th ed. Washington, DC:
American Society for Microbiology, pp.1152–72.
100. Juan I.M., John M. K., 1997. Whey Protein and Acetylated Monoglyceride
Edible Coatings: Effect on the Rancidity Process of Walnuts. J. Agric. Food
Chem. Vol.45, pp.2509-2513.
101. Kamper S.L., Fennema O.N., 1984, Water vapor permeability of an edible, fatty
acid, bilayer film. Journal of Food Science, Vol.49, pp.1482-1485.
102. Kaplan H.J. 1986. Washing, waxing, and color adding. In: Wardowdki WF,
Nagy S, Grierson W, editors. Fresh citrus fruit. Westport, Conn.: AVI
Publishing Co. pp.379.
103. Kazunari A., Shigeru D., Nobuhiro M., Shigehiko Y., Junzo S., 1992.
Self-Aggregates of Hydrophobized Polysaccharides in Water Formation and
Characteristics of Nanoparticles. Macromolecules. Vol.26., No.12.,
pp.3062-3068.
104. Kester J.J., Fennema O. R., 1986. Edible films and coatings: a review. Food
Technol Vol.40, Issue.12, pp.47–59.
105. Klaenhaemmer, T. R. 1993. Genetics of bacteriocins produced by lactic acid
bacteria. FEMS Microbiol. Rev. Vol.12, pp.39-85.
137 106. Köksel H, ahbaz F Özboy Ö., 1983, Influence of wheat-drying temperatures on
the birefringence and X-ray diffraction patterns of wet-harvested wheat starch.
Cereal Chem. Vol.70, Issue.4, pp.481-483.
107. Korgenski E.K., Daly J.A. 1998, Evaluation of the BIOMIC video reader for
determining interpretive categories of isolates on the basis of disk dif- fusion
susceptibility results. J Clin Microbiol. Vol.36, pp,302-4.
108. Krochta, J. M., De Mulder-Johnston, C. D., 1997, Edible and biodegradable
polymer films: Challenges and opportunities. Food Technol, Vol.51, pp.61-74.
109. Krochta, J.M., 2002. Proteins as raw materials for films and coatings:
Definitions, current status and opportunities in Protein-Based Films and
Coatings. Boca Raton, FL: CRC Press, pp.672.
110. Krogars K., Heinamaki J., Karjalainen M., Rantanen J., Luukkonen P., Yliruusi
J., Eur J., 2003, Development and characterization of aqueous amylose-rich
maize starch dispersion for film formation. Pharm. Biopharm. Vol.56, Issue.2,
pp.215-221.
111. Kulkarni S., De Santos F.A., Kattamuri S., Rossi S.J., Brewer M.S., 2011, Effect
of grape seed extract on oxidative, color and sensory stability of a pre-cooked,
frozen, re-heated beef sausage model system. Meat Science, Vol.88, pp. 139–
144.
112. Kumar, A., Vemula, P.K., Ajayan, P.M., John, G., 2008, Silver nanoparticle-
embedded antimicrobial paints based on vegetable oil. Nat. Mater. Vol.7,
pp.236–241.
138 113. Kumar, J., 2008, Lysostaphin: an antistaphylococcal agent. Appl. Microbiol.
Biotechnol. Vol.80, pp.555–561.
114. Lacroix M. 2009. Mechanical and Permeability properties of edible films and
coatings for food and pharmaceutical application. In: Ambuscade ME, Huber
KC. Edible Films and coatings for food applications. Springer Science Business
Media, LLC, New York, NY. pp.367-390.
115. Lahellec, C., D. Y. Fung, and F. E. Cunningham. 1981. Growth effect of sorbate
and selected antioxidants on toxigenic strains of Staphyloccoccus aureus. J.
Food Prot. Vol.44, pp.531-536.
116. Lawson L D., 1998, Garlic: a review of its medicinal effects and indicated active
compounds. In: Lawson L D, Bauer R, editors.Phytomedicines of Europe: their
chemistry and biological activity. Washington, D.C.: American Chemical
Society. pp.176-209.
117. Lee L.A., Puhr N.D., Maloney E.K., Bean N.H., Tauxe R.V., 1994, Increase in
antimicrobial-resistant Salmonella infections in the United States, 1989-1990, J.
Infect. Dis. VOl.170, pp.128-134.
118. Leroy, C., Delbarre, C., Ghillebaert, F., Compere, C., 2008, Effects of
commercial enzymes on the adhesion of a marine biofilm-forming bacterium.
Biofouling. Vol.24, pp.11-22.
119. Leskovac, V., Trivic, S., Wohlfahrt, G., Kandrac, J., 2005, Glucose oxidase
from Aspergillus niger: the mechanism of action with molecular oxygen,
139 quinones, and one-electron acceptors. Int. J. Biochem. Cell Biol. Vol.37,
pp.731-750.
120. Li, X. H., Xing, Y. G., Li, W. L., Jiang, Y. H., Ding, Y. L. 2010. Antibacterial
and physical properties of poly(vinyl chloride)-based film coated with ZnO
nanoparticles. Food Science and Technology, Vol.16, pp.225-228.
121. Liu, H., Chaudhary, D., Yusa, S., Tade, M.O., 2011. Preparation and
characterization of sorbitol modified nanoclay with high amylose
bionanocomposites. Carbohydrate Polymers. Vol.85, Issue.1, pp.97-104.
122. Lueck, E., 1976. Sorbic acid as a food preservative. Int. Flavors Food Addit.
Vol.7, pp.122-138.
123. Mahmoud, R., Savello, P.A. 1993, Solubility and hydrolyzability of films
produced by transglutaminase catalytic crosslinking of whey protein. J. Dairy
Sci. Vol.76, Issue.1, pp.29-35.
124. Maisnier-Patin, S., Forni, E., Richard, J., 1996. Purification, partial
characterisation and mode of action of enterococcin EFS2, an antilisterial
bacteriocin produced by a strain of Enterococcus faecalis isolated from a cheese.
Int. J. Food Microbiol. Vol.30, pp.255-270.
125. Mandic A., Dilas S.M., Cetkovic G.S., Canadanovic-Brunet J.M., Tumbas V.T.,
2008, Polyphenolic composition and antioxidant activities of grape seed extract.
International Journal of Food Properties, Vol.11, pp.713-726.
140 126. Marians, K. J., Hiasa, H., 1997, Mechanism of quinolone action. A
drug-induced structural perturbation of the DNA precedes strand cleavage by
topoisomerase IV. J Biol Chem Vol.272, Issue.14, pp.9401-9409.
127. Marri, L., Dallai, R., Marchini, D., 1996. The novel antibacterial peptide
ceratotoxin A alters permeability of the inner and outer membrane of
Escherichia coli K-12. Curr. Microbiol. Vol.33, pp.40-43.
128. Matope, G., Schlundt, J., Makaya, P.V., Aabo, S., Baggesen, D.L., 16 J.
Schlundt / International Journal of Food Microbiology 78 (2002) 3–17 1998.
Salmonella Enteritidis in poultry: an emerging zoonosis in Zimbabwe.
Zimbabwe Veterinary Journal 29, 132-138.
129. McElhaney R.N., 1982, The use of differential scanning calorimetry and
differential thermal analysis in studies of model and biological membranes.
Chem Phys Lipids. Vol.30, Issue.2-3, pp.229-259.
130. Mead P.S., Slutsker L., Dietz V., McCaig L.F., Bresee J.S., Shapiro C., 1999,
Food-related illness and death in the United States, Emerg. Infect. Dis.,
pp.5607-5625.
131. Miller K. S., Krochta J.M., 1997, Oxygen and aroma barrier properties of edible
films: A review. Trends in Food Science & Tech. Vol.8, Issue.7, pp.228-237.
132. Milner K. C., Shaffer, M. F. 1952. Bacteriologic studies of experimental
Salmonella infections in chicks. Journal of Infectious Diseases Vol.90,
pp.81-96.
141 133. Miteva, V., Stefanova, T., Budakov, I., Ivanova, I., Mitev, V., Gancheva, A.,
Ljubenov, M., 1998. Characterization of bacteriocins produced by strains from
traditional Bulgarian dairy products. Syst. Appl. Microbiol. Vol.21, pp.151-161.
134. Mohammed A. 2010, Chitosam application for active bio-based films
production and potential in the food industry: Review. Food Science and
Technology, Vol.43, Issue.6, pp.837-842.
135. Moir, C. J., and M. J. Eyles. 1992. Inhibition, injury, and inactivation of four
psychrotrophic foodborne bacteria by the preservatives methyl
p-hydroxybenzoate and potassium sorbate. J. Food Prot. Vol.55, pp.360–366.
136. Molobela, P., Cloete, T., Beukes, E.M., 2010, Protease and amylase enzymes for
biofilm removal and degradation of extracellular polymeric substances (EPS)
produced by Pseudomonas fluorescens bacteria. Afr. J. Microbiol. Res.4,
pp.1515-1524.
137. Moore, J. E., M. D. Barton, I. S. Blair, D. Corcoran, J. S. Dooley, S. Fanning, I.
Kempf, A. J. Lastovica, C. J. Lowery, M. Matsuda, D. A. McDowell, A.
McMahon, B. C. Millar, J. R. Rao, P. J. Rooney, B. S. Seal, W. J. Snelling, and
O. Tolba. 2006. The epidemiology of antibiotic resistance in Campylobacter.
Microbes and infection / Institut Pasteur 8:1955-1966.
138. Morillon V., Debeaufort F., Blond G., Capelle M., Voilley A., 2002. Factors
affecting the moisture permeability of lipid-based edible films: a review. Crit
Rev Food Sci Nutr. Vol.42, Issue.1, pp.67-89.
142 139. Mosen A. 2006, Beet-Sugar Handbook: Environmental Concerns of a
Beet-Sugar Factory. 1st ed., John Wiley & Sons, Inc., Hoboken, New Jersey.
140. Muriel-Galet, V., Cerisuelo, J. P., Lopez-Carballo, G., Lara, M., Gavara, R., &
Hernandez-Munoz, P. 2012. Development of antimicrobial films for
microbiological control of packaged salad. International Journal of Food
Microbiology, Vol.157, 195-201.
141. Muzzarelli, R.A.A., Jeuniaux, C., Gooday g.W., 1986, Chitin in Nature and
Technology. Plenum, pp.349-351.
142. National Committee for Clinical Laboratory Standards. 1996. Protocols for
evaluating dehydrated Mueller^Hinton agar. Approved Standard M6-A.Wayne,
PA: NCCLS.
143. Nissen-Meyer, J., Nes, I.F., 1997. Ribosomally synthesized antimicrobial
peptides: their function, structure, biogenesis, and mechanism of action. Arch.
Microbiol. Vol.167, pp.67-77.
144. Omojate Godstime C, Enwa Felix O , Jewo Augustina O, Eze Christopher O.
2014, Mechanisms of Antimicrobial Actions of Phytochemicals against Enteric
Pathogens – A Review Journal of Pharmaceutical, Chemical and Biological
Sciences ISSN: 2348-7658 Chapter 2, Section 2, pp.77-85.
145. Oren, Z., Shai, Y., 1996. A class of highly potent antibacterial peptides derived
from pardaxin, a pore-forming peptide isolated from Moses Sole fish. Eur. J.
Biochem. Vol.377, pp.303–310.
143 146. Oulahal-Lagsir, N., Martial-Gros, A., Bonneau, M., Blum, L.J., 2003,
Escherichia coli-milk’’ biofilm removal from stainless steel surfaces: synergism
between ultrasonic waves and enzymes. Biofouling. Vol.19, pp.159-168.
147. Pathania R, Zlitni S, Barker C, Das R, Gerritsma DA, Lebert J, et al.
2009, Chemical genomics in Escherichia coli identifies an inhibitor of bacterial
lipoprotein targeting. Nat Chem Biol Vol.5, pp.849-856.
148. Park S.Y., Marsh K.S., Rhim J.W., 2002. Characteristics of chitosan films as
affected by the type of solvent acid. Journal of Food Science, Vol.67,
pp.194-197.
149. Pascall M.A., Lin S.J., 2013, The Application of Edible Polymeric Films and
Coating in the Food Industry. Journal of Food Processing & Technology. Vol.71,
pp.95-101.
150. Pathania R, Zlitni S, Barker C, Das R, Gerritsma DA, Lebert J, et al.
2009, Chemical genomics in Escherichia coli identifies an inhibitor of bacterial
lipoprotein targeting. Nat Chem Biol Vol.5, pp.849-856
151. Paul G., 2008, Application to thermoplastics and rubbers. In: Principles and
Applications of Thermal Analysis. John Wiley & Sons. pp.207.
152. Pearson, J. P., Van Delden, C., Iglewski, B. H., 1999, Active efflux and
diffusion are involved in transport of Pseudomonas aeruginosa cell-to-cell
signals. J. Bacteriol. Vol.181, pp.1203-1210.
153. Petereit H.U., Meier C., Roth E., United States Patent. Patent No.: Us 7,160,558
B2, January 2007.
144 154. Pielichowski K., Leszczynska A., 2005, Structure-Property Relationships in
Polyoxymethylene/Thermoplastic Polyurethane Elastomer Blends, Journal of
Polymer Engineering. Vol.25, Issue 4, pp.359-373.
155. Prats G., Mirelis b., Llovet T., Munoz C., Miro E., Navarro F., 2000, Antibiotic
resistance trends in enteropathogenic bacteria isolated in 1985–1987 and 1995–
1998 in Barcelona, Antimicrob. Agents Chemother. Vol.44, pp.1140-1145.
156. Preeti S., Ali A.W., Karim, A., Horst-Christian L., 2012, The use of carbon
dioxide in the processing and packaging of milk and dairy products: A review.
International Journal of Dairy Technology. Vol.65, Issue.2, pp.161-177.
157. Proctor, V.A., Cunningham, F. E., Fung, D.Y.C., 1988, The chemistry of
lysozyme and its use as a food preservative and a pharmaceutical. Crit. Rev.
Food Sci. Nutr. Vol.26, pp.359-395.
158. Quattara, B., Simard, R. E., Holley, R. A., Piette, G. J. P., & Begin, A. 1997,
Antibacterial activity of selected fatty acids and essential oils against six meat
spoilage organisms. International Journal of Food Microbiology, Vol.37,
155-162.
159. Raija A., Han, J.H., 2003. Antimicrobial food packaging. In Novel Food
Packaging Techniques-1st ed. CRC Press, Boca Raton, Fl. Chap. 4.
160. Rantala, M., Nurmi, E. 1973. Prevention of the growth of Salmonella infantis in
chicks by the flora of the alimentary tract of chickens. British Poultry Science
Vol.14, pp.627-30.
145 161. Recio, M.C., Rios, J.L. 1989. A review of some antimicrobial compounds
isolated from medicinal plants reported in the literature 1978-1988. Phytoter.
Res., Vol.3, pp.117-125.
162. Recsei, P. A., Gruss, A. D., Novick, R. P., 1987, Cloning, sequence, and
expression of the lysostaphin gene from Staphylococcus simulans. Proc. Nat.
Acad. Sci. USA. Vol.84, pp.1127-1131.
163. Reddy, N. R., M. D. Pierson, and R. V. Lechowich. 1982. Inhibition of
Clostridium botulinum by antioxidants phenols and related com- pounds. Appl.
Environ. Microbiol. Vol.43, pp.835–839.
164. Reuter H.D., Koch H.P., Lawson L.D., 1996, Therapeutic effects and
applications of garlic and its preparations in Garlic. The Science and
Therapeutic Application of Allium Sativum L and Related Species. Williams
and Wilkins, Baltimore, pp.135-512.
165. Robert. R., 2003. Reserchers call for change of approach to prevent Listeria
infections. Center for Infectious Disease Research and Policy. News &
Perspective.
166. Robertson, G. L. 2006. Active and intelligent packaging. In Food packaging: pri
nciples and practice-2nd ed. CRC Press, Boca Raton, Fl. Chap. 14.
167. Rocourt J., Cossart P., In: M. Doyle, L. Beuchat, T. Montville (Eds.), 1997,
Listeria monocytogenes in Food Microbiology – Fundamentals and Frontiers,
ASM Press, Washington DC, pp. 337-352.
146 168. Rodrigue, D.C., Tauxe, R.V., Rowe, B., 1990. International increase in
Salmonella Enteritidis: a new pandemic? Epidemiology and Infection 105,
21-27.
169. Rodriguez-Saona, L.E., Allendorf M.E., 2011, Use of FTIR for Rapid
Authentication and Detection of Adulteration of Food. Annu. Rev. Food Sci.
Technol. Vol.2, pp.467-83.
170. Rotunda A.M., Jones, D.H., 2010, HIV-associated lipohypertrophy(buccal
fat-pad lipoma-like lesions) reduced with subcutaneously injected sodium
deoxycholate. Dermatol Surg. Vol.36, pp.1348-1354.
171. Ruiz J., Goni P., Marco F., Gallardo F., Mirelis B., Jimenez De Anta T., et al.,
1998, Increased resistance to quinolones in Campylobacter jejuni: a genetic
analysis of gyrA gene mutations in quinoloneresistant clinical isolates,
Microbiol. Immunol. Vol.42, pp.223-226.
172. Sandford C., Godwin M., Hardwick P., 1989. Administrative and Compliance
Costs of Taxation. Bath: Fiscal Publications.
173. Sawer, I.K., Berry M.I., Ford J.L. 1997. Effect of medium composition,
agitation and the presence of EDTA on the antimicrobial activity of cryptolepine.
Vol.25, Issue.3, pp.207-11.
174. Schlaad H., You L., Sigel R., 2009, Glycopolymer vesicles with an asymmetric
membrane. Chemical Communications, No.12, pp.1478-1480.
175. Schlundt J., 2002, New directions in foodborne disease prevention, International
Journal of Food Microbiology Vol.78, pp.3-17.
147 176. Schmidt H., Geitz C., Tarr P.I., Frosch M., Karch H., 1999, Non-O157:H7
pathogenic Shiga toxin-producing Escherichia coli: phenotypic and genetic
profiling of virulence traits and evidence for clonality, J. Infect. Dis. Vol.179,
pp.115-123.
177. Schuchat A, Swaminathan B, Broome C.V., 1991, Epidemiology of human
listeriosis. Clin Microbiol Rev Vol.4, pp.169-83.
178. Seong I.K., You H.B., 2001, pH-Induced Volume-Phase Transition of
Hydrogels Containing Sulfonamide Side Group by Reversible Crystal
Formation. Macromolecules. Vol.34, pp.8173-8178.
179. Seydim, A. C., & Sarikus, G. 2006. Antimicrobial activity of whey protein
based edible films incorporated with oregano, rosemary and garlic essential oils.
Food Research International, Vol.39, 639-644.
180. Shukla R., Cheryan M., 2001, Zein: The industrial protein from corn. Industrial
Crops and Products, Vol.13, Issue.3, pp.171-192.
181. Shulamit L., Michael S.S., 2008. Crystallinity and Cross-Linking in Porous
Polymers Synthesized from Long Side Chain Monomers through Emulsion
Templating. Macromolecules, Vol.41, pp.3930-3938.
182. Siracusa V., Rocculi P., Romani s., Rosa M.D., 2008, Biodegradable polymers
for food packaging: a review. Trends Food Sci Tech, 19:634-643.
148 183. Sisken B.F., Zwick M., Hyde J.F., Cotrill C.M., 1993, Maturation of the central
nervous system: comparison of equine and other species. Equine Veterinary,
Vol.25, Issue.14, pp.2042-3306.
184. Sivarooban, T., Hettiarachchy, N. S., & Johnson, M. G. 2008, Physical and
antimicrobial properties of grape seed extract, nisin, and EDTA incorporated
soy protein edible films. Food Research International, Vol.41, 781-785.
185. Skachkova V.K., Erina N.A., Chepel L.M., Prut E.V., 2003, Thermooxidative
stability and morphology of polypropylene-rubber-paraffin oil blends,
Vyssokomol Soedin Ser A i Ser B Vol.45, pp.2040-2046.
186. Snyder R.L., Bish D.L., 1989, Quantitative analysis. In: Bish DL, Post JE (Eds.)
Modern Powder Diffraction. Mineralogical Society of America, Washington,
DC, pp. 101–144.
187. Sofos, J. N., and F. F. Busta. 1981. Antimicrobial activity of sorbate. J. Food
Prot. Vol.44, pp.614–618.
188. Solgi, M., Kafi, M., Taghavi, T., Naderi, R., 2009, Essential oils and silver
nanoparticles (SNP) as novel agents to extend vase-life of gerbera (Gerbera
jamesonii cv. ‘Dune’) flowers. Postharvest Biology and Technology, Vol.53,
pp.155-158.
189. Solgi, M., Taghizadeh, M., Kafi, M., Taghavi, T.T., 2010, The New Method for
substitution of Environmental polluting silver containing by Plant Natural
Essences in the Postharvest Industry of Flowers. The 4th national Conference on
World Environment Day. Tehran, Iran.
149 190. Sperling, L.H., 2001. Introduction to Physical Polymer Science. 3rd Edition.
New York, Wiley.
191. Strom M.S., Paranjpye R.N., 2000, Epidemiology and pathogenesis of Vibrio
vulnificus. Microbes Infect. Vol.2, pp.177-188.
192. Suet-Yen S., Lee T.S., Tiam-Ting T., Soo-Tueen B., Rahmat A.R., Rahman
W.A.W.A., Ann-Chen T., Vikhraman M., 2013, Antimicrobial agents for food
packaging applications. Trends in Food Science & Technology Vol.33,
pp.110-123.
193. Tanaka T., Nagao S., Ogawa H., 2001, Attenuated total reflection fourier
transform infrared (ATR-FTIR) spectroscopy of functional groups
of humid acid dissolving in aqueous solution. Anal Sci, 1081-1084.
194. Thawien W., 2012, Protein-Based Edible Films: Characteristics and
Improvement of Properties. Agricultural and Biological Sciences, “Structure and
Function of Food Engineering, Chapter 3.” ISBN: 978-953-51-0695-1.
195. Threlfall E.J., 2000, Epidemic Salmonella typhimurium DT 104-a truly
international multiresistant clone, J. Antimicrob. Chemother. Vol.46, pp.7-10.
196. Tian, J., Ban, X., Zeng, H., He, J., Huang, B., Wang, Y., 2011, Chemical
composition and antifungal activity of essential oil from Cicuta virosa L. var.
latisecta Celak. International Journal of Food Microbiology, Vol.145,
pp.464-470.
150 197. US Food and Drug and Administration. 2003, Class II special controls guidance
document: antimicrobial susceptibility test (AST) systems; guidance for industry
and FDA. Rockville, MD: US Food and Drug Ad- ministration.
198. Valérie M., Frédéric D., Geneviève B., Martine C., Andrée V., 2010, Factors
Affecting the Moisture Permeability of Lipid –Based Edible Films: A Review.
Critical Reviews in Food Science and Nutrition. Vol.42, Issue.1, pp.67-89.
199. Van de Velde F.,Lourenço, N.D., Pinheiro H.M., Bakker M., 2002, Carrageenan:
A food-grade and biocompatible support for immobilization techniques. Adv
Synth Catal, No.344, pp.815-835.
200. Var Tiainen J., Motion R., Kulonen H., Rättö M., Skyttä E., Ahvenainen R.,
2004, Chitosan-coated paper: Effects of nisin and different acids on the
antimicrobial activity. Journal of Applied Polymer Science, Vol.94, Issue.3,
pp.986-993.
201. Veldhuizen E.J., Creutzberg T.O., Burt S.A., Haagsman H.P., 2007, Low
Temperature and binding to food components inhibit the antibacterial activity of
carvacrol against Listeria monocytogenes in streak taartare. J. Food Prot.,
Vol.70, Issue.9, pp.2127-2132.
202. Wells J.G., Davis B.R., Wachsmuth I.K., Riley L.W., Remis R.S., Sokolow R.,
et al., 1983, Laboratory investigation of hemorrhagic colitis outbreaks
associated with a rare Escherichia coli serotype, J. Clin. Microbiol. Vol.185,
pp.12-20.
151 203. Wen X.D., Roberto J.A.B., Sui S.T.H., Tara H.M. 2015. Antimicrobial volatile
essential oils in edible films for food safety. Science against Microbial
Pathogens. pp.1124-1134.
204. Whistler R.L., Daniel J.R., 1985. Carbohydrates. In O.R. Fennema (Ed.), Food
chemistry (2nd ed.) pp.69-137. New York: Marcel Dekker.
205. Willams, D.; Lemke, T., 2002, Foye's Principles of Medicinal Chemistry. 5th
ed.; Lippincott, Williams, & Wilkins: Baltimore, MD.
206. Xing, Y., Li, X. H., Zhang, L., Xu, Q., Che, Z., Li, W., et al. 2012. Effect of
TiO2 nanoparticles on the antibacterial and physical properties of
polyethylene-based film. Progress in Organic Coatings, Vol.73, 219-224.
207. Xu, Y.X., Kim, K.M., Hanna, M.A., Nag, D., 2005. Chitosan–starch composite
film: preparation and characterization. Industrial Crops and Products, Vol.21,
pp.185–192.
208. Yildirim, Z., Johnson, M.G., 1998. Detection and characterization of a
bacteriocin produced by Lactococcus lactis subsp. cremoris R isolated from
radish. Lett. Appl. Microbiol. Vol.26, pp.297-304.
209. Yoshida, H.; Nakamura, M.; Bogaki, M.; Ito, H.; Kojima, T.; Hattori, H.;
Nakamura, S., 1993, Mechanism of action of quinolones against Escherichia
coli DNA gyrase. Antimicrob Agents Chemother. Vol.37, Issue.4, pp.839-45.
210. Yudi, P., Vilas, S., & Sudip, R. 2005. Physical and antibacterial properties of
alginate-based edible film incorporated with garlic oil. Food Research
International, Vol.38, 267-272.
152
Appendix A
Figure A.1 The cytotoxicity test of JA-144 on Human Colon Cells (Provided by Dr. Esperanza Carcache de Blanco’s lab).
Figure A.2 The cytotoxicity test of TH-4 on Human Colon Cells (Provided by Dr. Esperanza Carcache de Blanco’s lab).
153 Figure A.3 The cytotoxicity test of TH-8 on Human Colon Cells (Provided by Dr. Esperanza Carcache de Blanco’s lab).
Appendix B
Figure B.1 An example of 96-wells micro-plate showing MICs results.
154