Fabrication of Zinc Oxide Nanoparticles and Films by Banana Peel Extract Food Waste and Investigation on Their Antioxidant and Antibacterial Activities

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Theses and Dissertations

2-1-2018

Fabrication of zinc oxide nanoparticles and films yb banana peel extract food waste and investigation on their antioxidant and antibacterial activities

Omar Hamed

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Recommended Citation

APA Citation Hamed, O. (2018).Fabrication of zinc oxide nanoparticles and films yb banana peel extract food waste and investigation on their antioxidant and antibacterial activities [Master’s thesis, the American University in Cairo]. AUC Knowledge Fountain. https://fount.aucegypt.edu/etds/422 MLA Citation Hamed, Omar. Fabrication of zinc oxide nanoparticles and films yb banana peel extract food waste and investigation on their antioxidant and antibacterial activities. 2018. American University in Cairo, Master's thesis. AUC Knowledge Fountain. https://fount.aucegypt.edu/etds/422

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School of Sciences and Engineering

Fabrication of Zinc Oxide Nanoparticles and Films by Banana Peel Extract Food Waste and Investigation on their Antioxidant and Antibacterial Activities

A Thesis Submitted to The Chemistry Master's Program In partial fulfilment of the requirements for The degree of Master of Science By:

Omar Hisham Mohamed Hamed

Under the supervision of:

Dr. Wael Mamdouh (Advisor) Associate Professor, Department of Chemistry, The American University in Cairo (AUC) & Dr. Hidayah Ariffin (Co-Advisor) Associate Professor, Department of Bioprocess Technology Universiti Putra Malaysia (UPM), Malaysia

December 27th, 2017

Table of Contents Page Number Abstract ...... vii List of Abbreviations ...... viii List of Figures ...... x List of Tables ...... xiv List of Equations ...... xv Chapter 1: General Introduction & Literature review ...... 2 1.1. The Concept of Waste ...... 2 1.2. Main Waste Streams ...... 4 1.3. Waste Hazards ...... 6 1.3.1. Health and Environment Problems ...... 7 1.3.2. Greenhouse Effect ...... 7 1.4. Waste Treatment ...... 8 1.4.1. Prevention ...... 9 1.4.2. Recycling ...... 10 1.4.3. Recovery ...... 11 1.4.4. Disposal ...... 11 1.5. Food Waste ...... 12 1.5.1. Food Waste Problems ...... 14 1.5.2. Management of Food Waste ...... 15 1.6. Banana Waste ...... 18 1.6.1. Overview on Banana Crop ...... 18 1.6.2. Waste Produced from Banana Cultivation and Processing ...... 19 1.7. Management of Banana Waste ...... 20 1.7.1. Conventional Uses of Banana Waste ...... 20 1.7.2. Innovative Applications for Banana Waste ...... 21 1.7.3. Source of Valuable Nutrients and Nutraceuticals ...... 22 1.7.4. Renewable Fuel ...... 25 1.7.5. Heavy Metal and Dye Absorbers ...... 27 1.7.6. Green Synthesis of Nanoparticles (NPs) ...... 27

1.8. Zinc Oxide Nanoparticles (ZnO NPs) ...... 30 1.8.1. The concept of Nanotechnology ...... 30 1.8.2. Properties and Applications of ZnO NPs ...... 32 1.9. Food Packaging ...... 33 1.9.1. Foodborne Illness ...... 33 1.9.2. Functions of Food Packaging ...... 34 1.9.3. Packaging Materials ...... 36 1.10. Packaging Techniques ...... 37 1.10.1. Nanotechnology in Food Packaging ...... 39 1.10.2. Enhancement of mechanical and barrier properties ...... 42 1.10.3. Intelligent Packaging ...... 44 1.10.4. Active Packaging ...... 46 1.11. Zinc oxide NPs in Antimicrobial Food Packaging ...... 49 1.11.1. Antibacterial Mechanism of ZnO ...... 50 1.11.2. Synthesis of ZnO NPs ...... 52 1.11.3. Green Synthesis of ZnO NPs ...... 55 Thesis Scope and Objectives ...... 57 Chapter 2: Materials & Methods ...... 59 2.1. Materials ...... 59 2.2. Synthesis Methods ...... 59 2.2.1. Extract Preparation ...... 59 2.2.2. Synthesis of ZnO NPs ...... 60 2.2.3. ZnOW NPs preparation: ...... 61 2.2.4. ZnO/surfactants NPs preparation: ...... 62 2.2.5. ZnO/BPE NPs preparation: (Green Synthesis) ...... 62 2.2.6. Film Preparation ...... 63 2.3. Characterization of the BPE ...... 65 2.3.1. Total Polyphenolics Assay (Folin Ciocalteu Test)...... 65 2.4. Characterization of NPs ...... 66 2.4.1. Transmission Electron Microscopy (TEM) ...... 66 2.4.2. Scanning Electron Microscopy (SEM) ...... 66

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2.4.3. Ultraviolet/visible (UV-Vis) spectroscopy analysis ...... 66 2.4.4. Size Stability & Zeta Potential analysis ...... 67 2.4.5. Fourier-Transform Infrared Spectroscopy (FTIR) analysis ...... 67 2.4.6. Powder X-ray Diffraction (XRD) analysis ...... 67 2.4.7. Investigation of the antioxidant Activities ...... 67 2.4.8. Investigation of the antibacterial activities ...... 68 2.5. Characterization of films ...... 68 2.5.1. Porosity Determination ...... 68 2.5.2. Investigation of film antioxidant Activity ...... 69 2.5.3. Investigation of film antibacterial Activity ...... 69 2.6. Statistical Analysis ...... 69 Chapter 3: Theoretical Background of Characterization Methods ...... 71 3.1 Folin Ciocalteu Assay ...... 71 3.2. Sonication ...... 72 3.3. TEM ...... 75 3.4. FESEM (SEM) ...... 78 3.5. UV-vis Spectroscopy ...... 80 3.6. Zeta Sizer ...... 82 3.7. FTIR Spectroscopy ...... 88 3.8. Accelerated Surface Area and Porosimetry System ...... 90 Chapter 4: Results and Discussion ...... 93 4.1. Characterization of the Banana Peel Extract ...... 93 4.1.1. Total Poly-phenolics Assay (Folin-Ciocalteu Assay) ...... 93 4.2. Characterization of ZnO NPs ...... 95 4.2.1. Size Measurement using TEM ...... 95 4.2.2. Morphology Assessment using Scanning Electron Microscopy (SEM) ...... 101 4.2.3. UV Absorbance Assessment using UV/Visible (UV/Vis) spectroscopy...... 106 4.2.4. Size Stability and Zeta Potential Assessment using Zeta Sizer ...... 111 4.2.5. Fourier-Transform Infrared Spectroscopy (FTIR) analysis ...... 118 4.2.6. Crystallinity Assessment using X-ray Diffraction (XRD)...... 130 4.2.7. Antioxidant Activity Assessment using the DPPH Assay ...... 131 4.2.8. Antibacterial Assessment using the Colony Count Method ...... 135 iv

4.3. Characterization of the ZnO NPs Films ...... 142 4.3.1. Porosity Assessment using ASAP system ...... 143 4.3.2. Antioxidant Activity Assessment using the DPPH Assay ...... 145 4.3.3. Antibacterial Activity Assessment using the Disk Diffusion Method (Kirby-Bauer Method) 146 Chapter 5: Conclusion and Future perspectives ...... 151 Chapter 6: Appendices ...... 153 Appendix I: Synthesis of ZnO NPs using fruit extracts ...... 153 Appendix II: Nanocellulose fibers synthesis ...... 155 Appendix III: Film Synthesis ...... 156 Appendix IV: Folin Test Calibration Curves...... 158 References ...... 159

Acknowledgments

First and foremost, I would like to express my sincere gratitude to my supervisor, Dr. Wael Mamdouh, for giving me the opportunity to work under his supervision and for his valuable guidance and continuous support and encouragement throughout this research work. He provided me with unlimited support and gave me his precious time and advice whenever I needed it.

Similarly, I would like to thank Dr. Hidayah Ariffin, for giving me the opportunity to conduct my research in her lab at UPM, Malaysia and for her support throughout my visit and sincere guidance in my research.

I am also thankful to all my professors at AUC at the department of chemistry from whom I have learnt a lot throughout the courses of my studies.

I also feel grateful to every one of my colleagues in Dr. Wael’s research group for their support, help and the time I spent with them. Special thanks to the researchers and colleagues here at AUC who participated in teaching me some research skills and provided me with continuous guidance during my work; for their care and guidance during my research work: Dr. Nahed Yacoub for the Accelerated surface area porosimetry (ASAP) experiments, Mr. Mahmoud Abdel Moez, and Ahmed Omaia for the FTIR experiments, and Mr. Emad Rafaat for the XRD experiments. Special thanks also to the researchers at UPM who helped me conduct my research: Dr Farideh Nemvar for her assistance in the nanoparticle synthesis, Dr. Rosnita A. Talib for her assistance in the film characterization and Dr. Harmaeen Ahmed Saffian for his sincere assistance as well.

I would like to acknowledge my family for their support; without them in my life I couldn’t have achieved this study. I would like to acknowledge my father, Hisham Hamed, for his continual support and his full presence. He has been great role model for me and he will always be my hero. Also, I would like to express my sincere appreciation to my mother, Amal, whom her prayers have been always with me and always being with me to face the difficulties through this study.

Finally, I would like to express my endless thanks to my friends at AUC: Amro Shetta, James Kegere, Sara Hassan and Ahmed Emad and my friend at UPM Faiz Norrahim.

Last, but by no means least, I thank God.

Abstract

Food waste is one of the major problems of today's world and thus there is a need to utilize it in innovative applications to reduce the problems associated with it. One of those is the utilization of banana peels in the green synthesis of nanoparticles where the extract of such peels was found to contain polyphenolic compounds that would serve as capping agents for the growing nanoparticles.

Our aim in this study was to investigate the use of banana peel extract (BPE) in the synthesis of zinc oxide nanoparticles (ZnO NPs) and to compare the prepared NPs with commercial bulk ZnO particles and also with ZnO NPs prepared using nonionic and anionic surfactants. All samples were characterized by using different characterization techniques such as transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), powder X-ray diffraction (XRD), zeta sizer and UV-vis spectrophotometry. The antioxidant and antibacterial activities of the ZnO NPs were also investigated to select the NPs that could be incorporated in a biodegradable food packaging film.

The ZnO NPs synthesized using the BPE were found superior to all NPs prepared in this study in most of the characterization steps. These NPs were found to have the smallest particle size (6 nm), as well as attaining this size for several days in aqueous solutions without significant size fluctuation. In addition, a more uniform spherical morphology was observed for these NPs, unlike the non-uniform flake like structures of the remaining NPs. Moreover, the capping of the BB extract components on these NPs rendered them antioxidant with a DPPH scavenging activity of 76.91%, even though ZnO NPs are known to produce reactive oxygen species (ROS). With regards to the antibacterial activity against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli), all ZnO NPs were found to be potent antimicrobial agents with minor differences amongst them against E. coli and slight differences against S. aureus.

ZnO NPs were then incorporated in a biopolymer (starch) for the synthesis of biodegradable films aimed for antimicrobial food packaging. The starch/ZnO NPs film composites were compared with commercial nylon/evoh film with regards to antioxidant and antimicrobial activity. It was found that the films with ZnO NPs derived from BPE exhibited DPPH scavenging activity of 20.9%, which was 50% higher than that of the commercial film. In addition, the films with ZnO NPs exhibited antibacterial properties towards S. aureus and E. coli when tested using disk diffusion method unlike the commercial nylon/evoh film which did not exhibit any antibacterial activities.

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List of Abbreviations Ag NPs: Silver nanoparticles ASAP: Accelerated surface area porosimetry ASR: Automobile shredder residue Au NPs: Gold nanoparticles BPE: Banana peel extract CFU: Colony forming unit DLS: Dynamic light scattering DPPH: 2,2-diphenyl-1-picrylhydrazyl radical DW: Dried weight E. coli: Escherichia coli eV: Electron volt EVOH: Ethylene vinyl alcohol FAO: Food and Agriculture Organization FC: Folin Ciocalteu FDA: US. Food and Drug Administration FESEM: Field emission scanning electron microscopy FTIR: Fourier Transform Infrared spectroscopy FOD: Fluctuation over days GAE: Gallic acid equivalent GDP: Gross domestic product GNP: Gross national product GRAS: Generally regarded as safe HOMO: Highest occupied molecular orbital HPLC: High performance liquid chromatography ISWM: Integrated solid waste management LUMO: Lowest unoccupied molecular orbital MAP: Modified atmosphere packaging MSW: Municipal solid waste NP: Nanoparticles PCS: Photon correlation spectroscopy viii

PVA: Polyvinyl alcohol PEG: Polyethylene glycol PVP: Polyvinylpyrrolidone ROS: Reactive oxygen species S. aureus: Staphylococcus aureus SDS: Sodium dodecyl sulfate SEM: Scanning electron microscopy TEM: Transmission electron microscopy TTIs: Time-temperature indicators UHT: Ultra heat treatment UV-Vis spectroscopy: UV-Visible spectroscopy WHO: World Health Organization XRD: powder X-ray diffraction

ZnO NPs: Zinc oxide nanoparticles

ZnOC: Commercial bulk zinc oxide particles

ZnOW: Chemically synthesized zinc oxide nanoparticles without using capping agents

ZnO/PVP: Chemically synthesized zinc oxide nanoparticles using PVP as a capping agent

ZnO/PEG: Chemically synthesized zinc oxide nanoparticles using PEG as a capping agent

ZnO/PVA: Chemically synthesized zinc oxide nanoparticles using PVA as a capping agent

ZnO/SDS: Chemically synthesized zinc oxide nanoparticles using SDS as a capping agent

ZnO/BP: Chemically synthesized zinc oxide nanoparticles using BPE as a capping agent

ZP: Zeta potential

List of Figures

Figure 1:An illustration showing the relationship between GDP per capita (per individual) versus MSW (municipal solid waste) per capita in different countries1 ...... 3 Figure 2:An illustration showing the composition of MSW in low, middle and high-income countries5 ...... 4 Figure 3:A general illustration for the Greenhouse effect16 ...... 8 Figure 4:An Illustration of the waste management hierarchy3 ...... 9 Figure 5: An illustration of closed-loop recycling of glass bottles18 ...... 10 Figure 6: An illustration of biodegradable waste hierarchy, with a focus on food waste in particular10 ...... 12 Figure 7: A simplified illustration of the food supply chain highlighting possible chances of food loss. [FSCW: Food supply chain waste]21 ...... 13 Figure 8: A graph showing variation in food losses and waste in different regions at consumption and pre-consumption stages23...... 14 Figure 9: An illustration of the Food Recovery Hierarchy30 ...... 16 Figure 10: An illustration of anaerobic digestion34 ...... 17 Figure 11: An illustration of the composting cycle36 ...... 18 Figure 12: An illustration of the main parts of the banana tree43 ...... 20 Figure 13: Schematic illustration showing the potential applications of banana by-products41 ...... 22 Figure 14: A photo of the banana tree inflorescence53 ...... 23 Figure 15: A schematic illustration of the generation of bioethanol70 ...... 25 Figure 16: A schematic illustration of the production of briquettes from biomass74 ...... 26 Figure 17: An illustration of the 12 principles of green chemistry86 ...... 29 Figure 18: A schematic illustration of the mechanism of green synthesis of NPs using food waste91 ...... 30 Figure 19: A general illustration of the different dimensions of the nanostructures94 ...... 31 Figure 20: A general figure illustrating the functions of Food Packaging113 ...... 35 Figure 21: Statistics showing the number of publications per year that were retrieved using Scopus (a database of peer-reviewed literature), searching for the terms 'food' and 'nanotechnology'129 ...... 40 Figure 22: A general Scheme illustrating the applications of nanotechnology in different areas of food science130 ...... 41 Figure 23: An illustration showing the torturous pathway concept; (a) gas molecules diffusing through the polymer in a straight pathway. (b) Gas molecules going along a longer torturous pathway due to the presence of nanofillers127 ...... 44 141 Figure 24: Oxygen Indicator system based on TiO2 NPs ...... 45 Figure 25: Controlled release nanosystems of antimicrobial compounds that are incorporated into the food packaging146 ...... 47 x

Figure 26: Antimicrobial food packaging, where antimicrobial agents are immobilized onto the packaging material146 ...... 48 Figure 27: Proposed mechanisms of the antibacterial activity of ZnO149...... 52 Figure 28: Different Morphologies of ZnO nanostructures(a, c & d: nanorods. b &g: nanosheets, e & f: prism like structures. h: nanospheres) 171 ...... 53 Figure 29: Synthesis of ZnO NPs using the mechanochemical processing149 ...... 54 Figure 30: Scheme for the preparation of BPE ...... 60 Figure 31: Scheme for ZnOW NPs preparation...... 62 Figure 32: Scheme for synthesis of ZnO/surfactants and ZnO/BPE NPs...... 63 Figure 33: Scheme for the preparation of starch film ...... 64 Figure 34: Scheme for preparation of S/ZnOC, S/ZnOW film, S/ZnO/PEG film, S/ZnO/BP3 film...... 65 Figure 35: Schematic diagram of cavitation caused by ultrasonic waves and agglomerate fracture206 ...... 73 Figure 36: Schematic illustration of the three types of sonicators; (A) Probe-type sonicator. (B) Bath-type sonicator, (C) Cup-type sonicator206 ...... 74 Figure 37: An illustration of the scattering events that can take place when an electron hits a sample208 ...... 77 Figure 38: A schematic illustration showing the emission of secondary electrons from (a) a coated sample (b) non-coated sample213 ...... 79 Figure 39: A schematic illustration of the UV/Visible spectrophotometer216 ...... 82 Figure 40: An illustration showing (A) Scattered light intensity fluctuations due to constructive and destructive interferences. (B) A correlogram showing the correlation decay against time219 ...... 84 Figure 41: Schematic representation of the components of a typical Dynamic Light Scattering system219 ...... 86 Figure 42: A schematic illustration showing a negatively charged particle and its electrical double layer.219 ...... 87 Figure 43: A schematic representation of an FTIR instrument224 ...... 89 Figure 44: A general illustration of an adsorption isotherm228 ...... 91 Figure 45: A graph showing the Folin- Ciocalteu test results performed on the banana peel extract prepared [E1 and E2] ...... 94 Figure 46: (a) TEM image of ZnOC NPs (b) TEM image of ZnOW NPs (c) Size distribution of ZnOW NPs ...... 95 Figure 47: TEM images of (a)ZnO/PVP NPs (c)ZnO/PEG NPs(e, f)ZnO/PVA NPs (g)ZnO/SDS NPs & Size distribution histogram of (b)ZnO/PVP NPs (d)ZnO/PEG NPs (h)ZnO/SDS NPs ...... 97

Figure 48: TEM images of (a)ZnO/BP1 NPs (c)ZnO/BP2 NPs (e)ZnO/BP3 NPs (g)ZnO/BP4 NPs & Size distribution hisograms of (b)ZnO/BP1 NPs (d)ZnO/BP2 NPs (f)ZnO/BP3 NPs (h)ZnP/BP4 NPs...... 99 Figure 49: TEM images of (a)ZnOC NPs, (b)ZnOW NPs, (c)ZnO/PVP NPs, (d)ZnO/PEG NPS, (e)ZnO/PVA NPs, (f)ZnO/SDS NPs ...... 100 Figure 50: TEM images of (a)ZnO/BP1 NPs, (b)ZnO/BP2 NPs, (c) ZnO/BP3 NPs, (d)ZnO/BP4 NPs...... 101 Figure 51: SEM images of (a)ZnOC NPs (b)ZnOW NPs ...... 102 Figure 52: SEM images of (a)ZnO/PVP NPs (b)ZnO/PEG NPs (c)ZnO/PVA NPs (d)ZnO/SDS NPs ...... 103 Figure 53: SEM images of (a)ZnO/BP1 NPs (b)ZnO/BP2 NPs (c)ZnO/BP3 NPs (d)ZnO/BP4 NPs...... 104 Figure 54: SEM images of (a)ZnO/BP3 NPs (b)ZnO/BP4 NPs ...... 105 Figure 55: UV-Vis spectra of ZnO NPs; (a)G1 and G2 (b) G3 (c)G4 ...... 107 Figure 56: General scheme illustrating the band gap energy241 ...... 109 Figure 57: A general scheme comparing the changes in the band gap of a material in the bulk and its nanoparticle counterpart.244 ...... 111 Figure 58: Five-day size stability study for ZnOW ...... 112 Figure 59: Five-day size stability study for (a)ZnO/PVP (b) ZnO/PEG (c) ZnO/PVA (d) ZnO/SDS ...... 113 Figure 60: Five-day size stability study for (a)ZnO/BP1 (b)ZnO/BP2 (c)ZnO/BP3 (d)ZnO/BP4 ...... 114 Figure 61: A graph from a study showing the particle size distribution of TiO2, SiO2 and ZnO in water246 ...... 115 Figure 62: A graph from a study showing changes in the particles size of TiO2, SnO2 and ZnO NPs after ultrasonication in water246 ...... 116 Figure 63: A graph from a study showing the colloidal stability of Fe/Au NPs synthesized using Pluronic F127, Pluronic F68, PEG 6k, PEG 10k, PEG 100l, Bovine serum albumin and Dextran over the course of 5 days247 ...... 117 Figure 64: Zeta Potential Values of ZnO samples ...... 118 Figure 65: A general scheme showing the proposed mechanism for the formation of ZnO crystal243...... 119 Figure 66: FTIR spectra of (a) ZnO Acetate (b)ZnOC particles (c)ZnOW NPs ...... 120 Figure 67: FTIR spectra of (a)pure PVP (b)ZnO/PVP NPs...... 121 Figure 68: FTIR spectra of (a)pure PEG (b)ZnO/PEG NPs ...... 123 Figure 69: FTIR spectra of (a)PVA (b)ZnO/PVA NPs ...... 124 Figure 70: FTIR spectra of (a)SDS (b)ZnO/SDS NPs ...... 126 Figure 71: FTIR spectra of (a)pure banana peel (b)ZnO/BP1 NPs (c)ZnO/BP2 NPs (d)ZnO/BP3 NPs (e)ZnO/BP4 NPs ...... 129

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Figure 72: XRD spectra of ZnO Samples ...... 130 Figure 73: A general illustration showing the three ZnO crystal structures (a) cubic rock salt (B1), (b) cubic zinc blende (B3), (c) hexagonal wurtzite. The black spheres represent O and the gray spheres represent Zn243 ...... 131 Figure 74: DPPH scavenging activity of the ZnO samples ...... 132 Figure 75: A general illustration of the mechanism of DPPH reduction by an antioxidant compound259 ...... 133 Figure 76: Colony count results for S.aureus (a) + ve control (b) ZnOW (c)ZnO/PVP (d)ZnO/PEG (e)ZnO/PVA (f)ZnO/SDS (g)ZnO/Bp1 (h)ZnO/BP2 (i) ZnO/BP3 (j)ZnO/BP4 ...... 136 Figure 77: Colony count results using E.coli (a) + ve control (b) ZnOW (c)ZnO/PVP (d)ZnO/PEG (e)ZnO/PVA (f)ZnO/SDS (g)ZnO/Bp1 (h)ZnO/BP2 (i) ZnO/BP3 (j)ZnO/BP4 ...... 137 Figure 78: Percent inhibition calculated from the colony count method [S.aureus] ...... 138 Figure 79: Percent inhibition calculated from the colony count method [E.coli] ...... 138 Figure 80: A general illustration showing the cell wall structure of (a)gram negative bacteria and (b) gram positive bacteria271 ...... 141 Figure 81: (a) S film (b)S/ZnOC film (c)S/ZnOW film (d) S/ZnO/PEG film (f)S/ZnO/BP3 film...... 143 Figure 82: ASAP results (a)BJH cumulative surface area of pores (b)BJH cumulative volume of pores (c) Diameter of pores ...... 144 Figure 83: DPPH scavenging activity of the prepared films ...... 146 Figure 84: Disk diffusion results using S.aureus (a) C film (b) S film (c) S/ZnOC film (d) S/ZnOW film (e) S/ZnO/PEG film (f) S/ZnO/BP3 film ...... 148 Figure 85: Disk diffusion results using E.coli (a) C film (b) S film (c) S/ZnOC film (d) S/ZnOW film (e) S/ZnO/PEG film (f) S/ZnO/BP3 film ...... 149 Figure 86: Photos of the pulps and peels of fruits used (a)banana (b)dragon fruit (c)rambutan ...... 153 Figure 87: SEM image of particles prepared using (a) dragon fruit's pulp extract (b) dragon fruit's peel extract (c) rambutan's peel extract (d) banana pulp extract (e)banana peel extract ...... 154 Figure 88: SEM images of nanocellulose fibers at different magnifications ...... 155 Figure 89: SEM image of (a)starch film (b) starch + homogenized ZnO film (c) starch + nanocellulose + homogenized ZnO film (d) starch + green synthesized particles film .. 157 Figure 90: Calibration curves of gallic acid solutions used in folin test (a) gallic acid dissolved in water for E1 analysis (b) gallic acid dissolved in acetone:water (1:1) for E2 analysis ...... 158

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List of Tables

Table 1: List of ZnO samples used in this study ...... 61 Table 2: Summary of TEM measurements of the ZnO NPs ...... 98 Table 3 :Calculated band gap energies for the ZnO Samples ...... 108 Table 4: Summary of Zeta Sizer Measurements in the 5-day study ...... 112 Table 5: A summary of antioxidant activities of ZnO NPs prepared using different extracts reported in this study and previous works ...... 134 Table 6: Disk diffusion results of the films...... 147 Table 7: Mechanical Testing results of nanocellulose films ...... 156

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List of Equations

Equations 1-5 [Generration of ROS by ZnO NPs]: Page 52 Equation 6 [Planck's Equation]: Page 81 Equation 7 [Beer Lambert Law]: Page 82 Equation 8 [Band Gap Energy Equation]: Page 108 Equation 9-12 [Mechanism of ZnO synthesis]: Page 117 Equation 13 [DPPH Scavenging Activity Equation]: Page 130

Chapter 1

General Introduction & Literature Review

Chapter 1: General Introduction & Literature review

1.1.The Concept of Waste

In the last few decades, the extensive use of Earth's resources has led to the generation of vast amounts of waste all over the world. It is estimated that 11.2 billion tons of solid waste are produced every year around the world1. The exploitation of Earth's resources has increased eight- fold in the last century2, especially in developed regions such as Europe where an average

European is reported to consume around 50 tons of resources per year, which is around three times the resources used per individual in developing countries3. The reason behind this is the interplay between two factors that govern waste generation; population and income growth. Of the two factors, it was found that the more powerful driver was the income level. Figure 1 shows the relationship between waste generation and GDP (Gross Domestic Product). On an average, an individual in a high-income country produces around 0.8 kg of waste per day, while an individual in a low-income country generates around 0.2 kg of waste per day which is quarter the amount generated in high-income countries1. It could be inferred from these numbers and Figure 1 that as a country becomes wealthier, waste problems arise, and the waste stream becomes more complex and varied. This was not the case in the past when biogeochemical processes were dominating, and all chemicals and resources were in a continuous cycle without generation of useless waste or depletion of the resources. Later, towns were developed and the natural resources were exploited by industrial methods, and chemicals were used in the environment and the production which led to the interruption of the natural cycles of raw materials, causing different materials to leave those cycles as waste4.

Figure 1:An illustration showing the relationship between GDP per capita (per individual) versus MSW (municipal solid waste) per capita in different countries1

When discussing waste, it should be noted that the term 'waste' is a rather ambiguous term that does not have a clear definition, as there is not a definite physical, biological or chemical criterion that distinguishes waste from other useful materials. In fact, there is not a single substance that only be waste with no potential to be converted into useful materials, because any matter could be utilized in some manner to become a useful resource. However, 'waste' is usually defined by legal authorities and organizational systems according to the economic profitability of a given substance to be used in a given technology. In that sense, waste could be generally defined as any product –

whether raw material or processed material – that no longer has a definite use in terms of further development4.

1.2.Main Waste Streams

Waste could be categorized according to its origin into the following:

1-Municipal Solid Waste (MSW): This category refers to waste that is generated from domestic consumption of materials, such as waste generated from households, shops, schools, offices, hotels and other institutions. It is commonly referred to as garbage or refuse waste. This type of waste includes food waste, paper, plastics, metals, textiles, glass, yard waste, etc. MSW varies in its composition and complexity from one country to another and it is greatly affected by the country's

GDP as seen in Figure 2. In low and middle-income countries, organic rich MSW dominates, while in high income countries most of the MSW is due to paper and plastics5.

Figure 2:An illustration showing the composition of MSW in low, middle and high-income countries5

2-Construction and Demolition (C&D) Waste: This type of waste is classified as high-volume waste and it is reported that is represents around 15% of the total waste that is produced in

developed countries. This per cent can increase in some countries such as Germany where it represents around 55 per cent of the total waste generated6.

3-End-of-life Vehicles: This type refers to waste generated from vehicles that have reached the end of their useful life. It was estimated that it accounts for 8-9 million tons of waste in Europe in

2010, with UK, France, Germany, Italy and Spain responsible for 75 per cent of it7. A subtype in this category is referred to as Automobile Shredder Residues (ASR) which includes the materials that remain from the vehicle after the reusable parts have been removed from shredded end-of-life vehicles. These materials include plastic, rubber, fiber, glass, foam, etc. ASR are generated in

Japan at a rate of 0.7 million tons per year8, and the US generates up to 5 million tons annually9.

4-Biomass waste: This type refers to biodegradable food, kitchen, garden and park waste. In

Europe, this type represents around one-third of the waste produced which amounts for about 118-

138 million tons annually.10

5-Hazardous waste: This type of waste needs to be treated in a special manner, even when present at small levels. These hazardous materials are usually mixed up with municipal and agricultural waste such as residual chemical pesticides, used batteries, paints, electronic equipment, fire extinguishers, refrigerators, agricultural fumigants, etc. It was reported that 8.5 million tons of waste that is considered hazardous move across international borders annualy11.

6-Health-care waste: This type is usually presented as one of the subcategories of hazardous waste.

It is estimated that around 0.5 kg and 3 kg of this type is genertaed annually per individual in low- income countries, including both hazardous and nonhazardous materials. In high-income countries, health-care waste associated with each individual might reach up to 6 kg per year12.

7-Electronic waste: This type of waste is experiencing a dramatic increase owing to the constantly growing demand for electrical goods. The total reported electronic waste generated globally

increased from 6 million tons in 1998 to 20-50 million tons in 200513. In 2004, a total of 315 million personal computers were reported to become outdated around the world and 130 million cellphones were found to completely lose their functions in 200513. This rise is not restricted to developed countries as it is estimated that outdated personal computers in will become significantly higher in developing regions rather than developped ones by the end of 2018 and that by 2030, outdated units will reach 400-700 million units in developing parts of the world, while developed regions will generate 200-300 million units14.

1.3.Waste Hazards

Each stream of waste is associated with a set of risks specific to it, but there are some general hazards that are common among most of these streams. The most serious hazard is imposing risks to human health and environment. This becomes more pronounced in cases where the collection and treatment of waste is absent or insufficient and in some situations where treatment and collection of waste is present sufficiently. In some developed countries, protests over waste treatment facilities take place where residents would reject incinerators and landfills because of concerns related to the safety of the individuals and the lack of trust in the responsible parties and authorities that should play the major role in ensuring that safety standards are enforced in the waste treatment and collection processes.

Developing countries suffer the most from waste disposal problems due to many reasons such as:

1- Deficient waste treatment and disposal infrastructure.

2- Inappropriate collection of waste

3- Weak enforcement of law related to waste collection and treatment

4- Limitation in financial resources

1.3.1. Health and Environment Problems

The most common method of waste collection and treatment that is found in developing countries is dumping in open, unsecured and uncontrolled sites, where waste materials are dumped and mixed with each other, burnt, and might even be grazed by stray animals. This leads to many serious problems in the environment as leaking hazardous substances would cause several health problems if they reached drainage systems, soils or animal feed. This uncontrolled manner of waste disposal have been associated with several health problems such as respiratory problems, hepatitis, cholera, malaria, yellow fever, typhoid, dysentery, skin and eye infections, plague and flea-born fever which can be carried by stray animals and rodents in these sites3.

1.3.2. Greenhouse Effect

Another major problem associated with the improper collection and treatment of waste is the greenhouse effect which is illustrated in Figure 3. The greenhouse effect is a natural phenomenon that is responsible for warming the Earth. When the sun rays fall on the Earth's surface, around one third of the incident solar energy is reflected back to space and the remaining radiation would be absorbed by the surface of the Earth and gases in the atmosphere. Most of this absorbed energy is remitted back to space at wavelengths which fall in the infrared region of the electromagnetic radiation spectrum. Most of this remitted energy will then be absorbed by certain gases in the atmosphere called the greenhouse gases. Examples of such gases are ozone, carbon dioxide, nitrous oxide and methane. The result of such effect is keeping the Earth warm, otherwise, the temperature on the Earth would fall to very low values15. This effect, however, is greatly enhanced when the levels of such gases rise in the atmosphere which would lead to climate change and global warming. This occurs due to the improper disposal of waste, especially waste that has a larger organic fraction. Anaerobic digestion of organic material would lead to the production of

large amounts of methane which rises to the atmosphere, enhancing the greenhouse effect. This enhanced greenhouse effect depends on the characteristics of waste (particle size, density, composition) and the condition of the dumping site (microbes, temperature, pH, nutrient, moisture)3.

Figure 3:A general illustration for the Greenhouse effect16

1.4.Waste Treatment

To avoid the hazards associated with waste, guidelines and regulations have been set to optimize waste treatment methods. In that sense, a strategic approach called integrated solid waste management (ISWM) has been proposed to achieve such goal. This approach aims at moving upstream in the waste management hierarchy as shown in Figure 4. ISWM aims at shifting waste management from conventional disposal methods towards the three Rs; reduce, recycle and reuse.

An even more preferred method is preventing the generation of waste and thus save the effort needed for subsequent treatment and disposal3.

Figure 4:An Illustration of the waste management hierarchy3

1.4.1. Prevention

Preventing the generation of waste is the most preferred strategy in waste management, as waste that is not produced does not need to be disposed of or recycled. This could be mainly achieved by improving manufacturing methods. For example, heavy metals and other banned substances could be substituted by more eco-friendly materials. In addition, packaging might be minimized

by avoiding interim packaging and reducing the thickness and size of final packaging. To guarantee the success of such procedures in preventing waste generation, the consumer behavior should be influenced to enhance the demand on green product and less packaging. Otherwise, the consumer would rely on old conventional products that produce large amounts of waste17.

1.4.2. Recycling

Recycling refers to the use of a material to generate secondary materials from it. It could be considered as a separate industrial process that has its own energy requirements, and other operational procedures such as collection, separation and transport. Recycling includes closed- loop and open-loop recycling. In the former, the material is used to produce secondary materials on the same level. For example, glass bottles could be used to produce glass bottles as seen in

Figure 5. Open-loop recycling, however, refers to the use of a material to produce secondary materials in a different product life cycle and usually, the new products are of less quality and functionality. An example of open-loop recycling is the use of office paper to produce toilet paper, or the use of plastic bottles to produce plastic drainage tubes17.

Figure 5: An illustration of closed-loop recycling of glass bottles18

1.4.3. Recovery

Another approach to waste treatment is recovering energy from it by thermal treatment such as incineration. Incineration involves the combustion of solid waste in the presence of large amounts of oxygen at very high temperatures (above 850 0c) leading to the production of combustion gases

(carbon monoxide, carbon dioxide, water vapor, nitrogen dioxide), ash and heat. In case of incomplete combustion or low levels of oxygen, carbon monoxide is generated in larger amounts.

The main objectives of incineration are the fast reduction of the volume of waste and the utilization of recovered energy or heat for other purposes such as heating and production of electrical energy19.

1.4.4. Disposal

Disposal of waste is the least preferred approach in waste treatment despite being the most commonly used in many countries. The term 'waste disposal' is also referred to as landfilling, both of which mean the burying of waste in mining voids or unused quarries. This approach requires large areas to be reserved for landfilling and might lead to adverse effects on human health and environment. These adverse effects are mainly due to contamination of soils, aquifers and ground water due to leaching of chemicals and heavy metals. In addition, if waste is mainly made up of organic compounds e.g. food waste, anaerobic digestion might take place leading to the generation of large amount of methane which will be released into the atmosphere enhancing the greenhouse effect. Moreover, landfills attract vermin like mice, flies and insects which might lead to serious diseases. In modern landfills, some strategies might be employed to reduce such hazards17.

1.5.Food Waste

The abovementioned hazards and treatment methods are quite common among the different waste streams, but still, each waste stream might have its own risks that are associated with it and preferred treatment regimens. Food waste is regarded as one of the important categories of waste.

The term 'food waste' could be misleading and ambiguous which might lead to confusion when analyzing results from different studies. Figure 6 illustrates hierarchy of food wastage showing that it falls originally under bio-waste which lies under biodegradable waste.

Figure 6: An illustration of biodegradable waste hierarchy, with a focus on food waste in particular10

Food wastage includes several components:

1-Food losses: This refers to wholesome edible products that were originally generted to be consumed by man but instead were not eventually consumed and was lost in one of the production or transporation processes unintentionally because of lack of technology, poor infrastructure, drawbacks in transportion systems, storage or packaging materials and processes, insufficient

skill20s or consumption by pests21. These losses can therefore take place along the entire food supply chain as illustrated in Figure 7.

Figure 7: A simplified illustration of the food supply chain highlighting possible chances of food loss. [FSCW: Food supply chain waste]21

2-Food Residuals: This type refers to the parts of food that are mostly inedible by man such as peels, skins, bones, leaves, shells, etc22.

3-By-products : This type refer to the edible products that can be generated during food processing, but are not generated initially to be consumed by man and were fed to animals instead. The type of waste also includes animal by-products which refer to the remnants of animals, whether entire bodies or part of animals such as embryos, ova, semen, etc22.

4-Food Waste: The term 'food waste' in this hierarchy refers food that is discarded intentionally despite being suitable for consumption. This includes food that is spoiled, spilled, wilted or bruised

as well as whole food that are unopened and free of any flaws. It is estimated that around 1.3 billions tons of edible food that was initially produced to be consumed by man is lost annually23.

This happens in both developed and developing countries with varying amounts as shown in

Figure 8. Food is discarded intentionally by consumers in medium and high-income countries, despite being suitable for consumption. In low-income countries, food waste is not usually generated at the consumer level like high-income countries due to the increasing need of food, but it is rather lost in earlier stages of the food supply chain and thus, would most probably be related to the 'food losses' category mentioned above23.

Figure 8: A graph showing variation in food losses and waste in different regions at consumption and pre-consumption stages23.

1.5.1. Food Waste Problems

Many problems are associated with food wastage. One of those problems which is associated with all four categories of food wastage is enhancing the greenhouse effect due to emission of greenhouse gases because of anaerobic digestion of food in landfill sites or other disposal sites.

This leads to climate changes and global warming, as well as spread of disease carried by rodents and animals feeding on such waste20. In a study in UK, it was estimated that diverting disposed food from landfills would cause a net reduction in greenhouse effect that would be equivalent to removing away one out of every five vehicles off the road24.

Another problem which is mainly associated with the 'food waste' category is the depletion of natural resources that were used to produce food that was later thrown away intentionally while still being suitable for consumption. This also raises social and ethical concerns as it is estimated that in 2010-2012, 870 million people globally were faced with chronic undernourishment25, while

1.3 billion tons of food that is perfectly convenient for consumption is thrown away every year26, and 90 million tons of which are discarded in Europe alone where 16 million citizens depend on charitable organizations for receiving food aid27. In the US, 38 million tons of food waste were thrown away in landfills in 2014, while 13 per cent of American households suffered from problems in providing enough food for their members28.

Finally, food waste causes economic problems as food waste in developed countries sums to around USD 680 billion and USD 310 billion in developing ones26. These figures represent depleted resources and wasted investments which leads to the reduction of the producer's income and an increase in the consumer's expenses20.

1.5.2. Management of Food Waste

The same methods that were mentioned earlier in waste treatment could be applied for food waste management as well. In addition, a food recovery hierarchy has been adopted specifically for food wastage to address the problems related to it. This food recovery hierarchy (as shown in Figure 9) also starts with the reduction of waste as the most preferred method for food waste management.

The second tier in the food recovery hierarchy is feeding hungry people by donating food to authorized parties such as food banks and shelters. The third tier is diverting food waste to animal feed to reduce the costs needed for animal feed, thus saving money for farmers and companies29.

Figure 9: An illustration of the Food Recovery Hierarchy30

The fourth tier is involving food waste in an industrial process for fuel conversion and energy recovery. This could be done, for example, by anaerobic digestion (Figure 10). In anaerobic digestion, food waste is digested by microbes in the absence of oxygen creating two products; biogas (mostly methane and carbon dioxide) and solid or liquid residue called the digestate. The methane gas is the primary component of natural gas and thus could be utilized in generating electricity and heat and could be processed into biofuel as well. The digestate is usually rich in 16

nutrients and could be used as a fertilizer31. Anaerobic digestion has several advantages such as reducing the amount of waste sent to landfills which would prevent the environmental problems associated with landfills. In addition, it was estimated that processing food waste using anaerobic digestion would create additional profit that was amounted to USD 172 per ton of food waste32.

However, anaerobic digestion is faced with some challenges such as high capital costs, low-cost of competitor methods, and the fluctuation in performance caused by the variability in food waste with regards to nutrient content, moisture content, organic loading rate, pH, etc33.

Figure 10: An illustration of anaerobic digestion34

The fifth tier in the food recovery hierarchy is composting (Figure 11). In composting, food waste is broken down by microorganisms, snails, earthworms and snails in the presence of oxygen into a soil-like material called compost that is later used as an organic fertilizer to improve soils for the next generation crops and it can sometimes be used instead of soil10. Composting helps reduces the emission of methane from food waste, reduces the need for artificial fertilizers, promotes higher agricultural crop yield, can aid in reforestation by improving the quality of soil and can save money

that would be spent on fertilizers or on treatment methods that would be needed to combat air or water pollution35.

Figure 11: An illustration of the composting cycle36

1.6.Banana Waste

1.6.1. Overview on Banana Crop

The amount of waste produced from different food commodities depends on how much a commodity is cultivated and consumed. Bananas are one of the most important crops that are cultivated and consumed worldwide. According to the Food and Agriculture Organization (FAO), bananas rank fourth on the list of the most cultivated food crops after wheat, rice and maize in developing countries37. Accurate estimates regarding banana production are not easy to obtain since banana cultivation is usually conducted by smallholder farmers, but available data shows that the production of bananas globally has increased at an annual rate of 3.7 percent between 2000 and 2015, reaching 117.9 million tons in 2015, up from 68.2 million tons in 200038. In addition,

bananas have also been the number one selling items in Walmart stores for several years up until

201539.

Bananas are one of the most diverse crops, as there are around 1000 varieties of bananas produced worldwide, but currently the most popular banana is the Cavendish type banana which represents around 47% of global production. The Cavendish type banana was the substitute of the Gros

Michel type which dominated earlier until the 1950s when Panama disease destroyed most of the

Gros Michel bananas40. Bananas belong to the family Musaceae, and the different banana varieties are different species within this family according to their 'ploidy' that results from the hybridization between two diploid species; Musa acuminata and Musa balbisiana. The term 'banana' also refers to the edible banana which is eaten directly as dessert (dessert cultivar), whereas 'plantains' (the cooking cultivar) refer to bananas that need to be cooked before eating and it is considered staple food in many African countries like Uganda41.

1.6.2. Waste Produced from Banana Cultivation and Processing

Since bananas are only cultivated for their fruits, several tons of wastes and by-products are associated with banana cultivation and consumption. The main by-product generated from banana consumption is the peel. However, there are also many by-products that are generated due to banana cultivation such as the leaves, pseudo stem, inflorescence, fruit stalk (rachis) and rhizome

(highlighted in Figure 12). These are different parts of the banana tree that are left over after harvesting the fruit, because the bananas eaten today do not have seeds, so farmers must cut the whole plant to allow the young suckers (young budding plant) to replace the mother plant41. All in all, 80% of the total banana plant mass will be regarded as waste after harvesting. Around 220 tons of waste are generated per hectare annually from banana cultivation42. The most common practice

to get rid of such enormous amount of waste is open fire burning, which of course will cause serious environmental problems such enhancing the greenhouse effect.

Figure 12: An illustration of the main parts of the banana tree43

1.7.Management of Banana Waste

1.7.1. Conventional Uses of Banana Waste

Such enormous amount of waste requires innovative ideas to utilize this abundant resource into valuable products. These by-products or wastes generally have a limited commercial value and are regarded as agricultural waste. Farmers usually would leave the pseudostem and leaves after

harvesting so that they would rot and replenish nutrients in the soil. In some regions in Southeast

Asia, young shoots, inflorescence and pseudostem piths might also be consumed as vegetables by the indigenous people in these regions, but to a limited extent due to their undesirable taste when compared with other leafy vegetables44. Moreover, the banana leaves are sometimes used as wrapping materials for traditional foods. A better alternative to the abovementioned applications is utilizing banana waste as animal feed, thus saving the cost needed for conventional animal feed.

However, this application requires additional processing to remove the water content that reduces the nutritional density greatly45. Although banana waste might be poor in essential nutrients to be used for such application, it is at the same time high in fiber content46.

1.7.2. Innovative Applications for Banana Waste

There are also some innovative ideas that might render these by-products valuable as shown in

Figure 13. Banana by-products can serve as renewable resource or biomass that can be turned into other raw materials and products that in turn can be recyclable and biodegradable because of using a renewable resource. Therefore, they could be greatly utilized in what is called 'green technology', which means a process or application that is environmental friendly and conserve the natural environment and resources, while posing the minimum threat to living species on Earth47. To encourage green technology, it should be kept independent from the currently available agro-food commodities, because this might lead to other problems such as food insecurity. For example, if corn is used to drive green technology, this might eventually lead to food insecurity and unsustainable energy return. Banana by-products however are perfect for adaptation in green technology since they are readily available, of no real value in the existing agro-food commodity market and no hazardous phytochemicals were reported in them48. This can also serve as an additional revenue to farmers since most of the banana crop is cultivated by smallholder farmers.

Figure 13: Schematic illustration showing the potential applications of banana by-products41

1.7.3. Source of Valuable Nutrients and Nutraceuticals

The main reason why banana waste is valuable and could be employed in 'green technology' is their composition. Banana waste contains a large amount of nutrients, bioactive compounds and nutraceuticals that could be used for numerous applications such as:

1- Starch: The pith of the pseudostem that is removed during fruit selection, could be processed

into edible starches. It was reported that banana starches have a low amylase content and high

resistance to heating and attack by amylase enzyme and have been proven superior to corn

starch which would give a potentially higher value in the market49.

2- Pectin: Banana peels were reported to have a high pectin content that could be produced via

acid extraction and precipitation using ammonium salts or alcohol. In a study, it was reported

than the pectin content of banana peels was much higher than that in plantain peels and both

of them showed similar or slightly higher pectin content when compared to other fruit peels50.

Both pectin and starch are commonly used as thickening agents, gelling agents and stabilizers

in the food industry41.

3- Anthocyanins: Anthocyanins are the pigments that are responsible for the red and violet colors

of the banana inflorescence which includes the flowers that will develop later into fruits

(Figure 14)51. Anthocyanins are abundant in the banana inflorescence bracts and their reported

concentration was 14-32 mg anthocyanin/100 g bracts. This is slightly higher than the

commercial anthocyanin extracted from red cabbage, which means that it may serve as a cheap

alternative to produce natural colorant for the food industry52.

Figure 14: A photo of the banana tree inflorescence53

4- Dietary Nutrients: Several studies have been made to study the nutritive value of banana by-

products54,55. Some of them are already consumed in some parts of the world, such as the

inflorescence which have consumed as vegetables and salad for a long time, and the banana

pith from the pseudostem which has also been consumed as vegetables in places like Malaysia,

India and SriLanka due to its high content of starch, sugars and minerals44. The peels were also

reported to have a high potassium content, and a dietary fiber content that is slightly higher

than that found in oats, barley and rice brans56. In a study that aimed at producing low calorie

food products with high fiber content, it was found that the incorporation of banana peels into

biscuits at a ratio of 10% did not affect the overall aroma, taste or color while adding the benefit

of having the high dietary fiber content57. The peels were reported to have a dietary fiber

content of 40-50%, proteins and amino acids with a percentage of 8-11% and lipids and fatty

acids with a percentage of 2.2 to 10.9%54.

5- Nutraceuticals & Bioactive Compounds: A nutraceutical is a food product or part of a food

product that could provide medical and health benefits such as the prevention and treatment of

disease58. Banana by-products were found to contain a wide range of compounds having

nutraceutical properties such as different polyphenolics such as gallocatechin, cinnamic acid,

caffeic acid, etc. These compounds were found in banana male flower, pseudostem and peel59.

Polyphenolics are proven to have antimicrobial60, antioxidant61, neuroprotective62,

chemopreventive, anticancer63 and antiproliferative activities64. Another class of nutraceuticals

that are found in the banana by-products are phytosterols that have been proven to lower blood

cholesterol65 and reduce the risk of coronary heart diseases66.

1.7.4. Renewable Fuel

One of the innovative applications for banana by-products is the production of bio-fuel. Fossil fuels have always been the main source of energy throughout the years, and every day the energy demand is booming exponentially, while the production of fossil fuels and the discovery of new reserves are not increasing with the same rate as the demand. In addition, the fossil fuels have always been debatable due to their environmental effects. Therefore, there is a need to use more sustainable fuel to gradually substitute the existing ones. One of the proposed approaches to obtain sustainable fuel was the conversion of agricultural waste into renewable fuel such as ethanol and methane via microbial fermentation of biomass waste67.

Ethanol is used in the industry both as a solvent and a renewable fuel. Bio-ethanol is currently being produced from food crops such as corn. This is done in two steps as shown in Figure 15.

First, macromolecules such as cellulose and polysaccharides are broken down into sugars, and then these sugars are fermented using either bacteria or yeast and converted into ethanol68. Banana peels have also been proven to be suitable for the production of bio-ethanol and thus can reduce the cost of using staple food crops like corn and wheat69.

Figure 15: A schematic illustration of the generation of bioethanol70

Methane is also used as a fuel in many industries and household kitchens as it is the main component of natural gas. It is produced usually from natural gas fields, but it can also be produced by fermentation of organic matters such as agricualtural biomass and manure. This is done via an anaerobic digestion of the bioamss such as banana waste by microorganims as desrcribed earlier71.

In addition, banana peels can also be used to directly produce energy by forming banana briquettes.

Briquettes are produced via a densification process to enhance the energy content and reduce the voluem and moisture content (Figure 16). They are usually made from sawdust and coal cake, but agricutural residues have been proposed as an alternative to overcome the shortage of wood-based products. It was reported that when banana peels are bound with molasses could be used as raw material to produce banana briquettes72. It was even found than banana briquettes when compared to other briquettes made from sawdust, coconut fiber and palm fiber have a much lower burning rate while having the same strength when similar densification paramtertes are applied73.

Figure 16: A schematic illustration of the production of briquettes from biomass74

1.7.5. Heavy Metal and Dye Absorbers

Heavy metals and synthetic dyes pose a great threat to human health as they contaminate the drinking water, and thus absorbers such as activated carbon are used to remove them. Banana pseudostems were reported to be efficient absorbents (after treatment) of lead (II)75, mercury (II)76, chromium (III)77, chromium (IV)78, methyl red79 and methylene blue80.

1.7.6. Green Synthesis of Nanoparticles (NPs)

One of the novel approaches that have been proposed to utilize banana by-products and other food waste is using them as capping and reducing agents in the synthesis of nanoparticles instead of the conventional chemical methods. This is referred to as the green synthesis of nanoparticles.

Conventional chemical methods that are used to synthesize nanoparticles as well usually involve the use of hazardous chemicals such as organic solvents, and they require harsh conditions such as high temperatures and pressures which lead to a higher cost of synthesis. Therefore, interest in developing environmentally friendly and sustainable methods is growing significantly in order to substitute the conventional methods already used81. Recently, several ‘green chemistry’ books describing green processes have been published82–84. Green chemistry refers to chemistry which aims at maximizing the efficiency and minimizing the dangerous effects on the environment and human health. Although no reaction can be perfectly ‘green’, scientists try to abide to the 12 principles of green chemistry as much as possible to decrease the undesirable effect of the chemical industry and research. The 12 principles, as shown in Figure 17, are as follows:

1- The synthetic methods should be designed to as to prevent the formation of waste in the first

place rather than treating or cleaning up the waste after the reaction.

2- The method should be designed to incorporate as many atoms from the reactants into the final

product.

3- Substances that are used and generated from the synthetic methods should have little or no

toxicity on the environment and human health

4- The products of the chemical reaction should have their efficacy preserved while having

reduced toxicity simultaneously.

5- Solvents and mass separating agents should be avoided in the reaction as much as possible,

and if used, they should be safe.

6- Energy requirements of the reaction should be considered with regard to their economic and

environmental impacts and should be minimized as much as possible.

7- The raw materials used in the chemical reaction should be from renewable resources rather

than depleting ones whenever this is technically and economically suitable.

8- Derivatives such as protecting agents and blocking groups should be avoided as much as

possible since they require additional reagents and generate waste.

9- Catalysis is preferred over stoichiometric reactions to increase selectivity, reduce waste, energy

demands and reaction times.

10- The products of the reaction should be degradable so as not to persist in the environment and

the degradation products should be safe as well.

11- Analytical methods should be developed to monitor the chemical reactions as they occur and

prevent in advance the release of any hazardous substances.

12- Accidents such as releases, fires and explosion that could result from the reaction should be

prevented by selecting the non-hazardous substances.

By adhering to one or more of these principles, the overall negative effect of the chemical reactions could be reduced and thus become under the umbrella of “green chemistry”85.

Figure 17: An illustration of the 12 principles of green chemistry86

The banana by-product that have been studied for such application is mainly the banana peel.

Several nanoparticles have been successfully synthesized using banana peel extract such as silver

(Ag) NPs87,88, gold (Au) NPs89 and Pd (NPs)90. Some of the compounds that present in the banana peel extract such as polyphenolics act as capping agents in the process of the NP formation leading a smaller size and a more uniform size distribution. A schematic illustration of the overall mechanism can be seen in Figure 1891.

ZnO NPs are one of those NPs that have wide range of applications and have been synthesized using other fruit waste but has not yet been studied using the banana peel extract.

Figure 18: A schematic illustration of the mechanism of green synthesis of NPs using food waste91

1.8.Zinc Oxide Nanoparticles (ZnO NPs)

1.8.1. The concept of Nanotechnology

Before reviewing the properties and applications of ZnO NPs, it is necessary to define nanotechnology first. Nanotechnology is the conscious synthesis of objects that are in the nano- range to be used for different scientific and technological purposes. To be considered as a nanomaterial, the object should have at least one of its dimensions below 100nm. This limit is mostly agreed upon between scientists by consensus, although there is still no scientific evidence showing the disappearance of properties associated with nanomaterials above 100nm. Thus, it was suggested that an object is considered a nanomaterial if it exhibited the nano-properties even it its dimensions were greater than 100nm92. Nanomaterials can be classified according to their 30

dimensions into: zero-dimensional nanomaterials if all three dimensions where below 100nm (such as quantum dots and nanoparticles), one-dimensional nanomaterials if two dimensions where below 100nm (such as nanotubes, nanowires and nanofibers), two-dimensional nanomaterials if only one dimension was below 100nm (such as ultrathin films) and three-dimensional nanomaterials if all dimensions where not in the nano-range but the material itself was assembled from nano-sized elementary structures (such as nanocomposites) (Figure 19)93.

Figure 19: A general illustration of the different dimensions of the nanostructures94

When objects have one of their dimensions in the nano-range, they were found to exhibit new properties that differ from their bulk counterparts. This change in properties is due to two effects; surface and quantum effects. Reducing the size of an object to the nanoscale results in a larger fraction of surface atoms when compared to bulk atoms. In other words, the surface-to-volume ratio of the object increases and this can be advantageous in properties such as catalysis, surface 31

functionalization and other inherent properties that will become more pronounced such as antibacterial and antioxidant activities95.

1.8.2. Properties and Applications of ZnO NPs

ZnO has a broad range of applications owing to its properties. It is regarded as II-VI semiconductor since Zn belongs to group two and O belongs to group six in the periodic table. It thus has unique optical, electrical conductivity, chemical sensing and piezoelectric properties. It also exhibits a wide band gap which reaches around 3.3 eV96. It was first studied for its antimicrobial effect in the early 1950s, but its actual use as an antimicrobial was reported in 1995 when a study was conducted on it together with MgO and CaO and their antimicrobial activities against several bacterial strains were proven97–99. In the nanoscale, the antimicrobial activity of ZnO increases significantly due to the increased surface-to-volume ratio as it can enter the bacterial cell and interacts with its core which would lead to the death of the bacterial cell. ZnO nanoparticles also possess high optical absorption in the UV region which was found beneficial for its antibacterial activity and its application in cosmetics as a UV absorbant100.

In the food industry, ZnO can be consumed as a source of zinc. ZnO NPs is also used in the antimicrobial food packaging to combat foodborne pathogens. They have been embedded in a wide range of materials such as glass, paper, chitosan, polypropylene and others. Paper films coated with ZnO NPs were reported to exhibit antibacterial activity against E. coli when the films were irradiated with UV light and it was found that increasing the exposure time resulted in higher antibacterial activity. In the absence of light, the antibacterial activity was still expressed but was less than before101. In another study, ZnO NPs were coated onto glass and it was found to exhibit an antimicrobial activity against E. coli and reduced its colony count by 89%, and against S. aureus but to a less extent, reducing it by only 15%. In addition, antifungal activity of ZnO nanorods was

also reported102. ZnO nanorods were coated onto glass surface and were found to have an antifungal activity when tested with Candida albicans and the antifungal activity was found to persist after 2 months of storage103. Active microbial food packaging is a form of food packaging where the packaging material can interacte with product or the headspace inside leading to the inhibition of microbial growth104. It thus reduces the amounts of preservatives that would need to be added to inhibit microbial growth.

1.9.Food Packaging

1.9.1. Foodborne Illness

Food packaging in general is vital to protect food against microbial contamination. Many people are dying every year because of consuming unsafe food which leads to illness either due to microbiological or chemical contamination. According to the World Health Organization, around

1 in 10 people suffers from foodborne illnesses every year and around 420,000 die due to contaminated food105. Several factors contribute to unsafety of food such as using contaminated water, improper food handling and food production processes, the poor storage of food due to inadequate infrastructure especially in developing countries, and poorly enforced regulations. In addition, as animal husbandry practices increase to maximize production, pathogens’ prevalence increases as well, which might contribute to the unsafety of food as well. In addition, the growth of the population in many countries have increased the risk of communicable diseases. Global warming also contributes to this issue as it affects insect vectors causing them to be hosts to new human pathogens. Pathogens themselves are becoming more virulent as well and resistance to antibiotics is increasing everyday106.

The effects of foodborne illnesses on individuals are quite variable. One might suffer from mild self-limiting symptoms such diarrhea, nausea and vomiting, but on the other hand life threatening

conditions might also occur such as kidney and liver failure, neural disorders, paralysis, cancers and premature deaths. The major hosts for most of the foodborne pathogens are those with weak immune systems such as infants, pregnant women, the elderly and immune-compromised patients107. .

In a study108 that was performed in 2011, it was estimated that 9.4 million cases of foodborne illness were caused by 31 different pathogens in the United States. An additional 36.4 million illnesses were domestically acquired through these 31 pathogens as well. The 9.4 million foodborne illnesses result in 55,961 hospitalizations and 1351 deaths each year. Of the 31 pathogens that were discovered, norovirus was found to be the major contributor to foodborne illnesses108.

1.9.2. Functions of Food Packaging

There are several ways to combat foodborne illnesses, one of which is food packaging. Packaging is not just a means of preserving food and other products, it also aims at facilitating the use of the product and communicating with the consumer. It is for these reasons that packaging in general is considered one of the largest industries around the world as it represents around 2% of Gross

National Product (GNP) in developed countries109,110. The basic functions of food packaging can be classified into four main ones (as shown in Figure 20): (i) protection of food from the external environment, (ii) communication and informing the consumer with the required information regarding the packaged commodity, (iii) providing enhanced end use convenience features for the consumer, and (iv) containment of the food product. The communication and convenience functions can be considered as marketing tools for the packaging, but the principal aim of packaging is the containment of the food product and protecting and preserving it by extending its shelf life and maintaining the quality of the packaged food111. The shelf life of a food product

is the time period during which it will preserve its sensory, chemical, physical and microbiological traits and would therefore be safe to consume112.

Figure 20: A general figure illustrating the functions of Food Packaging113

In order to develop a packaging system, one must be aware of the factors that affect the shelf life of the food product which are mainly classified into three factors: (i) the properties of the package,

(ii) the characteristics of the product such as the physical, chemical and microbiological nature of the product, and (iii) the external factors such as the environment that the product is exposed to during different stages such as storage and distribution114. By understanding these three factors, one can have a clear picture of the deteriorative reactions that can influence the food product and accordingly design an appropriate packaging system. Of these deteriorative reactions, microbial contamination and lipid oxidation are considered some of the most important deteriorative reactions associated with food products, especially with multi-ingredient products such as ready- made sandwiches. Lipid oxidation occurs when molecular oxygen reacts with unsaturated lipids

forming lipid peroxides. This leads to the development of rancid flavor and it was reported that some of the end products of lipid oxidation are genotoxic and cytotoxic as well115.

1.9.3. Packaging Materials

To fulfill the required objectives from packaging, different materials and techniques are used. A successful packaging system can be attained by (i) selecting the appropriate material that is suitable for the product characteristics, (ii) does not cause environmental issues, (iii) of reasonable price for the consumer and (iv) abides to the marketing considerations of the product116. Some of the popular packaging materials are paper, paperboard, glass and metal. However, the main category of materials that are used in the packaging industry is plastics. There are many kinds of plastics that are utilized in food packaging such as polyolefins, polyesters, ethylene vinyl alcohol, polyamides, polyvinyl chloride, and polystyrene117. The majority of nowadays packaging is dominated by plastics. Metal, glass and paper do have some good barrier properties, but plastics seem to be more appealing to the food industry owing to their cost effectiveness, light weight, formability and versatile characteristics. Therefore, 40% of the plastics are consumed only by the packaging industry, with half of that for food packaging alone. Although plastics offer many advantages, their use can pose many problems. Firstly, plastics are petroleum-based products and thus they are not sustainable. Secondly, plastics cause a huge waste problem since they are not biodegradable and thus causing an environmental issue. Thirdly, many plastics have weak barrier properties to gases and humidity. Different products have different needs with regards to gas barrier properties and matching the barrier performance of plastics with specific products can be challenging. A solution to this drawback was using polymer blends and multilayered composite structures in plastic packaging. In this manner, the barrier properties of plastics could be somehow

optimized to match the specifications of each product. However, their high expense and the difficulty in recovering these materials still posed environmental problems.

On the other hand, biopolymers were possible candidates as an alternative to plastics. They are biodegradable and thus would not cause the landfill problem caused by plastics and they are quite cheap and readily available. However, their weak barrier and mechanical properties prevented their use in food packaging.

1.10. Packaging Techniques

Some novel techniques are used in packaging to ensure a microbiologically safe environment for the food product. One of these techniques is modified atmosphere packaging (MAP), where the atmosphere inside the packaging is modified to preserve the packaged food. The three main gases that need to be controlled in such technique are oxygen (O2), carbon dioxide (CO2) and

118 nitrogen(N2) . Oxygen is known to promote several deteriorative reactions in different food products such as lipid oxidation and microbial growth. Therefore, oxygen must be kept at very low concentrations in most of food products. Carbon dioxide on the other hand can be beneficial at certain levels since it dissolves forming carbonic acid that lowers the pH and thus, inhibiting the growth of microorganisms111. In addition, carbon dioxide is also reported to inhibit enzymatic reactions in bacteria119. Nitrogen however is relatively inert, and it is used mostly to prevent the pack from collapsing to maintain an acceptable pack shape, and it also acts as a filler gas to reduce the proportions of other gases. Many ready-to-serve products are packed in modified atmosphere

120 using CO2/N2 gas mixes with 25-50 % CO2 and 50-75% N2 to maximize the shelf life . Each product could require specific gas compositions to maintain its shelf life. A drawback of such technique is encountered in products that contain different ingredients where large amounts of air

could be retained in interior spaces. This might lead to inadequate exposure of all components to the modified atmosphere designed for the food commodity121.

Another technique that can be used for packaging food products is aseptic packaging, in which the food product is sterilized and then filled in a sterile packaging under aseptic conditions. The main application of such technique is ultra-heat treated (UHT) products packed in cartons122. This technique requires several steps such as sterilizing the food product before filling, sterilizing the packaging materials before filling, sterilizing lines of production, air, gases and machine zones involved in the packaging process, as well as maintaining sterility throughout the entire operation111.

In addition to the above-mentioned techniques, some post-processing strategies have been developed to reduce the risk of microbial contamination such as pasteurization. This technique is usually used to reduce the risk of Listeria monocytogenes, Clostridium botulinum and Bacillus cereus. It is worth mentioning that Listeria monocytogenes can survive low pH environments, high salt concentrations and low water activities. Moreover, it can also grow under refrigeration conditions, and it was also found to survive different cleaning and disinfection routines. In this regards, it can be considered a major risk in ready-made meals since it might survive different disinfection steps and might contaminate the product during peeling, slicing or filling the meal in the packaging123. Therefore, pasteurization techniques might be used to reduce such risks. There are different forms of post processing treatments such as thermal pasteurization, high pressure processing and irradiation. In thermal pasteurization, an in-package surface heat treatment is applied in the form of steam, dry heat or infrared sources on the food product to increase the shelf life111. If heat treatment might affect the food product, high pressure processing might be used, where the packaged food product is placed in a high-pressure vessel and then the pressure is raised

to high values causing the damage of microbial cell membranes and collapse of vacuoles and cessation of microorganism movement124. The last technique is irradiation where the packaged product is subjected to gamma, X-ray or electron beam irradiation. These radiations would damage the genetic material of the microorganisms, forming lesions and breaks in the DNA strand leading eventually to death of the microorganism125. Although it is an effective method, however, its use is limited due to safety concerns regarding radiated food products and the possible formation of free radicals as result of radiation126.

1.10.1. Nanotechnology in Food Packaging

Another technology that is showing great potential in the field of food packaging is nanotechnology. Nanotechnology is a growing trend in many industries and it has been expected that it would influence around $3 trillion across the global economy by 2020, which would require an average of 6 million employers in different fields127. With regard to food packaging, the applications of nanotechnology in it was estimated in 2008 to be worth $4.13 billion128. A clear indication of the increasing interest in applying nanotechnology to the food sector has also been demonstrated by reviewing the number of scientific publications in recent years that have been retrieved using a database of peer-reviewed literature (Scopus) and searching for the terms ‘food’ and ‘nanotechnology’ whether in the title, abstract or keywords (Figure 21).

Figure 21: Statistics showing the number of publications per year that were retrieved using Scopus (a database of peer-reviewed literature), searching for the terms 'food' and 'nanotechnology'129

The applications of nanotechnology to the food sector in general is quite variable as seen in Figure

22. First, it can be used in agriculture to formulate more efficient pesticides and fertilizers. It could also be used to improve the health of animals though the formulation of more targeted vaccines, and new techniques to detect pathogens. Second, it can be used to improve nutrition by formulating nutraceuticals with higher stability and bioavailability. Nanotechnology could also be used in food processing such as encapsulation of flavor and aromas and improving the textural properties of food. The final area in the food sector where nanotechnology is applied is food packaging, which is probably the most active area in food science that have witnessed nanotechnology applications130.

Figure 22: A general Scheme illustrating the applications of nanotechnology in different areas of food science130

The different categories of food industry applications of nanotechnology include: (i) improving the mechanical and barrier properties using nanocomposites, (ii) active packaging and (iii) intelligent packaging. Each category refers to one of the basic functions that were mentioned earlier (Figure 20). Improving the mechanical and barrier properties and active packaging aims at improving the protection and preservation functions by different mechanisms. Intelligent packaging however aims at enhancing the communication function by providing more accurate data to the consumer regarding the properties of the packed product131.

1.10.2. Enhancement of mechanical and barrier properties

It was mentioned earlier that plastics are the dominating materials that are used in packaging due to their various advatnages mentioned earlier. Their drawbacks however regarding the landfill problems and unsustainability have led scientists to think of alternative solutions to overcome these environmental problems. Biopolymers were the available candidate owing to their biodegradability and availability, but their poor barrier and mechanical properties lagged their use in industry. In addition, plastics had another drawback since no pure polymer would have all the desired mechanical and barrier properties required for different food applications. This led to the need for polymer blends to achieve the desired properties. Although polymer blending was proven useful in that sense, it had limitations such as the higher production and material cost, and the need to use other additives which complicated regulatory operations since additional approvals would be required130.

Nanotechnology was a potential candidate to overcome these drawbacks using polymer nanocomposites which are used to improve the properties of both plastics and biopolymers, especially their physical and barrier properties. A composite material is one that consists of two or more phases, the first of which is called the continuous phase, which is usually present in greater amount, and the other phase is the dispersed phase which often referred to as the filler. In nanocomposites, the filler is an organic or inorganic material that has one of its dimensions in the nano-range (less than 100 nm). In contrast to polymer blending which required significant amounts of materials, nanocomposites require low levels of nanofillers which are sufficient to change properties of the packaging material without having a major influence on the transparency or the density of the continuous phase. One of the first nanocomposites that were used in the packaging industry are the nano-clay composites where clay nanoparticles are incorporated in very small

amounts in different polymers. This was found to enhance the mechanical and barrier properties such as stiffness, toughness, thermal stability, tensile strength and shear strength. For example, when clay nanoparticles were incorporated in soy protein, the oxygen permeability was reduced 6 times using 8% of clay132. Others have reported that when clay nanoparticles were incorporated in soy protein isolate, the tensile strength increased from 2.26 MPa to 15.60 MPa by increasing the percentage of clay nanoparticles from 0 to 15%133. In addition to enhanced mechanical and barrier properties, clay is readily available, biodegradable and quite cheap and that made nanoclay based composites capture considerable interest among the scientific community134. Other materials have also been incorporated as nanofillers and were found to enhance the mechanical and barrier

135 136 137 properties as well such as silicon dioxide (SiO2) , nanocellulose fibers , carbon nanotubes

138 and titanium dioxide (TiO2) . How these nanofillers are thought to improve the barrier properties of packaging material is reported to be the creation of a torturous pathway for gas diffusion (Figure

23). The filler particles are impermeable to gases and thus the gas molecules would have to diffuse around them instead of taking the straight path that they used to take in the absence of the nanofillers. This torturous path that occurs in the polymer due to the nanofillers allows the manufacturer to achieve a larger effective film thickness using small amounts of polymer when compared to that used in polymer blending139. All in all, polymer nanocomposites could be of great advantage in the food packaging industry due to the cost savings, waste reduction and smaller amounts of polymer that would be utilized. In addition, the use of these polymer nanocomposites might allow the use of biodegradable polymers in the food packaging industry which suffer from poor mechanical and barrier properties140.

Figure 23: An illustration showing the torturous pathway concept; (a) gas molecules diffusing through the polymer in a straight pathway. (b) Gas molecules going along a longer torturous pathway due to the presence of nanofillers127

1.10.3. Intelligent Packaging

Nanotechnology has also enhanced another function of the food packaging which is the communication function through intelligent packaging. This enables the consumer to have a clear idea about the real quality of food in real time. Instead of writing on the label ‘Best Before’, an indicator which is fabricated using nanotechnology would interact with the food itself or the headspace generating a response that correlates with the actual quality of the food. This might also be useful for producers during food distribution and production as they could monitor in real time the status of the food and decide which actions should be taken along the entire production and distribution process113.

One example of intelligent packaging are oxygen indicators which are used to monitor the concentration of oxygen in the headspace in modified atmosphere packages using TiO2 nanoparticles as the indicators. Such an indicator (Figure 24) would contain TiO2 NPs, an electron donor such as glyceraldehyde and a redox dye such as methylene blue. Upon exposure to UV radiation, the electrons in the valence band in TiO2 NPs would be elevated to the conduction band 44

which would lead to the creation of electron holes in the valence band. The electron donor would then donate electrons to fill the electron holes preventing the excited electrons from going back to the valence band. This would lead to the buildup of excited electrons in the conduction band which would then be available to reduce the methylene blue dye to a leuko state where methylene blue is colorless. At this stage, the system is activated and upon exposure to oxygen afterwards, the reduced methylene blue becomes oxidized again and the blue color appears again with an intensity that reflects the amount of oxygen in the system141.

141 Figure 24: Oxygen Indicator system based on TiO2 NPs

There are other examples of intelligent packaging as well such as systems developed to detect spoilage and pathogens in the contained food. These systems utilize antibodies that are conjugated to quantum dots that exhibit fluorescence emission blue shifts on binding to the bacterial surfaces.

The advantage of such conjugation is that quantum dots are stable against photo bleaching, have long decay life-time, highly sensitive and have high fluorescence efficiency142. In addition, the thermal history of the packaged food during different stages such as handling, storage and

distribtuion could be monitored by time-temperature indicators (TTIs). For example, silver nanoplates have been used for such application. These nanoplates have sharp edges and in the visible radiation spectrum they would display strong in-plane resonance mode. During storage, the corners of the nanoplates become increasingly round causing a gradual blue shift of the spectrum, changing the color gradually from cyan to blue over time and the level of the developped blue color would depend on the temperature143. Freshness indicators are also used in intelligent packaging to provide the consumer, producer and retailer real time information of the product.

These are mainly based on the detection of microbial metabolites that are produced on spoilage of food such as amines. A method was developed for such an application where a 1-10 nm silver and/or copper coating was deposited on plastic or paper packaging. The transition metal would react with volatile sulphides and the coating changes color144.

1.10.4. Active Packaging

Besides improving mechanical and barrier properties and intelligent packaging, nanotechnology have also been used to combat foodborne illnesses and enhance the protection of food from microbial contamination by active packaging. Active packaging refers to a form of packaging where active materials are directly in contact with food, leading to the modification of the atmosphere of the food or the food composition itself145. Examples of such active materials include antimicrobial compounds, oxygen and moisture absorbers, and preservatives are included in the packaging material. Antimicrobial packaging is a subtype of active packaging is where the packaging play a role in retarding or inihibting the growth of microgorganisms through direct contact with product or the headspace inside104. The incorporation of nanomaterials into the packaging material to be used for combating microbial infection can be divided according to their mechanism of action into two main groups: a system where nanocomposite materials are

incorporated into the packaging material and are in direct contact with the food to exert their antimicrobial effect directly on the food (Figure 26), and a system where nanocapsules are attached to the packaging material are release the antimicrobial component into the headspace of the package where it will interact with the surface of the product (Figure 25).

Figure 25: Controlled release nanosystems of antimicrobial compounds that are incorporated into the food packaging146

In the first system (Figure 25), the nanomaterials are not in direct contact with the food, but rather they are in the form of nanocapsules that release their components into the headspace of the packaging and these capsules are held in place by being enclosed in the interior of the package.

This type of systems can exert direct and indirect antimicrobial activity. Indirect antimicrobial activity can be exploited by having nanocapsules that incorporate carbon dioxide absorbing/emitters, moisture scavengers and oxygen scavengers. These exert their activity by altering the internal atmosphere and thus, inhibiting microbial growth. In, nanosystems with direct

antimicrobial activity, the capsules would incorporate antimicrobial compounds that are volatile such as essential oils which would be released into the headspace147–149.

Figure 26: Antimicrobial food packaging, where antimicrobial agents are immobilized onto the packaging material146

In the second system (Figure 26), the antimicrobial compound is immobilized in the form of nanocomposite materials. The antimicrobial compound would gradually diffuse into the food matrix, eliminating the need to use excessive amounts of preservatives and antimicrobial compounds directly added to the food product149. Different polysaccharides, organic compounds and metal oxides have been used for such application. For example, silver nanocomposites were reported to decrease the microbial count of Salmonella spp. by up to eight orders of magnitude when compared to the control. It is hypothesized that silver’s antimicrobial activity is due to the adhesion of silver to the microbial cell surface causing its rupture, increasing the permeability of the cell membranes, and causing the degradation of lipopolysaccharides150. Chitosan has also been

reported to be a possible candidate for such application as well as it was found to demonstrate antimicrobial activity as well due to the electrostatic interaction between chitosan molecules which are positively charged and the cell membrane molecules of bacteria which are negatively charge, and this interaction causes an increase in the membrane permeability150. In addition, zinc oxide was incorporated in a polyethylene nanocomposite and was found to reduce the spoilage of orange juice without affecting its sensory properties151. Inorganic nanoparticles such as metal oxides are superior over molecular antimicrobials in terms of the ease with which they could be incorporated into the polymer in order to form functional antimicrobial materials, unlike molecular ones that might require the presence of certain functional groups in the polymer used152. Inorganic compounds are also preferred over organic compounds like essential oils owing to the sensitivity of organic compounds to intense processing conditions that are employed in different stages and due to the development of microorganism resistance to them as well. Inorganic compounds in the nano-size however exhibit significant antimicrobial activities in minute amount because of the increased surface-to-volume ratio153 and they are more stable in conditions employed during food processing such as high temperatures and pressures99.

1.11. Zinc oxide NPs in Antimicrobial Food Packaging

Among the inorganic NPs, zinc oxide (ZnO) was found to have several advantages over other metal oxides. In a study where the antibacterial activtites of ZnO, copper oxide (CuO), and ferric oxide (Fe2O3) NPs were evaluated against four different bacterial species- E. coli, , S. aureus,

Pseudomonas aeruginosa and Bacillus subtilis - ZnO NPs showed the greatest antimicrobial activity147. When compared to Ag NPs which were also proven to have antibacterial properties as well, ZnO NPs have some advantages. In terms of fate in the body, ZnO can easily decompose into Zn+2 ions and get used by the body as an essential nutrient. Silver however accumulates over

time by chronic uptake of silver products leading to argyria (permanent bluish-gray discoloration of the skin) in the skin or argyrosis in the eyes154. In addition, ZnO is recognized by the United

States Food and Drug Administration (FDA) as a generally recognized as safe (GRAS) compound

155, unlike silver which has a limit of 0.05 mg Ag+/kg of food that was shown to have no observable effects in humans156. ZnO is also widely used in different food applications such as preventing the spoilage of meat and fish by incorporating it into the linings of food cans and it has also been used as a supplement for zinc. Silver, on the other hand, is not used in the food industry except in the form of nanoparticles in food contact material and most of its use is limited to the healing of chronic wounds and burns.

In an attempt to investigate the activity of ZnO NPs when in contact with food, a group of researchers used orange juice as the food matrix and a packaging that is made up of low density polyethylene packaging embedded once with Ag and once with ZnO NPs. They found that the shelf life of the orange juice was prolonged for up to 28 days without having undesirable effects on the sensory properties of the juice using the ZnO coated film. The silver coated one however, had some undesirable effects on the sensory properties, although it prolonged the shelf life for a longer period than the former film158. A similar experiment was performed using the same films, but the orange juice was inoculated this time with 8.5 log CFU/mL (colony forming unit/mL) of

Lactobacillus plantarum. Both films managed to reduce the rate of the microbial growth, but the silver coated one showed a higher antibacterial activity159.

1.11.1. Antibacterial Mechanism of ZnO

To explain the mechanism of action of ZnO as an antibacterial agent, several theories (Figure 27) have been proposed and the exact mechanism remains unknown. The first theory is the release of

antimicrobial Zn2+ ions. ZnO NPs would release Zn2+ ions which are believed to inhibit active transport systems in the bacteria, and disrupt the amino acid metabolism and enzyme system160.

In the second theory, ZnO NPs are thought to interact directly with the microorganism through electrostatic interaction causing damage to the integrity of the cell161. The bacterial cells acquire a negative charge in biological pH values to the excess carboxylic groups that become dissociated leading to a negative charge on the surface of the cell. ZnO NPs however would acquire a positive charge and thus would become attracted to the bacterial cell leading to the disruption of the bacterial cell membrane162. In one study when ZnO NPs were investigated against E.coli cells, significant damage to the cell wall of the bacteria was detected causing the internalization of the

ZnO NPs into the cells and this led to further damages in the cell wall and intracellular content leakage163.

The third theory suggests that ZnO NPs generate reactive oxygen species (ROS) such as hydroxyl radical, hydrogen peroxide and superoxide and these are responsible for the antibacterial activity.

Their release is thought to be a result of the activation of ZnO NPs by visible light and UV radiation such that when incident radiation has an energy that exceeds the band gap of ZnO (~3.3 eV), electrons move from the valence band to the conduction band and as a result, anelectron hole (h+) is formed in the valence band and a free electron (e-) is formed in the conduction band164. Water molecules would then react with the electron hole causing it to separate to .OH and H+. Oxygen molecules dissolved in water would react with the free electron forming superoxide anion radicals which would then react with H+ forming hydrogen peroxide anions. Hydrogen peroxide anions would then react with H+ ions forming hydrogen peroxide molecules165. The chemical equations explaining the generation of the reactive oxygen species can be summarized as follows (equations

1-5) 165:

− + ZnO + hν → ZnO + ecb + hvb [Equation1]

− .− ecb + O2 → O2 [Equation 2]

+ . + hvb + H2O → OH + H [Equation 3]

.− + − O2 + H → HO2 [Equation 4]

− + − HO2 + H → e → H2O2 [Equation 5]

Only the hydrogen peroxide molecule can penetrate the bacterial cell membrane since it is uncharged unlike hydroxyl radicals and superoxide. After penetrating the cell, oxidative stress is triggered in the cell and finally the cell dies165. One of the downsides to this mechanism is that it requires the presence of UV radiation to initiate the reactions needed for the formation of the ROS, while numerous studies have reported that ZnO NPs showed antimicrobial activity even in dark conditions166–168.

Figure 27: Proposed mechanisms of the antibacterial activity of ZnO149

1.11.2. Synthesis of ZnO NPs

ZnO NPs can be synthesized by many techniques and the synthesis parameters can be modified to obtain the desired size and morphology of ZnO NPs. Different ZnO nanostructures have been successfully synthesized such as nanotubes, nanorods, nanospheres, nanoneedles, flower-shaped 52

nanostructures and nanorings (Figure 28)169. Each morphology has different optical, electrical and physicochemical properties leading to a wide variety of applications170. The methods used for synthesis are either physical or chemical ones.

Figure 28: Different Morphologies of ZnO nanostructures(a, c & d: nanorods. b &g: nanosheets, e & f: prism like structures. h: nanospheres) 171

An example of the physical methods is the mechano-chemical processing (Figure 29). In this simple technique, a ball milling is used to mill the precursors (zinc chloride and sodium carbonate) leading to the production of zinc carbonate and sodium chloride. The chemical reaction occurs because of the local heat and pressure produced by the ball mill which serves as a low temperature chemical reactor. These products are then heat treated to decompose zinc carbonate into ZnO, which is latter washed and dried and then finally obtained as nanoparticles with a size that is influenced by different synthetic factors such as the heat treatment temperature and the milling time. It was reported that as the milling time increased, the size of the ZnO is reduced, while as the temperature increased, the size of the nanoparticles increased172. This method is suitable for

large scale production of ZnO NPs since it is a rather simple method with a low cost. In addition, it does not involve the use of organic solvents so it is a more attractive option from an environmental point of view173.

Figure 29: Synthesis of ZnO NPs using the mechanochemical processing149

Different chemical methods could be used as well for the synthesis of ZnO NPs such as precipitation, sol-gel, and hydrothermal synthesis. The chemical methods allow better control of the morphology and the particle size174. A simple example of the chemical methods is precipitation where a precursor such as zinc acetate dihydrate solution is added to sodium hydroxide and then the solution is heated and stirred leading to the precipitation of ZnO which is then separated from the solution and dried for further experiments175.

In the chemical methods, several parameters need to be controlled to obtain the desired size and morphology such as the reaction time, temperature, concentration of precursors, stirring speed, calcination temperature, etc. One of the methods used to further control the size and morphology is by using size-directing agents or capping agents. These capping agents are usually surfactants such as polyvinyl alcohol (PVA)176, polyvinylpyrrolidone (PVP)177, polyethylene glycol (PEG)178, sodium dodecyl sulfate (SDS)179, etc.

1.11.3. Green Synthesis of ZnO NPs

As mentioned earlier, several methods have been designed to produce ZnO NPs using green chemistry instead of conventional wet chemical methods that use up depleting expensive precursors and require the use of harsh conditions such as high temperatures and pressures. This green approach involves the use of natural reagents such as those derived from plants or using microorganisms. Different parts of the plants could be used such as the stem, leaf, root, seed or fruit. The main components that are utilized in the nanoparticles production are believed to be the polyphenols which act as capping and reducing agents in the process180. In one study, ZnO NPs have a size of 20 nm were successfully produced using palm pollen grains. The palm pollens were believed to act as the reaction medium for the formation of ZnO and as a stabilizer as well181. In another study, the aqueous extract of Sargassum muticum (S.muticum) (brown marine macroalgae) was mixed with zinc acetate dihydrate and heated to 100 °C and this led to the formation of ZnO NPs with a size ranging from 30 to 57 nm182. Other natural extracts have been reported as well such as Moringa oleifera leaf extract183, aloe barbadensis miller leaf extract184,

Eucalyptus globulus leaf extract185 and many others.

Food and agricultural waste have also been used for the green synthesis of ZnO NPs. Several waste materials have also been used to prepare ZnO NPs such as rambutan peel extract186 and lemon peel extract 187. These extracts were reported to be successful in the green synthesis of ZnO NPs owing to their polyphenolic content, and it was mentioned earlier than the banana peel is rich in these polyphenolics as well.

Thesis Scope and Objectives

The aim of this work is to develop a protocol to synthesize stable ZnO NPs using the BPE extract and fully characterize the produced NPs with regards to their morphology, size distribution, zeta potential, stability, antioxidant and antibacterial activities. These NPs will then be compared to others prepared using nonionic and anionic surfactants, as well as commercial bulk ZnO particles to select the NPs that could be incorporated in starch films to be used in antimicrobial food packaging. The films will also be characterized with regards to their antioxidant and antibacterial activities to investigate whether the NPs would retain their activities or not and will be compared to a nylon/evoh commercial film that is widely used in Egyptian households and restaurants.

Chapter 2

Materials & Methods

Chapter 2: Materials & Methods

2.1.Materials

Zinc acetate dihydrate (ACS reagent, molecular weight 219.5 g/mol, purity = 99.5-101.0%) purchased from Lobachemie (India) was used in the synthesis of ZnO NPs as well. Commercial

ZnO (ACS reagent, molecular weight 81.38 g/mol, purity ≥99.0%) purchased from ACROS organics (U.S.A) was used to compare with the prepared ZnO NPs. The Folin-Ciocalteu phenol reagent (2N) and the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) reagent (molecular weight 394.32) , the three nonionic surfactants – polyvinyl alcohol (molecular weight: 125,000), poylvinylpyrollidone (molecular weight: 40,000) and polyethylene glycol (molecular weight:

35,000)– , Sodium lauryl (dodecyl) sulfate (SDS) (molecular weight: 288.38) were all purchased from Sigma-Aldrich (Germany. Gallic acid anhydrous (molar mass 170.12 g/mol) used in the Folin test was purchased from Merck (USA) Sago starch was purchased from a local market in Malaysia.

The bananas used were of the Giant Cavendish type (Musa acuminate) and were purchased from a local Egyptian market.

2.2.Synthesis Methods

2.2.1. Extract Preparation

Extracts were prepared using the peel of Cavendish type bananas according to the previous works188 (Figure 30 ). The bananas were first washed thoroughly to remove possible contaminants.

The peels were then separated from the pulps, and then the peels were cut into small pieces. 50 grams of the peel were heated with 1 liter of distilled water (E1) and another 50 grams were heated with 1 liter of acetone: water (1:1) solution (E2) to 80 °C for two hours. The two obtained extracts

(E1 & E2) were then filtered using 125 mm Whatman filter paper. The filtered solutions were then stored in 4°C for further use. 59

Figure 30: Scheme for the preparation of BPE

2.2.2. Synthesis of ZnO NPs

ZnO NPs were prepared using the precipitation method according to previously described protocols184,189–191. In this study, 4 classes of ZnO particles were analyzed as seen in table 1, one class was purchased (G1), and the remaining three classes (G2, G3 & G4) were chemically synthesized as described in the following sections.

Table 1: List of ZnO samples used in this study

Group Number Sample(s) Description

G1 ZnOC Purchased commercial bulk ZnO particles (commercial) G2 ZnOW Chemically synthesized ZnO NPs prepared (without surfactants or without using surfactants or BPE BPE) G3 ZnO/PVP, ZnO/PEG, Chemically synthesized ZnO NPs prepared (with surfactants ZnO/PVA, ZnO/SDS using surfactants (PVP, PEG, PVA, SDS) capping) as capping agents G4 ZnO/BP1, ZnO/BP2, Chemically synthesized ZnO NPs prepared (with BPE capping) ZnO/BP3, ZnO/BP4 using BPE with increasing amounts as capping agent.

2.2.3. ZnOW NPs preparation:

To prepare ZnOW NPs, 50 mL of 0.1 M solution of Zn acetate was prepared and then drops of 2M

sodium hydroxide (NaOH) solution were added until reaching a pH of 12 (around 3.5 ml of 2M

NaOH). The solution was heated and stirred at 80oc for 2 hours at 1000 rpm and the obtained

precipitate was then obtained by centrifugation at 10,000 rpm for 10 minutes and then washed

using water and ethanol to remove impurities. The precipitate was then dried by lyophilization

using Bio-Base Freeze Dryer-Table Top Type (China). The obtained precipitate was then used for

later characterization and film preparation (Figure 31).

Figure 31: Scheme for ZnOW NPs preparation

2.2.4. ZnO/surfactants NPs preparation:

To prepare ZnO/NPs using surfactants, the same procedure described above was followed with slight modification according to previous works (Figure 32)192. The four surfactants were added at a ratio of 10:1 (0.1M zinc acetate: surfactant) (w:w) before adding NaOH and were completely dissolved. Further steps were followed as described above.

2.2.5. ZnO/BPE NPs preparation: (Green Synthesis)

For the preparation of ZnO NPs using the BPE, different amounts of BPE extract were used as follows:

1- ZnO/BP1 NPs: 0.1 M Zn acetate solution: BPE = 5:1 (v:v)

2- ZnO/BP2 NPs: 0.1 M Zn acetate solution: BPE = 5:2 (v:v)

3- ZnO/BP3 NPs: 0.1 M Zn acetate solution: BPE = 5:3 (v:v)

4- ZnO/BP4 NPs: 0.1 M Zn acetate solution: BPE = 1:1 (v:v)

The extract was added dropwise to the 0.1 M Zn acetate solution according to the abovementioned quantities and then NaOH was added dropwise until reaching a pH of 12 as described earlier and

the resultant solution was stirred and heated at 800C for 2 hours. According to pervious works193, the polyphenolic compounds are relatively stable at such temperature. Table 1 summarizes the

ZnO samples used in this study.

Figure 32: Scheme for synthesis of ZnO/surfactants and ZnO/BPE NPs

2.2.6. Film Preparation

The films were prepared using the solvent cast method as described in previous works. In this study, six different films were used as follows:

1- Commercial film: A film that was purchased from a local market and is used frequently

in households and restaurants for packaging fresh food. It was made of nylon/ethylene

vinyl alcohol (EVOH). (C film)

2- Starch film: film prepared using starch and glycerol only. (S film)

3- S/ZnOC film: ZnOC were incorporated in the starch film as described in the following

paragraph.

4- S/ZnOW film: ZnOW NPs were incorporated in the starch film.

5- S/ZnO/PEG film: ZnO/PEG NPs were incorporated in the starch film.

6- S/ZnO/BP3 film: ZnO/BP3 NPs were incorporated in the starch film.

To prepare the starch film, 4 g of starch were added to 100 mL of distilled water, together with 1.2 g of glycerol (30% w/w of starch used) which was used as plasticizer. This solution was then stirred on a hot plate until reaching the gelatinization temperature (80 oC) for around 30 minutes.

Later, the gelatinized solution was poured in 150 mm polystyrene petri dishes and left for 3 days at room temperature to dry. The dried films were then peeled off and used for further steps (Figure

33).

Figure 33: Scheme for the preparation of starch film

For the preparation of the other films having NPs, 0.2 g of the NPs (5% w/w of grams of dry starch used were first suspended in 100 mL of water, then sonicated for 10 minutes using Q500 probe sonicator operating at 50 watts. 4 g o starch were then added to the sonicated suspension together 64

with 1.2 g of glycerol and the next steps were the same as those followed in the starch film preparation (Figure 34).

Table 2 summarizes the films used in this study.

Figure 34: Scheme for preparation of S/ZnOC, S/ZnOW film, S/ZnO/PEG film, S/ZnO/BP3 film

2.3.Characterization of the BPE

2.3.1. Total Polyphenolics Assay (Folin Ciocalteu Test)

The phenolic content of the prepared banana peel extracts was measured using the Folin Ciocalteu method194,195. 0.5 mL of the extract were added to 2.5 mL of 10-fold diluted Folin reagent and 2 mL of 7% sodium carbonate aqueous solution. A calibration curve was prepared using gallic acid

(reference solution) with different concentrations (0.01 mg/mL, 0.02 mg/mL, 0.03 mg/mL, 0.04 mg/mL, 0.05 mg/mL, 0.06 mg/mL, 0.07 mg/mL, 0.08 mg/mL, 0.09 mg/mL and 0.1 mg/mL). 0.5

mL of these dilutions were added instead of the extract to the Folin reagent and sodium carbonate.

The prepared solutions were incubated for 2 hours and then the absorbance was measured at 765 nm using the CARY 500 UV–Visible Spectrophotometer.

2.4.Characterization of NPs

2.4.1. Transmission Electron Microscopy (TEM)

The size of the ZnO NPs samples was measured using TEM. 0.01% (w/v) suspensions of ZnO NPs in distilled water were first prepared and sonicated using the Q500 sonicator (Qsonica, LLC, USA).

A few drops were then taken on a copper grid and then the grid was positioned in the TEM Hitachi

H-7650 (Japan) for size measurements.

2.4.2. Scanning Electron Microscopy (SEM)

The morphology of the ZnO NPs samples was assessed using the Leo Supra 55 (Zeiss Inc.,

Germany) (ZEISS) field emission electron microscope. Each powdered sample was gold sputtered for 40 seconds at 15 milliampere using 2 targets before examination to render the surface of the particles conducting and prevent accumulation of charges on the surface, thus giving better contrast.

2.4.3. Ultraviolet/visible (UV-Vis) spectroscopy analysis

The UV-vis absorbance was measured for the ZnO NPs to measure the band gap energy and confirm the successful synthesis of NPs. First, 0.01% (w/v) suspensions of ZnO NPs in distilled water were prepared and sonicated using the Q500 sonicator. Then, 2 ml were taken in a quartz cuvette and analyzed using the CARY 500 UV–Visible Spectrophotometer.

2.4.4. Size Stability & Zeta Potential analysis

The size stability and zeta potential of the ZnO NPs produced were assessed using the Malvern

Zetasizer NanoZS90 (UK). 0.01% (w/v) suspensions of ZnO NPs in deionized water were prepared and sonicated as well using the Q500 sonicator. 1 ml of each sample was then placed in a zeta potential cuvette for zeta potential measurement. To assess the size stability, a 5-day study was conducted where each day 1 mL of the previously sonicated suspension was placed in a plastic cuvette and analyzed for its size.

2.4.5. Fourier-Transform Infrared Spectroscopy (FTIR) analysis

To study the chemical composition of the synthesized samples, FTIR was used. Around 2 mg of each sample was added to 200 mg of KBr (spectroscopic grade) and the two were mixed together.

This mixture was then subjected to a pressure of around 20 kPa to produce transparent pellets.

Infrared spectra of the pellets were then obtained using Thermo Scientific Nicolet 8700 FTIR

(USA).

2.4.6. Powder X-ray Diffraction (XRD) analysis

The crystallinity and purity of the prepared ZnO NPs samples was assessed using XRD (Bruker

AXS D8, Germany).

2.4.7. Investigation of the antioxidant Activities

To assess the antioxidant activity of the ZnO NPs samples, the DPPH assay was used as conducted in previous works196. 100 µM solution of DPPH was first prepared in the dark. Then 10 mg of each

ZnO NPs sample was added to 1.5 mL of the prepared DDPH solution and left for 2 hours. The suspensions were then centrifuged, and the supernatant was then placed in a plastic cuvette and absorbance was measured at 517 nm using the CARY 500 UV–Visible Spectrophotometer.

2.4.8. Investigation of the antibacterial activities

A slightly modified version of previous works was used to test the antibacterial activity of the ZnO

NPs197. S. aureus and E. coli pre-cultures prepared by incubating aerobically for 24 hours at 37°C in nutrient broth. After overnight growth, 2mL of the bacterial culture were taken in falcon tubes containing 0.5 mg of the nine ZnO NPs samples for an overnight incubation. The ZnO NPs samples were previously kept under UV light for sterilization. Serial dilutions were prepared from the previously incubated culture until reaching a dilution of 10-7. Then 200µL of the seventh dilution were spread on nutrient agar and cultured at 37°C for 24 hours. In addition, a positive control was prepared without the addition of ZnO powder, and a negative control was prepared using medium without adding bacterial culture to it to check for sterility of the media used in the experiment.

After 24 hours, the colonies were counted and compared to the positive control colonies to calculate the percent inhibition.

2.5.Characterization of films

2.5.1. Porosity Determination

To assess the porosity of the synthesized films, the accelerated surface area porosimetry (ASAP) method was used where each film was cut into very small discs (around 0.2 mm discs). These discs were then inserted in the ASAP 2020 sample holder glass tube. After mounting the tube in the

ASAP 2020 equipment, it was degassed below 50 mmHg for 30 minutes at 30 oC and then it was heated to 80 oC for 6 hours with a heating rate of 10oC/minute. Nitrogen adsorption occurred at -

196 oC to measure the surface area and the pore size distribution. The porosity was analyzed using the Barrett-Joyner-Halenda (BJH) equation.

2.5.2. Investigation of film antioxidant Activity

The antioxidant activity of the films was assessed similarly to the ZnO NPs. Films were cut into

2-cm discs and were placed in the 1.5 mL of 100 µM DPPH for 24 hours. Absorbance was measured for the resultant DPPH solutions, together with the control DPPH solution.

2.5.3. Investigation of film antibacterial Activity

To assess the antibacterial activity of the films, the disk diffusion method was used as this has been reported in several previous studies to be the suitable method for such assessment. The films were cut into discs having a diameter of 34 mm. First, a pre-culture was prepared for both S. aureus and

E. coli. Then 2 mL of both pre-cultures was added to nutrient broth, and vortexed. 100 µL of the resulting solution were then spread on solidified agar. Finally, the discs were placed carefully on the top of the agar after having been sterilized using the UV lamp in the laminar flow. The plates were then incubated overnight.

2.6. Statistical Analysis

GraphPad Prism software was used for statistical analysis where one-way ANOVA was carried out followed by Tukey's test and statistical significance was expressed at P<0.05.

Chapter 3

Theoretical Background of Methods

Chapter 3: Theoretical Background of Characterization Methods 3.1 Folin Ciocalteu Assay

The Folin Ciocalteu (F-C) method is a simple spectroscopic method that can be used to determine the phenolic content in products instead of chromatographic methods. It was developed from the

Folin Denis reagent that was used to determine tyrosine in proteins back in the 19th Century198.The

F-C reagent is usually purchased from suppliers but it can also be prepared in the laboratory by dissolving 100 g of sodium tungstate (VI) dihydrate and 25g of sodium molybdate (VI) dihydrate with 700mL distilled water, 100mL concentrated hydrochloric acid and 50mL of 85% phosphoric acid added to it 150g of lithium sulphate hydrate199. The F-C reagent is stable if it is protected from reductants and light. The reagent consists of a mixture of phosphomolybdic and phosphotungstic acids where the oxidation states of molybdenum and tungsten are 6+. Upon reaction with a reductant such as phenolic compounds, the oxidation states are between 5 and 6 and the molybdenum blue and tungsten blue are formed. These products show broad light absorption with a maximum absorbance at 765 nm. In order to accelerate this reaction, the medium is made basic by adding sodium carbonate or sodium hydroxide as the reaction is quite slow in acidic medium200.

The method was also adapted for the colorimetric determination of proteins as the reagent can also react with tyrosine and tryptophan. This colorimetric determination of proteins is called the Lowry method201. A drawback of the F-C method is the ability of the F-C reagent to react with substances other than phenolic compounds as well. It was found to react with tertiary aliphatic amines, tertiary amine-containing biological buffers, hydrazine, hydroxylamine, inorganic reducing agents, etc202.The most problematic interference was found to be caused by sugars in the sample being analyzed. However, the F-C method was found to be advantageous in the sense that it has an equivalent response to different phenolic substances in the sample being analyzed making it suitable for measuring the total phenolic content, instead of measuring only a fraction of the 71

phenolic compounds which is the case with other methods such as High Performance Liquid

Chromatography(HPLC)203. In fact, HPLC was used in an experiment to analyze components of the Eucalyptus leaf litter and it estimated a total phenolic content that was only 10% of the phenolic content estimated by the F-C method204.

3.2. Sonication

Sonication or ultrasonic dispersion are terms that are given to the process disruption of particle agglomerates through the application of sound waves having frequencies that are not audible. It is a rather inexpensive convenient method that is simple to operate. The basic principle of sonication is described in Figure 35. Sound waves are caused to travel through a liquid medium in high and low-pressure cycles that are alternating at high frequency values (20-40 kHz). In the low-pressure cycle, microscopic vapor bubbles are formed in a process called cavitation. After that, these bubbles collapse in the high-pressure cycle (compression). This collapse leads to the production of localized shock wave that give rise to very high temperatures that might reach up to 10,000 k, pressure spurts and liquid jet streams with speeds that could reach 400 km/h. This huge energy output is the basis of the sonication process205.

In ultrasonic devices, electrical power is transformed by a piezoelectric transducer (probe) into vibrational energy. This transducer can be adjusted by an applied AC electric field. Depending on the way by which the ultrasonic waves are generated in the liquid suspension, sonication is divided into two main types; direct sonication and indirect sonication.

Figure 35: Schematic diagram of cavitation caused by ultrasonic waves and agglomerate fracture206

In the former type, the ultrasound probe is immersed into the mixture directly. An example of direct sonication is the probe sonicator (Figure 36A). However, in indirect sonication, the sample is in a container that is immersed into the liquid where the ultrasonic waves are propagating. The physical barriers to wave propagation are thus much less in direct sonication, leading to the delivery of a greater effective energy yield into the mixture. An example of indirect sonication is the bath sonicator (Figure 36B) where the transducer element or the probe is attached to a metal tank in which a bath holds the suspension of interest. The ultrasonic waves are thus conducted to the surface of the tank first and then into the bath holding the mixture. In the cup sonicator (Figure

36C), another type of indirect sonication, the transducer is inverted in the end of a plastic cup where the container holding the sample is immersed205.

Figure 36: Schematic illustration of the three types of sonicators; (A) Probe-type sonicator. (B) Bath-type sonicator, (C) Cup-type sonicator206

It should be noted that the delivered energy is not totally consumed in the disruption of the agglomerates. This entails that the power display in the instrument does not reveal the real extent of energy that is used to disrupt the particles. A significant amount of energy might be lost due to several reasons such as ultrasonic degassing, thermal losses and certain chemical reactions such as radicals’ formation. The amount of energy that is responsible for the actual dispersion or the separation of agglomerates is consumed in breaking the Van der Waals forces that are holding together the nanoscale or micrometer size particles. Usually, the nanoscale particles form aggregates with larger forces than micrometer size particles and thus would require large energies such as those encountered in sonication to separate. Micrometer size particles however form weaker aggregates that can be easily dispersed with stirring or moderate mixing206.

3.3. TEM

TEM is a microscopy technique that is used to visualize samples that cannot be visualized using the light microscope by using a beam of high energy electrons that interact with an ultra-thin specimen through a series of scattering events. The samples can range from a few micrometers to a few nanometers owing to the high resolution achieved in this technique, and therefore the technique is used in visualizing viruses, sub-organelles, and macromolecules207.

TEM is a microscopy technique, which differs from spectroscopic techniques. In spectroscopy, spectra of the sample under study are obtained to analyze the interaction of electromagnetic radiation with the sample. The spectra would show some parameters such as intensities of radiation that is transmitted, absorbed or scattered as a function of the wavelength, frequency, wave number, etc. These spectra can then be used to interpret some structural features in the sample208.

In microscopy however, the sample is visualized by illuminating it with light and magnifying the image obtained to see the sample. A microscope is any instrument that shows us images revealing detail finer than 0.1 mm. The human eye can see with a resolution of about 0.1-0.2 mm depending on the eye condition and illumination. To see images with a higher resolution, a microscope is needed.

The resolution is the shortest distance between closely spaced points at which the two points can be seen as separate entities, and it depends mostly on the wavelength of the radiation used to visualize the object. In the first type of microscopes, the light microscope, radiation used is in the visible region with a wavelength of about 400-700 nm. This restricts the resolution in light microscopes to about 0.2 micrometers. A system of lenses is used to manipulate the radiation and magnify the images of the specimens under study to about 10 times and 100 times. The discovery

of the light microscope allowed visualization of samples that could not be seen before such as human cells, bacteria, etc209. UV radiation could be used instead of visible light to obtain higher resolution in a modification of the light microscope, the fluorescence microscope. However finer detail and higher resolution could not be achieved using the light microscope as the resolution was restricted by the wavelength.

In 1924, De Broglie introduced the wave-particle dualism and mentioned that all moving matter has wave properties with the wavelength being related to the momentum. He suggested that electrons, like photons and other particles, have wave – particle dualism as well and so they can act as the beam of photons in light microscope. This led scientists to think of a microscope that utilizes a beam of electrons accelerated in an electric field to obtain very small values of the wavelength in order to achieve much better resolution than the light microscope, and this led to the discovery of the electron microscope, in which the wavelength can reach about 2.5 pm in 200 kv electric field that is used commonly in a TEM207. There are several types of electron microscopes, the two most popular are the TEM and the scanning electron microscope (SEM).

Electrons are negatively charged particles. So, when they hit a solid sample, they would interact with the electric potential exhibited by the sample. This entails that electrons interact with both the positively charged nuclei and the negatively charged electron clouds as well. This interaction leads to the deflection of the electrons from their path and a series of scattering events. Scattering is the main interaction that takes place between the electron and a sample and based on the scattering events which can be seen in Figure 37, many signals can be obtained208.

Figure 37: An illustration of the scattering events that can take place when an electron hits a sample208 The reason why scattering is very significant is that it is essential electron microscopy in general.

The human eye cannot visualize objects unless it visible light interacts with it; either though reflection or refraction, and the light microscope also depends on the same idea. Similarly, a sample cannot be visualized in electron microscopes unless it interacts with electrons and scatters them. Therefore, objects which do not scatter electrons are invisible in electron microscopes

(sometimes invisibility is required in some images in TEM). As electrons pass through a specimen, they are scattered by the different scattering centers in the specimen which are either the positive nuclei or the negative electron cloud. The non-uniform distribution of the electrons emerging from the sample can be used to predict different information related to the sample such as chemical and structural properties.

Some electrons are transmitted without scattering or with very little scattering as seen in the figure.

Others are scattered either elastically or inelastically. Elastic scattering involves no loss of the electron energy, that is, no energy was transferred between the electron and the sample that it hits.

Inelastic scattering however, involves some transfer of energy between the electron and the sample. This energy loss might lead to a series of events such as ejection of electron from the inner shell of the sample or ejection of valence electrons. Whether the electron is scattered elastically or inelastically, the scattering might be forward or backward scattering as well. The forward scattering entails that the electron beam is transmitted below the sample, which requires a thin sample that is transparent to electrons to allow them to pass with or without scattering.

Transmission electron microscope, as the name suggests, is based on that forward scattering in visualizing the sample and depends mainly on the electrons that do not diverge greatly from the incident electron beam as TEM is constructed to gather these electrons207.

3.4. FESEM (SEM)

The principle underlying the SEM instrument is quite similar to that of the TEM, in that both rely on the interaction of electrons with the sample and the events that follow that interaction. In SEM however, the signal that is of importance is the generation of secondary electrons (the primary electrons are the incident electrons generated by the electron gun). These secondary electrons can be realized using the scanning electron microscope to give information regarding the topography and morphology of the samples210. The generation of a secondary electron is one of the inelastic events that can take place when a beam of electrons interacts with a sample. An inelastic event takes place when an incident electron interacts with the electric field of the specimen. This causes an energy transfer to take place to the specimen and electrons could be released because of such energy transfer and these electrons are called secondary electrons. The secondary electrons are low

energy electrons (less than 50 eV) and thus, those that are generated in deep regions of the specimen are quickly absorbed, and only those that are generated near the surface are emitted211.

In SEM, the sample must be conductive or coated with a conductive layer such as gold or platinum.

Otherwise charging will take place and the images will be distorted. This would take place because a nonconductive sample will cause incident electrons to stop on one spot rather than flowing through the specimen stage, and a negative charge will be accumulated in the irradiated point which will result in a negative potential at this point. This means that number of secondary electrons emitted will be small because the incident electrons falling on the sample are small and are concentrate to specific regions only as seen in Figure 38. However, if the sample is conductive, the electrons will flow through the specimen stage and the number of electrons flowing to the specimen will be equal to those exiting the sample leading to a clear image of the sample212.

Figure 38: A schematic illustration showing the emission of secondary electrons from (a) a coated sample (b) non-coated sample213

3.5. UV-vis Spectroscopy

The UV and Visible radiation constitute a small portion of the electromagnetic spectrum. Like other forms of electromagnetic radiation, the energy of the UV and visible radiation can be calculated using equation 6214:

E = h ∙ ν [Equation 6]

Where E is the energy of the UV and visible radiation, h is Plank’s constant and v is the frequency

The energy of the visible radiation therefore ranges from 36 to 72 kcal/mol, and the energy of the

UV radiation can reach 143 kcal/mol. When a molecule absorbs radiation with such energy, electronic changes occur, where electrons can transfer from a ground state to an excite state depending on the amount of energy that is absorbed. Light or radiation is only absorbed by a molecule if it has enough energy to move it to higher energy levels. Each absorbing species has its own electronic nature and thus would have distinct electronic transitions that would result in absorbance bands at wavelengths that are characteristic to this species. Vibrational and rotational energy levels however are usually superimposed on the electronic energy levels causing the absorbance bands to be broadened. Based on this principle, UV/Vis spectroscopy is used in spectroscopic analysis and chemical identifications214. It can be used for qualitative analysis, where a spectrometer would record the absorbance of the sample at different wavelengths and would then plot the absorbance versus the wavelength in what is known as a spectrum. From the spectrum, the wavelength at which maximum absorbance occurred (called λmax) can be identified.

This wavelength is characteristic for different chemical species. In addition, different molecules would display a certain pattern in the spectrum and this could be used as a fingerprint to match it

with unknown samples. Moreover, UV/Vis spectroscopy can be used for quantitative analysis of certain chemical species using the Beer Lambert law shown in equation 7215:

A = εbc [Equation 7] where A is the absorbance, ε is the extinction coefficient which is characteristic number for each substance under a certain set of conditions, b is the path length through which radiation passes which is usually the length of the cuvette that holds the sample in the device, and c is the concentration.

From this law, it can be deduced that the amount light that would be absorbed by a sample is directly proportional to the number of absorbing species in the sample. This principle made this technique a common method used for quantitative analytical methods215.

Usually a UV/Vis spectrophotometer (Figure 39) consists of a source of radiation, a dispersing device (or a monochromator), a sample holder and a detector. The source of radiation is usually either a deuterium lamp which gives off a good intensity continuum in the UV region and fair intensity in the visible region. Another source of radiation is the tungsten-halogen lamp which yields a fair intensity over the entire visible region and part of the UV region. A dispersing device, or a monochromator, is then needed to select only part of the radiation emitted by the source to reach the sample. This dispersing device can be a grating or a prism, together with an entrance and exit slit. The sample is then usually held in a cuvette. The cuvette should be made from a material that would not absorb radiation in the selected wavelength region. Therefore, for the visible radiation, plastic cuvettes can be used as they would not absorb radiation, while quartz cuvettes will be used for analysis involving UV radiation as plastic ones would absorb in the UV region.

Finally, the detector is a device that would convert the light signal into an electrical signal. The

most commonly used detectors are the photomultiplier tube detector and the photodiode detector215,216.

Figure 39: A schematic illustration of the UV/Visible spectrophotometer216

3.6. Zeta Sizer

The Zeta sizer is a device that is utilized to analyze the size distribution and stability and the zeta potential of NPs.

The technique upon which the Zeta sizer depends to measure the size is called dynamic light scattering (DLS). DLS is a technique that can be utilized to estimate the particle size in the sub- micron region.

The basis upon which this technique depends in measuring the particle size is the Brownian motion exhibited by the particles in a given solvent. By measuring the Brownian motion, the size of a particle can be predicted as small particles generally have larger Brownian motion and are

bombarded much easier by solvent molecules, while larger particles exhibit a much slower

Brownian motion. The velocity of such motion is interpreted as the translational diffusion coefficient (D) upon which the calculation of the diameter of the particles depend217.

This velocity is measured by estimating by analyzing the rate at which the intensity of a scattered light varies upon detection by a suitable optical device. As the particles move in the dispersion, the intensity of the scattered light fluctuates causing constructive and destructive interferences of the scattered light which leads to the fluctuation of intensities overtime. This fluctuation in intensity is measured against short decay time intervals as shown in Figure 40217. In the case of small particles, the intensity of scattered light tends to fluctuate much more rapidly that in the case of larger ones. The intensities at different time intervals are measured and compared by a correlator. The correlator is used to analyze the extent of likeness between two signals or one signal with itself at different time breaks. In large particles, the signal will be changing slowly and the correlation between scattered intensities are predicted to persist for much longer times. However, in small particles, the Brownian motion is much faster, causing the intensity fluctuations to be much larger and thus, the correlation will decrease rapidly. This relationship between correlation of intensities and time are displayed in a correlogram (Figure 40). The size is then obtained from this correlation function using certain algorithms218.

Figure 40: An illustration showing (A) Scattered light intensity fluctuations due to constructive and destructive interferences. (B) A correlogram showing the correlation decay against time219

The DLS system is comprised of six components as shown in Figure 41. A laser is needed to serve as the light source that would illuminate the sample in the cell. This laser source would provide a stable beam of coherent monochromatic light. The sample is placed usually in disposable plastic cuvettes. The scattered light from the sample is then measured using a detector that is positioned at either at certain angles depending on the model of each instrument. Most modern DLS instruments use the APD (avalanche photodiode) detectors. In order to decrease the intensity of the laser that would reach the sample, an attenuator is incorporated and this leads to the reduction of the intensity of scattering. This is needed as the intensity of the scattered light can only be interpreted by the detector within a definite range. If light reached the detector at a high intensity, the detector will not be able to analyze them due to saturation. The correlator will then compare

the intensities of scattered light at consecutive time breaks to calculate the rate of variation in intensity. This will then be transferred to a computer where the user could examine the date and obtain needed information regarding the size. DLS could be quite advantageous if compared to other techniques, as it is a noninvasive technique that requires minimal sample preparation and no calibration is required for it. In addition, DLS instruments are quite much more compact and affordable and they offer user friendly interfaces that allows users to perform comprehensive data analysis219.

The zeta sizer that is used in measuring the particle size is also utilized to estimate the zeta potential of the particles in a colloidal dispersion. A charged particle in a dispersion will have counter ions close to its surface resulting from their preparation methods and using capping agents that are normally charged or neutral. This will result in the development of an electrical double layer around each particle. This layer is composed of two parts as seen in Figure 42.

Figure 41: Schematic representation of the components of a typical Dynamic Light Scattering system219 The inner part is termed the Stern layer, in which ions are held strongly to the surface of the particle. The outer part however is more diffuse, and it contains ions that form a stable entity with the particle, such that if the particle moves across the solvent, the ions inside the boundary will travel with it. This boundary is called the slipping plane or the hydrodynamic shear and the potential at it is called the zeta potential. The actual charge on the surface of the particle (called the Nernst potential) cannot be measured and only the zeta potential could be predicted220.

Figure 42: A schematic illustration showing a negatively charged particle and its electrical

double layer.219

The significance of the zeta potential is that it can be used to predict the stability of colloidal systems such as a solid dispersed in a liquid or a liquid dispersed in a liquid such as emulsions.

Particles with low or zero zeta potential values will tend to flocculate forming aggregates. While particles with zeta potential values that are at least +30mV or -30mV will form stable suspensions as they are prevented from coming together221.

The basic principle that underlies measuring the zeta potential is the measurement of the electrophoretic mobility of the particles which is achieved through the application of an electrophoresis experiment on the sample and the subsequent measurement of the velocity of the particles, and the application of the Henry equation to obtain the zeta potential value222. The 87

cuvettes used for zeta potential measurements are disposable plastic cuvettes that have gold platted copper electrodes on both of its sides. This cuvette could be used for both DLS and zeta potential measurements. Like DLS measurements, a laser beam is also used to determine the zeta potential

(Figure 42). Two beams will be generated from that laser beam after splitting where one of them will fall onto the sample and the other one will be regarded as a reference beam223. The beam falling onto the sample will be scattered and then it will combine the other splitted beam to measure the Doppler shift which would be used to predict the particle velocity caused by the applied electric field and finally the zeta potential could be determined by a series of mathematical equations219.

3.7. FTIR Spectroscopy

Fourier Transform Infrared Spectroscopy (FTIR) is a technique that is used mainly for qualitative analysis as it is capable of identifying functional groups that are present in the sample being analyzed. By “infrared” we mean any electromagnetic radiation that lies between 0.7 μm and 1000

μm. The mid-IR region (between 2.5 μm and 25 μm) is mostly the region of interest in chemical analysis as it involves frequencies that correspond to the fundamental vibrations of almost all of the functional groups in organic molecules. The radiation in this region does not have enough energy cause electronic excitation, but rather it leads vibrational excitation of the functional groups. The covalent bonds will either experience stretching or bending according to the atoms involved. Therefore, almost all organic compounds absorb IR radiation that would correspond to the energies of the vibrations brought about by the energy of the incident radiation. An IR spectrometer, like a UV/Vis spectrometer, would provide an absorption spectrum that would show the functional groups in the sample and could serve as a fingerprint for each single compound223.

Figure 43: A schematic representation of an FTIR instrument224

A typical FTIR spectrometer (see Figure 43) consists of a source of radiation that would generate

IR radiation. Instead of a monochromator that is used in UV/Vis spectrometers, FTIR spectrometers consist of an interferometer. This interferometer consists of a beam splitter, a compensating plate and two mirrors; one mirror is stationary and the other one is movable. The light from the source would pass through the interferometer and gets split and each beam will be sent in a direction towards one of the two mirrors. The beam that travels to the stationary mirror will reflect back and then fall onto the beam splitter, while the other one travels to the moving mirror causing its total path length to be variable as opposed to that reflected by the stationary mirror. When the two beams recombine, constructive and destructive interference take place due to the difference in the path lengths and this creates an interferogram that would show the variation in transmitted intensity versus time. This intensity versus time spectrum can then be transformed into an intensity versus frequency spectrum by the use of a mathematical function called Fourier transform. This function is done for every point on the interferogram. An advantage of FTIR spectroscopy is that it measures all wavelength simultaneously instead of moving from one

wavelength to another, which would reduce the time needed to obtain the spectrum and improve the signal-to-noise ratio224,225.

3.8. Accelerated Surface Area and Porosimetry System

There are several methods that could be used to investigate the surface and porosity of particles and membranes such as small angle X-ray scattering, microscopy and gas adsorption. Gas adsorption involves the exposure of the particles or membranes to vapors or gases at different pressure conditions and evaluating their uptake onto the particles or membranes. Usually surface atoms differ from bulk ones with regards to their energy, as the atoms lying on the surface are usually more energetic than their bulk counterparts, which makes them more prone to interaction with external factors. Thus, gas molecules and liquid molecules could adhere to the surface and get adsorbed either physically or chemically. The gas adsorption techniques that are used to analyze the surface area and porosity of materials rely on the physical adsorption phenomenon that takes place between gas or vapor molecules with surface molecules of the material being investigated. The gas or vapor molecules are called the adsorptive, the solid surface is called the adsorbent, and once the gas is adsorbed onto the adsorbent they collectively form the adsorbate or the adsorption complex.

To investigate the surface area, the gas molecules will be pumped onto the solid surface at increasing pressure and constant temperature until saturation, and then the pressure will be reduced to allow desorption to take place. An isotherm is then obtained showing the relationship between the amount of gas adsorbed and the pressure and then this isotherm will be mathematically described using different equations to determine the surface area and porosity226.

Of which, the Brunauer, Emmett and Teller (BET) equation is utilized to estimate the surface area of porous materials, where nitrogen is exposed to the material under investigation at 77k (liquid nitrogen conditions) and the surface area is measured from the monolayer of nitrogen molecules formed on the solid surface and from the knowledge of the cross-sectional area of nitrogen molecules (16.2 A2/molecule)227. The area of isotherm that is of importance in the BET analysis is the first region of the isotherm as shown in Figure 44.

Figure 44: A general illustration of an adsorption isotherm228

The Barrett-Joyner-Halenda (BJH) analysis could be used to investigate the porosity, pore size and total pore volume using the desorption branch of the hysteresis loop as shown in Figure

44229. This is done using a modified form of the Kelvin equation to relate the amount of gas desorbed from the surface and the pores of the material as the relative pressure decreases to the size of the pores. The pores can be classified according to their sizes into micropores having a diameter that is less than 2 nm, mesopores having a diameter ranging from 2 – 50 nm and macropores having a diameter that is larger than 50 nm.

Chapter 4

Results and Discussion

Chapter 4: Results and Discussion

4.1.Characterization of the Banana Peel Extract

4.1.1. Total Poly-phenolics Assay (Folin-Ciocalteu Assay)

The Folin-Ciocalteu (FC) test was performed to confirm the extraction of polyphenolic compounds from the banana peel used in the green synthesis of ZnO NPs to control their size and stability.

The FC results (Figure 45) have shown that the two extracts prepared, once using water as the extracting solvent and once using water: acetone (1:1) mixture as the extracting solvent, contained polyphenolic compounds and the calculated amounts in the peel were found to be 235.53 mg GAE

(gallic acid equivalent) /100 g banana peel and 239.61 mg GAE/ 100 g banana peel respectively.

These results were found to be quite less than some of the amounts calculated in previous works which reported that the total phenolic contents in banana peels ranged from 0.9 to 3.0 g GAE /100 g of the dry weight of the peel230,231. However, these reported studies calculated the polyphenolic content in the dry weight of the peel unlike our study in which the peels were used directly without drying.

The reason behind drying the peels in some published studies is to protect the polyphenolic contents from the possible enzymatic action caused by polyphenol oxidase in the peel when the peel is stored for a long period. This was also the reason why acetone was used in the extraction process as it was reported that acetone inhibits the activity of the polyphenol oxidase enzyme and thus preserves the polyphenolics present232. However, the difference between the two extracts (the one prepared using water: acetone (1:1) and without acetone) was not found to be significant which infers that this enzymatic action did not affect the phenolic content.

Figure 45: A graph showing the Folin- Ciocalteu test results performed on the banana peel extract prepared [E1 and E2]

In addition, many different factors are known to influence the efficiency of the extraction of polyphenolic compounds from plant material such as the polarity of the solvent and the polyphenolic contents to be extracted, the extraction time, the extraction temperature, the pH of the extract, the liquid-to-solid ratio233,234, the cultivar used, and the ripeness stage. The factors could also contribute to the difference between the calculated amounts and reported ones.

The phenolic contents that were identified in the banana peel in previous studies were gallocatechin

(160 mg/ 100 g Dry Weight [DW])231 , delphinidin, cyanidin235, catecholamines236, beta carotene, alpha carotene237, stigmasterol, campesterol, cycloartenol, cycloeucalenol and 24-methylene cycloartanol238.

4.2.Characterization of ZnO NPs

The synthesized ZnO NPs were characterized together with the ZnOC. A total of 9 samples were available for characterization as described earlier in table 1.

4.2.1. Size Measurement using TEM

TEM images were taken for the ZnO samples to determine the actual size of the NPs in addition to studying the morphology and the particle size distribution. Although TEM is considered the most accurate tool to determine the particle size, it still suffers from the small sample size it uses which might pose a challenge to draw statistically conclusive remarks regarding the particle size239. It was also used to predict the morphology of the NPs, although SEM was more accurate for such purpose. When possible, ImageJ analysis software was used to draw a histogram showing the particle size distribution for the samples.

Figure 46: (a) TEM image of ZnOC NPs (b) TEM image of ZnOW NPs (c) Size distribution of ZnOW NPs

ZnOC showed a rather prism like structure (Figure 46a) and accurate size measurements were thus not possible. However, the lengths of the structures when measured were found to be larger than 100 nm. On the other hand, ZnOW NPs showed a spherical/sheet like morphology. Size measurements were done using ImageJ and the particle size was found to range between 18 to 30 nm, and most of the NPs showed a diameter of 20-24 nm.

The NPs prepared using the nonionic surfactants showed the largest sizes among all NPs;

ZnO/PVP NPs did not show a uniform particle size distribution with two peaks appearing in the histogram at 29.8 and 41 nm (Figure 47a), ZnO/PEG NPs had a size ranging from 10 to 110 nm most NPs lied between the 30-40 nm range (Figure 47c) and ZnO/PVA NPs were rod like in shape and thus a histogram was not displayed for them (Figure 47e). The reason for the increase in size in the samples prepared using the nonionic surfactants is probably due to the adsorption of the surfactants on the ZnO NPs and since they are of high molecular weight, they showed a size larger than the other samples. ZnO/SDS NPs however, we found to be of similar dimensions of ZnOW, having a size range of 10-50 nm and most of the NPs were in the 20-25 range (Figure 47g).

ZnO/SDS also showed rod-like structures like ZnO/PVP and ZnO/PVA NPs, as well as spherical like particles like ZnOW and ZnO/PEG NPs

Figure 47: TEM images of (a)ZnO/PVP NPs (c)ZnO/PEG NPs(e, f)ZnO/PVA NPs (g)ZnO/SDS NPs & Size distribution histogram of (b)ZnO/PVP NPs (d)ZnO/PEG NPs (h)ZnO/SDS NPs 97

ZnO/BP1 NPs were also of comparable size to ZnO/SDS NPs and ZnOW NPs, showing a size ranging from 10 to 45 nm and most were in the 20-25 range (Figure 48a). However, most of the particles were spherical in shape and almost no rods were observed unlike ZnO/SDS NPs.

The remaining ZnO NPs prepared using the BPE showed a smaller size than other samples;

ZnO/BP3 showing the smallest size ranging from 4 to 12 nm (Figure 48e). The ZnO/BP2 and

ZnO/BP4 NPs showed relatively smaller sizes as well; ZnO/BP2 NPs had a size ranging from 6 to

22 nm (Figure 48c) and ZnO/BP4 NPs ranged from 10 to 24 nm (Figure 48g).

Table 3 summarizes the TEM findings of all nine samples and Figures 49 and 50 show the NPs at similar magnifications for comparison.

Table 2 Summary of TEM measurements of the ZnO NPs

Sample NPs size range Predominating size TEM Morphology (nm) (nm) ZnOC NA NA Prism-like ZnOW 18-30 20-24 Nanosheets/spheres ZnO/PVP 24-44 30-32 & 40-42 Nanosheets/spheres + nanorods ZnO/PEG 10-110 30-40 Nanosheets/spheres ZnO/PVA NA NA Nanorods ZnO/SDS 10-50 20-25 Nanosheets/sphere + nanorods ZnO/BP1 10-45 20-25 Nanosheets/spheres ZnO/BP2 6-22 10-12 Nanospheres (flowerlike bundles) ZnO/BP3 4-12 6-7 Nanospheres ZnO/BP4 10-30 16-18 Nanospheres

Figure 48: TEM images of (a)ZnO/BP1 NPs (c)ZnO/BP2 NPs (e)ZnO/BP3 NPs (g)ZnO/BP4 NPs & Size distribution hisograms of (b)ZnO/BP1 NPs (d)ZnO/BP2 NPs (f)ZnO/BP3 NPs (h)ZnP/BP4 NPs 99

Figure 49: TEM images of (a)ZnOC NPs, (b)ZnOW NPs, (c)ZnO/PVP NPs, (d)ZnO/PEG NPS, (e)ZnO/PVA NPs, (f)ZnO/SDS NPs

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Figure 50: TEM images of (a)ZnO/BP1 NPs, (b)ZnO/BP2 NPs, (c) ZnO/BP3 NPs, (d)ZnO/BP4 NPs

4.2.2. Morphology Assessment using Scanning Electron Microscopy (SEM)

The morphology of the NPs was further assessed using SEM. ZnOC particles also showed a prism- like structure (Figure 51a), similar to the one observed in the TEM image.

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Figure 51: SEM images of (a)ZnOC NPs (b)ZnOW NPs

ZnOW NPs had a flake-like shape that was predominant with very little spheres (Figure 51b). The flake-like structure of the particles also appeared in the ZnO NPs prepared using the four surfactants, with very little spherical particles.

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Figure 52: SEM images of (a)ZnO/PVP NPs (b)ZnO/PEG NPs (c)ZnO/PVA NPs (d)ZnO/SDS NPs

The flakes disappeared in the ZnO NPs prepared using the BPE (Figure 53). ZnO/BP1 and

ZnO/BP2 NPs were very similar and showed uniform conical shaped particles. The ZnO/BP3 NPs were the most spherical in shape and were grouped together in large flower-like structures.

ZnO/BP4 NPs were similar to ZnO/BP3 NPs in shape, but they were grouped in star-like structures rather than large spheres.

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Figure 53: SEM images of (a)ZnO/BP1 NPs (b)ZnO/BP2 NPs (c)ZnO/BP3 NPs (d)ZnO/BP4 NPs

It is evident that the BPE has made the NPs more uniform in morphology with even the last amounts used. As the amount of the extract increased, more spherical NPs were observed and the

NPS get grouped into different nanostructures such as the flower-like nanostructures and nano- stars which were more evident in ZnO/BP3 NPs and ZnO/BP4 NPs at a much lower magnification

(Figure 54).

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Figure 54: SEM images of (a)ZnO/BP3 NPs (b)ZnO/BP4 NPs

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4.2.3. UV Absorbance Assessment using UV/Visible (UV/Vis) spectroscopy

UV-visible spectroscopy was utilized to analyze the optical properties of ZnO and predict the band gap energy in order to confirm the successful preparation of ZnO NPs. The spectra that were obtained are shown in Figure 55. It is clearly evident that all of the prepared ZnO samples showed sharp surface plasmon resonance bands at slightly different wavelengths. The surface plasmon resonance is one of the effects of the interaction of metal particles with electromagnetic radiation.

The oscillating electric fields in an incident radiation induces an oscillation to take place in the conduction band electrons in the metal particles and this oscillation is in resonance with the incident oscillating radiation. The maximum amplitude of such oscillation is reached at a certain frequency which is referred to as surface plasmon resonace240.

The synthesized ZnO samples also showed a blue shift when compared to the commercial ZnO sample which indicates the successful preparation of ZnO NPs. The blue shift was more obvious in the ZnO samples prepared using the BPE which suggests that their sizes were smaller than those prepared using the other surfactants.

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Figure 55: UV-Vis spectra of ZnO NPs; (a)G1 and G2 (b) G3 (c)G4 107

The band gap energies were also calculated using the cut off wavelength for each sample using the following equation241:

c Band Gap Energy = h × [Equation 8] λ where h is Planck's constant (6.626 x 10-34 J.s), c is the speed of light (3.0 x 108 meter/sec and λ is the cutoff wavelength. The calculated energy is expressed in terms of eV (electron volt) where 1 eV=1.6 x 10-19 Joules241. The band gap energy values are presented in table 4.

Table 3 Calculated band gap energies for the ZnO Samples

Sample Cut-off Wavelength Calculated Band Gap Energy (nm) (eV) ZnOC 380 3.37 ZnOW 356 3.49 ZnO/PVP 354 3.51 ZnO/PEG 355 3.5 ZnO/PVA 344 3.51 ZnO/SDS 347 3.58 ZnO/BP1 347 3.58 ZnO/BP2 344 3.61 ZnO/BP3 340 3.65 ZnO/BP4 362 3.43

The highest band gap energies correlated to the samples that showed larger blue shifts which was explained to be due to the size effect. The band gap refers to the region that lies between the bottom of the conduction band and the top of the valence band (Figure 56). As the size of a semiconductor such as ZnO decreases, this band gap increases due to the shift of the absorption edge to shorter wavelengths242. This happens due to what is known as the “quantum confinement” which refers

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to the changes in the atomic structure that is brought about by the effect of the nano-size on the energy band structure243.

Figure 56: General scheme illustrating the band gap energy241

The energy band structure of materials is made up of two bands; the valence band and the conduction band. The valence band refers to the space or the orbitals that lies in the outermost shell of atoms or molecules. It is also known as the “Highest Occupied Molecular Orbital

(HOMO)” and it is the region where the probability of finding the outermost electrons is the highest. This band represents the ground state and the electrons lie in this band attached to the nucleus and thus they remain immobilized unless they will have to move later on to the conduction band.

The conduction band lies at a higher energy level than the valence band. It usually does not contain electrons in the ground state of materials, but it represents the region or the space where the 109

electrons from the valence band can travel to upon excitation. It is thus referred to as the “Lowest

Unoccupied Molecular Orbital (LUMO)”. Once electrons reach the conduction band, they become free to move and are able to conduct electricity. The band gap is usually expressed in electron volts

(eV) which is a small energy unit that refers to the energy that is gained by an electron that was accelerated across a potential difference of one volt. As this band gap energy increases, more energy will be needed to move the electron from the valence band to the conduction band. In conductors, the valence band and the conduction band are overlapping and thus electrons can travel freely. However, in insulators this band gap energy is very large and thus electrons cannot move from the valence band to the conduction band. In semiconductors like ZnO, the band gap energy is narrow, and a small amount of energy is needed to elevate electrons from the valence band to the conduction band.

When the size of the material changes, the band structure changes as well as seen in Figure 57. In bulk material, the bands are actually a continuum of energy levels of many atoms and molecules.

As the size decreases to reach nano scales, these band become narrower and more discrete as each particle is made up of only a finite number of atoms and thus the number of overlapping energy levels in the bands decrease. When the size is further decreased till the particle is made up of only one atom, the band will consist of very discrete energy levels that can be represented by a single line as seen in Figure 57. Therefore, the band gap increases as the particle size decreases and this explains why ZnO NPs exhibit a higher band gap energy than bulk ZnO and why a blue shift is observed in the UV spectrum of ZnO NPs244.

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Figure 57: A general scheme comparing the changes in the band gap of a material in the bulk and its nanoparticle counterpart.244

4.2.4. Size Stability and Zeta Potential Assessment using Zeta Sizer

The hydrodynamic diameter of the ZnO NPs was measured using the Malvern Zeta sizer.

Measurements were taken for five days to study the stability of the size of the NPs in aqueous solutions (Figures 58-60). The values are summarized in Table 5.

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Figure 58: Five-day size stability study for ZnOW

Table 4: Summary of Zeta Sizer Measurements in the 5-day study

Sample Starting Size (nm) Last-measured Size Fluctuation over (nm) days (FOD) (%) ZnOW 773.45 2940.5 28.19 ZnO/PVP 1316.5 2832.5 18.23 ZnO/PEG 952.55 2124 20.65 ZnO/PVA 344.55 3232 87.76 ZnO/SDS 950.55 2034.5 18.19 ZnO/BP1 463.9 3641.5 80.85 ZnO/BP2 239.9 324.3 6.84 ZnO/BP3 281.45 336.15 3.86 ZnO/BP4 642 960.9 9.8

It could be interpreted from table 5 that the starting hydrodynamic diameter was somewhat similar between the first five samples, except for ZnO/PVA which showed the smallest starting diameter.

The percent fluctuation decreased significantly in the ZnO/PVP, ZnO/PEG and ZnO/SDS as compared to the ZnO NPs prepared without using surfactants. However, it increased in the 112

ZnO/PVA NPs. The stability was somewhat improved using PVP, PEG and SDS as capping agents, as compared to PVA.

Figure 59: Five-day size stability study for (a)ZnO/PVP (b) ZnO/PEG (c) ZnO/PVA (d) ZnO/SDS

The starting size was significantly smaller in the NPs prepared using BPE when compared to those prepared with and without surfactants. In addition, the percent fluctuation was found to be smaller in ZnO/BP2, ZnO/BP3 and ZnO/BP4, with ZnO/BP3 showing the smallest fluctuation (3.86%).

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Figure 60: Five-day size stability study for (a)ZnO/BP1 (b)ZnO/BP2 (c)ZnO/BP3 (d)ZnO/BP4

The size that is estimated by DLS is usually quite different from the actual size of the particles determined by TEM because in DLS, the size measurement is based on Stockes-Einstein equation which depends on the hydrodynamic radius in estimating the size245. The hydrodynamic radius is the radius of an imaginary sphere that would have similar diffusion coefficient if place in the same viscous environment of the particles being measured. The actual size was reported to be best estimated using TEM as it determines the actual radius of the particles, despite depending on a statistically small sample compared to DLS239. Another reason for the difference between the size estimated by DLS and TEM is the shape irregularity which was shown in TEM and FESEM as most particles were shown to have rod like structures or flake-like structures and this will affect

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the calculations that are used in DLS to estimate the particle size239. In addition, the DLS measurements depend on the concentration of the sample and therefore, different sizes will be reported for different concentrations239. ZnO NPs at concentrations 1, 10, and 100 mg/L were found to have a size of 2990, 6820 and 11400 nm respectively246.

Another factor that is probably the main reason for this discrepancy in size is the particle aggregation. Metal oxide NPs in general are known to aggregate rapidly in aqueous solutions and especially ZnO. This was reported in a study where TiO2, SiO2 and ZnO NPs were analyzed using

DLS to compare the sizes reported by the manufacturer (25, 40 & 20 nm respectively) to the size measured using DLS. It was found that all three showed much larger sizes when they were analyzed using DLS as shown in Figure 61246.

Figure 61: A graph from a study showing the particle size distribution of TiO2, SiO2 and ZnO in water246

A stability study was also performed on the three metal oxide NPs after disrupting them using ultrasonication. The ultrasonication managed to reduce the size of the TiO2, SiO2 and ZnO to

146, 225 and 244 nm respectively. The TiO2 and SiO2 NPs were still in the nanoscale after one day of the sonication, but TiO2 NPs reached the micrometer scale after 25 hours. The ZnO NPs 115

however aggregated much faster than the previous ones and reached in the micrometer scale in less than two hours as seen in Figure 62246.

This was also observed in another study where PEG 6k, PEG 10k and PEG 100k were used as surfactants for the synthesis of iron oxide NPs with gold core (Fe/Au NPs). The PEG 6k and PEG

10K managed to stabilize the size of the NPs for the first two days unlike PEG 100k which resulted in particles with a diameter of around 600 nm from the first day despite having a larger degree of polymerization that was expected to lead to larger adsorbed layer thickness and thus longer ranged steric forces. Later on, micron sized particles were observed which implied that the dominating motion in the system is the sedimentation of large aggregates (Figure 63).

Figure 62: A graph from a study showing changes in the particles size of TiO2, SnO2 and ZnO NPs after ultrasonication in water246

This was hypothesized to be due to the particle-to-polymer size ratio. When the polymer has a comparable size to the NP, the adsorbed layers of the polymer would be less dense than other smaller polymers and this will lead to fewer adsorbed chains per particle. Although this might be expected to cause a smaller size and better stability, the results did not confirm this and thus it was

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concluded that bridging flocculation might be the reason for the large sizes measured and the lack of stability. Bridging flocculation is a situation where polymer chains attached loosely to a particle would bridge two or more particles together247. This was also reported in another study where poly(styrene sulfonate) (PSS) was used in the synthesis of quasi-single domain magnetic NPs with

Fe0 cores and magnetic shells. It was found that smaller PSS chains were more effective steric stabilizers than larger PSS chains248.

Figure 63: A graph from a study showing the colloidal stability of Fe/Au NPs synthesized using Pluronic F127, Pluronic F68, PEG 6k, PEG 10k, PEG 100l, Bovine serum albumin and Dextran over the course of 5 days247

Zeta potential measurements for our prepared ZnO NPs were also collected for the 9 samples to study the effect of the charge on the stability of the NPs in aqueous solutions. As shown in Figure

64, ZnO NPs prepared with and without surfactants all showed positive zeta potential values.

However, ZnO NPs prepared using BPE with different amounts all showed negative zeta potential values with ZnO/BP2, ZnO/BP3, ZnO/BP4 showing the highest values when compared to the 117

remaining samples. This might be the reason behind the stability of the ZnO/BP2 and ZnO/BP3

NPs in water as the high negative charge implies that polyphenolics in BPE are adsorbed on the

NPs and prevented them from aggregating.

Figure 64: Zeta Potential Values of ZnO samples

4.2.5. Fourier-Transform Infrared Spectroscopy (FTIR) analysis

The FTIR spectra for the prepared ZnO NPs are shown in Figures 66-71. As evident from the spectra, a peak at 430 – 470 cm-1 is common in all of them. This peak corresponds to Zn-O stretching band249 which confirms the successful synthesis of ZnO. The possible mechanism for the formation of ZnO from the precursors used is shown in equations (9-12)243.

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푍n(CH3COO)2. 2H2O + NaOH → Zn(OH)2 + 2CH3COONa + 2H2O [Equation 9]

2+ − 2− 푍n(OH)2 + 2H2O = Zn + 2OH + 2H2O = [Zn(OH)4] [Equation 10]

2− 2− [푍n(OH)4] → ZnO2 + 2H2O [Equation 11]

2− − 푍nO2 + 2H2O → ZnO + 2OH [Equation 12]

Basically zinc acetate like other zinc salts would produce colloids of zinc hydroxide [Zn(OH)2]

250 under alkaline conditions . When excess NaOH is added, the Zn(OH)2 dissolves forming zincate

2- ion [Zn(OH)4 ]. The zincate ion is considered the basic growth unit for ZnO formed later on.

Zn(OH)2 also decomposes at high temperatures to give ZnO and water. The mechanism of the reaction could be visualized in Figure 65.

Figure 65: A general scheme showing the proposed mechanism for the formation of ZnO crystal243

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Figure 66: FTIR spectra of (a) ZnO Acetate (b)ZnOC particles (c)ZnOW NPs

The FTIR spectra of the NPs together with those of the pure surfactants and BPE could also be used to study the capping of the NPs by the surfactants and the components of the BPE by determining the possible functional groups that were involved in the capping process.

The spectrum of the PVP-synthesized ZnO NPs (Figure 67b) shows a broad band near 3424 cm-1 which correlates to the stretching vibration of -OH group. This band is probably due to the moisture absorbed onto the NPs since its position is quite similar in both the ZnO/PVP and pure PVP spectra

(Figure 67a). The band at 1640 cm-1 is attributed to stretching vibration of C=O group. This group is speculated to have a high affinity for zinc ions and thus would be responsible for the capping of

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the NPs. This can be confirmed by the shift in this band from 1655 cm-1 in pure PVP to 1640 cm-

1 in ZnO/PVP which implies that an interaction has taken place.

Figure 67: FTIR spectra of (a)pure PVP (b)ZnO/PVP NPs

Several bands are also appearing in the pure PVP spectra ranging from around 1080 to 1360 cm-1 and this is the range where the band for the stretching vibration of C-N group appears. These bands 121

have disappeared in the ZnO/PVP spectrum except for a small band near 1025 cm-1 which is probably the shifted band of the C-N group implying that it was involved in the capping as well.

This suggests that the C=O and the C-N groups of the PVP have a high affinity for ZnO NPs due to their O and N atoms and that this these groups are responsible for the capping of the NPs251.

In the spectrum of the PEG synthesized ZnO NPs (Figure 68b), the band at 3420 cm-1 is also attributed to the stretching vibration of the OH group. This -OH group is probably due to the adsorbed moisture and the -OH groups in the PEG skeleton as well.

The peaks at 1060 cm-1, 1224 cm-1, and 1409 cm-1in the ZnO/PEG spectrum are most probably due to the stretching vibration of C-OH, the asymmetric stretching of C-O-C,and the alkyl C-H bending vibration respectively. The small band near 854 cm-1 is probably attributed to the C-C skeletal vibrations. This pattern was also observed in previous studies251,252 using PEG to cap NPs and it reveals that PEG stabilized the ZnO NPs through steric and electrostatic interactions of -OH groups in the PEG structure.

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Figure 68: FTIR spectra of (a)pure PEG (b)ZnO/PEG NPs

In the spectrum of the PVA-synthesized ZnO NPs (Figure 69b), the band at 3417 cm-1 is also attributed to the stretching vibration of the -OH group which is due to the adsorbed moisture and the -OH groups found in the PVA skeleton.

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Figure 69: FTIR spectra of (a)PVA (b)ZnO/PVA NPs

The band shifted from 3455 cm-1 in the pure PVA spectrum (Figure 69a) to 3417 cm-1 in the

ZnO/PVA spectrum. This might be due to the increased interaction between the -OH groups of

PVA with the ZnO NPs as the -OH group is found in each repeating unit in the PVA skeleton. The band at 1407 cm-1 in the ZnO/PVA spectrum is probably attributed to the CH bending vibration which was originally at 1438 cm-1 in the pure PVA spectrum. The band at 2929 cm-1 in the

ZnO/PVA spectrum is due to the stretching vibration of the methylene groups in the PVA skeleton.

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This proves that the ZnO NPs were encapsulated by PVA especially through hydrogen bonding with the -OH group in its skeleton251,253.

The spectrum of the SDS-synthesized ZnO NPs (Figure 70b) also shows the same band at 3423 cm-1 which corresponds to the stretching vibration of the -OH group. The band has shifted from

3471 cm-1 in the pure SDS spectrum (Figure 70a) to 3424 cm-1 in the ZnO/SDS spectrum which might be due to hydrogen bonding between the SDS molecule and the ZnO NPs. The bands at

2854 and 2921 cm-1 in the ZnO/SDS spectrum corresponds to the stretching vibration of CH bond.

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-1 2- The band at 1068 cm is characteristic for the SO4 group. Its intensity is much smaller in the

ZnO/SDS spectrum than the pure SDS spectrum.

The bands at 995 cm-1 and 831 cm-1 in the pure SDS spectrum correspond to the S-O-C vibration which have disappeared in the ZnO/SDS spectrum except for a smaller band at 906 cm-1.

Figure 70: FTIR spectra of (a)SDS (b)ZnO/SDS NPs 126

In addition, the band at 1213 cm-1 in the ZnO/SDS spectrum corresponds to the stretching vibration of the S=O group which had slightly shifted from 1222 cm-1 in the pure SDS spectrum and decreased in intensity. These shifts might imply that the electron clouds of S=O and S-O have coordinated with Zn forming ZnO/SDS complex254.

Finally, the ZnO NPs synthesized using the BPE have shown similar spectra as seen in Figure 71.

The BPE, as mentioned earlier, contains a mixture of components such as gallocatechin, delphinidin, alpha and beta carotene, catecholamines and others. According to the spectra of the

BPE (Figure 71a) and those of ZnO NPs synthesized using BPE with different ratios (Figures

71b, c, d, e), several observations could be pointed out to predict the successful capping of the

NPs. The band at 1625 cm-1 in the BPE spectrum is attributed to the stretching vibration of C=O group255. This band has shifted in all four spectra in figures 91-94 to around 1575 cm-1. The band at 3405 cm-1 is common among all of the spectra and it is attributed to the stretching vibration of the O-H group. In the region around 1400 cm-1, multiple weak bands are observed in the ZnO/BP3 and ZnO/BP4 spectra (Figures 71 c,d). These bands are probably attributed to the stretching vibration of the aromatic C=C group. They might have been absent in ZnO/BP1 and ZnO/BP2 because the amount the extract used in their synthesis was less. The band near 1035 cm-1 in the four spectra in figure 91-94 is probably attributed to the C-O-C stretching vibration. It had shifted from 1050 cm-1 in the BPE spectrum.

It is obvious from the above observations that ZnO has a significant binding affinity to several functional groups which were found in the surfactants and the BPE such as C-O, C-N, COO, OH, etc. Thus, there were not huge differences between the spectra of all of the synthesized NPS as

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ZnO would behave similarly no matter which surfactant was used due to its constant binding pattern to specific functional groups. In addition, the surfactants used did not have a lot of differences in their structures, and so it was only possible to differentiate between the NPs by comparing the fingerprint region which was found in the range of 750 – 1800 cm-1.

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Figure 71: FTIR spectra of (a)pure banana peel (b)ZnO/BP1 NPs (c)ZnO/BP2 NPs (d)ZnO/BP3 NPs (e)ZnO/BP4 NPs

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4.2.6. Crystallinity Assessment using X-ray Diffraction (XRD)

The XRD spectra for the prepared ZnO samples together with commercial ZnO are shown in

Figure 72. All samples showed the same peaks which were also found in commercial ZnO, indicating the purity of the prepared ZnO NPs.

Figure 72: XRD spectra of ZnO Samples

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The sharp peaks in Figure 72 also indicate a crystalline structure. No other peaks from starting materials were found which also implies that the surfactants and the components of the BPE did not affect the orientation of the samples. This has also been observed in other studies that have used PVP256, PEG256, PVA176 and SDS257 as capping agents in the synthesis of ZnO NPs.

The peaks come in agreement with the JCPDS file for ZnO (JCPDS 36-1451) which corresponds to the hexagonal wurtzite structure of ZnO. ZnO is a II-VI binary compound semiconductor since it is composed of Zn (from group 12 (IIb) in the periodic table) and O (from group VIA in the periodic table). It usually adopts one of three crystal structures; wurtzite B4, rock salt B1, zinc blende B3, as shown in Figure 73. Under ambient conditions, the wurtzite structure is the most stable phase of the three243.

Figure 73: A general illustration showing the three ZnO crystal structures (a) cubic rock salt (B1), (b) cubic zinc blende (B3), (c) hexagonal wurtzite. The black spheres represent O and the gray spheres represent Zn243

4.2.7. Antioxidant Activity Assessment using the DPPH Assay

The antioxidant activity of the ZnO samples was assessed using the DPPH assay. After measuring the absorbance of the control DPPH solution and the supernatant of the DPPH/ZnO solution, the

DPPH scavenging activity was calculated by substituting the absorbance values in the following equation258: 131

퐴 [1 − 푠] 푥 100 [Equation 13] 퐴푐

(As: supernatant DPPH, Ac: control DPPH)

Figure 74: DPPH scavenging activity of the ZnO samples

The results were then tabulated in Figure 74, which shows than G1, G2 and G3 had a negative

DPPH scavenging activity, while G4 showed positive DPPH scavenging activity values which entails that only ZnO NPs prepared using BPE were shown to have antioxidant activity. DPPH is a nitrogen-centered free radical containing both π system and an unpaired electron in the central nitrogen atom, having a deep violet color in solution. Upon interaction with an antioxidant compound the violet color changes to a pale yellow color due to the transfer of electrons from the antioxidant compound to the odd electron present on the nitrogen atom in DPPH moiety as illustrated in Figure 75 and this results in decreasing n π * transition intensity at 517 nm186. This 132

electron donation results in a stable DPPH molecule and thus the DPPH assay can be used to predict the antioxidant activity of a compound by measuring its ability to scavenge DPPH free radicals according to its hydrogen donation capacity and the result is expressed in term of %DPPH scavenging activity185.

Figure 75: A general illustration of the mechanism of DPPH reduction by an antioxidant compound259

ZnO is known to be a photoactive compound that generates reactive oxygen species (ROS) upon when exposed to UV radiation260,260,261. This explains why G1, G2 and G3 showed negative values, as ZnO is expected to increase the oxidative stress in the DPPH assay. These results were also similar to previous works that studied the ROS generation from ZnO NPs used in different sunscreens when ZnO NPs were proposed as phototoxic elements that results in oxidative stress and thus, several strategies were proposed such as surface coating of the ZnO NPs or adding supplementary minerals and vitamins to reduce the oxidative stress caused by ZnO260,262–264.

In this study, ZnO NPs were surface-coated using BPE, whose components are known to have antioxidative potential as described earlier. The capping of the constituents of the BPE was proven efficient in preventing the oxidative stress causing by the ZnO NPs in solution according to Figure

74. In addition, the antioxidant activity varied significantly between the members of G4, were it was found to increases as the amount of BPE used in the synthesis process increased. This pattern

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was observed in ZnO/BP1, ZnO/BP2 and ZnO/BP3. However, in ZnO/BP4, the antioxidant activity started to decrease again, which might entail that the concentration used in ZnO/BP3 was the optimum one and led to the most efficient capping. This could be a potential substitute to the

ZnO NPs used in sunscreens, and it can also be of advantage in food packaging applications as

ZnO will have dual activity as it will act both as an antimicrobial agent preventing microbial contamination and as an antioxidant agent which would be of advantage in lipid rich food which are prone to oxidation.

Table 5: A summary of antioxidant activities of ZnO NPs prepared using different extracts reported in this study and previous works

Extract Used %DDPH Time of References scavenging activity incubation Polygala tenuifolia root 45.47% 20 minutes 265 extract Coptidis Rhizoma extract 52.34% NA 266

Allium sativum extract 20% 2 hours 196 (garlic) Rosmarinus Officinalis 82% 2 hours 196 extract (rosemary) Ocimum basilicum extract 50% 2 hours 196 (basil) Eucalyptus globulus leaf 63.88% 30 minutes 185 extract Banana peel extract 35.93% 2 hours This study [ZnO/BP1] Banana peel extract 45.65% 2 hours This study [ZnO/BP2] Banana peel extract 76.91% 2 hours This study [ZnO/BP3] Banana peel extract 58.39% 2 hours This study [ZnO/BP4]

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In other studies, ZnO NPs were also prepared using different natural extracts and was also found to exhibit an antioxidant activity. Table 6 gives an overview of these studies' results, with the parameters used in each study that might be the reason for the differences that might be present.

4.2.8. Antibacterial Assessment using the Colony Count Method

The antibacterial activity of the ZnO samples was measured using the colony count method to confirm the antibacterial activity of the prepared NPs and to compare between the different samples with regards to their antibacterial activity. The plates were viewed after 24 hours of plating the bacteria (Figures 76 and 77) and the percent inhibition was calculated by comparing the colonies on the plates were ZnO NPs were added to the positive control and the results were plotted in Figures 78 and 79.

In general, the antibacterial activity of the ZnO NPs prepared was more pronounced in the E. coli strain as seen from Figure 77. Moreover, the difference between the antibacterial activities of the nine samples against E. coli is non-significant since they all showed a percent inhibition of 94-

98%, including the ZnO NPs prepared without using any additives. When it comes to S. aureus, some differences could be pointed out. Both the ZnO NPs prepared using surfactants and BPE showed higher antibacterial activity than the ZnO NPs prepared without using additives. In addition, when comparing the ZnO NPs prepared using surfactants to those prepared using BPE, it is obvious that the ones prepared using the surfactants in general showed a higher antibacterial activity with ZnO/PEG, ZnO/PVA and ZnO/SDS showing the highest activities. The ones prepared using BPE showed a slightly lower antibacterial activity except for ZnO/BP3 NPs which showed the highest activity among them. There was also no significant difference between the antibacterial activity of ZnO/BP3, ZnOC, ZnO/PEG, ZnO/PVA and ZnO/SDS, yet they were all significantly higher than the remaining NPs. 135

Figure 76: Colony count results for S.aureus (a) + ve control (b) ZnOW (c)ZnO/PVP (d)ZnO/PEG (e)ZnO/PVA (f)ZnO/SDS (g)ZnO/Bp1 (h)ZnO/BP2 (i) ZnO/BP3 (j)ZnO/BP4

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Figure 77: Colony count results using E.coli (a) + ve control (b) ZnOW (c)ZnO/PVP (d)ZnO/PEG (e)ZnO/PVA (f)ZnO/SDS (g)ZnO/Bp1 (h)ZnO/BP2 (i) ZnO/BP3 (j)ZnO/BP4

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Figure 78: Percent inhibition calculated from the colony count method [S.aureus]

Figure 79: Percent inhibition calculated from the colony count method [E.coli]

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Possible factors affecting the antibacterial activities:

There are two factors that might influence the antibacterial activity; (i) the size of the particles and

(ii) their surface charge.

For the former, the sizes were determined earlier using TEM and it was found that the ZnO/BP2 and ZnO/BP3 had the smallest sizes (11.1 and 6.5 nm). It was also found that the ZnO NPs prepared using the surfactants had the largest particle size (ZnO/PVP: 29.8-41 nm, ZnO/PEG:

36.29 nm and ZnO/SDS: 26.03nm). It was reported earlier that as the particle size of the ZnO NPs decrease, the antibacterial activity increases267. This effect was found to be more pronounced in E. coli than S. aureus.

Mechanisms of antibacterial actions of ZnO NPs

The theory proposed behind this is that as the particle size decreases, the surface area increases, and thus the amount of H2O2 generated at the surface of the particles increases and this was one of the proposed mechanisms for the antibacterial activity of ZnO NPs as mentioned earlier. It was also reported that E. coli is much more sensitive to H2O2 than S. aureus due to the thin peptidoglycan layer found in gram negative bacteria. This explains why the decrease in particle

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size would have a much more pronounced effect in the antibacterial activity against E. coli rather than S. aureus267.

There is another proposed theory explaining the effect of the particle size on the antibacterial activity. As the particle size decreases, the penetration of the NPs into the bacterial cell and mitochondria becomes much more easier causing cell death by apoptosis268.

Accordingly, this might explain the increased overall activity of the NPs against E. coli rather than

S. aureus. The lack of significant differences amongst the antibacterial activities of the nine samples against E. coli might indicate that decreasing the size beyond a certain value will not further improve the antibacterial activity. In the case of S. aureus, ZnO/BP3 which had the smallest size showed higher antibacterial activity as opposed to the other ZnO NPs prepared using BPE.

This might be related to the small particle size of ZnO/BP3. However, this effect was opposed in

ZnO NPs prepared using surfactants which have shown a larger particle size compared to ZnO

NPs prepared without using surfactants. The reason suggested for this is the rod-like shape of the

ZnO NPs prepared using surfactants which was found in the TEM images. It might be that this morphology aids in the penetration of the NPs into the bacterial cell. The other proposed reason is that decreasing the size beyond a certain value might cause the antibacterial activity to decrease instead of increasing as reported earlier.

Moreover, there is another factor that might interplay with the particle size and explains the results reported in this study and that is the surface charge161. It was mentioned earlier that one of the proposed antibacterial mechanisms of ZnO NPs is the electrostatic interaction between the NPs and the bacterial cell wall causing damage to the integrity of the cell. According to this theory, as the electrostatic interaction between the NPs and the bacterial cell wall increases, the NPs

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accumulated onto the bacterial cell causes damage to the integrity of the cell membrane and subsequent death. Both gram positive and gram-negative bacteria have a negative charge on their outer layers. In gram negative bacteria, the outer membrane contains phospholipids and lipopolysaccharides which impart a strong negative charge to the outer surface of gram negative bacteria (Figure 80)269. Gram positive bacteria do not have an outer membrane, but they have a thick peptidoglycan layer in their cell where lipoteichoic acid which is anchored to the cell membrane by diacylglycerol (Figure 80). The lipoteichoic acid is an anionic glycopolymer having phosphate in their structure and thus imparts a negative charge to the surface of gram positive bacteria270.

Figure 80: A general illustration showing the cell wall structure of (a)gram negative bacteria and (b) gram positive bacteria271

Accordingly, positively charged ZnO NPs would have a more pronounced effect that negatively charge ones. In this study, it was found that ZnO NPs prepared with and without surfactants all have shown a positive zeta potential, unlike those prepared using BPE which had negative zeta potential. This might explain the difference between the antibacterial activities observed against

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S. aureus where ZnO/PEG, ZnO/PVA and ZnO/SDS all showed higher antibacterial activity as compared to ZnO/BP1, ZnO/BP2 and ZnO/BP4. This effect is not pronounced in ZnO/BP3 which had an antibacterial activity comparable to those prepared using surfactants.

Thus, the effect of the particle size would explain the pronounced antibacterial activity of ZnO/BP3 and the effect of the surface charge would explain the pronounced antibacterial activity of

ZnO/PEG, ZnO/PVA and ZnO/SDS against S. aureus. These effects were not pronounced at all in the antibacterial activity against E. coli suggesting a higher overall antibacterial activity of ZnO

NPs against E. coli and the lack of discrepancies among the nine samples might be an interplay of both the effect of the particle size and the surface charge.

4.3.Characterization of the ZnO NPs Films

The films were prepared according to the protocol described earlier. They can be seen collectively in Figure 81. Of the 9 NPs prepared, only 3 were chosen for the film synthesis: ZnOW, ZnO/PEG and ZnO/BP3. The aim of the film preparation was to investigate their antioxidant and antibacterial activities and to study whether the ZnO NPs will be able to exert their activities or not. ZnO/PEG and ZnO/BP3 were chosen because they showed the most positive results in the antibacterial testing, and ZnO/BP3 also exerted the highest antioxidant activity when compared to the rest of the NPs prepared using BPE. ZnOW and ZnOC were also incorporated in films to act as references.

Starch film was used to serve as a negative control for further characterization steps. The purchased nylon film was also used for comparison purposes.

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Figure 81: (a) S film (b)S/ZnOC film (c)S/ZnOW film (d) S/ZnO/PEG film (f)S/ZnO/BP3 film

4.3.1. Porosity Assessment using ASAP system

The results of the porosity assessment as seen in Figure 82 show that the films were mostly mesoporous (S, S/ZnO/BP3, S/ZnO/PEG, commercial film) and only S/ZnOW film was macroporous. All pores were far from the range of bacterial diameter (0.2 -2 µm).

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Figure 82: ASAP results (a)BJH cumulative surface area of pores (b)BJH cumulative volume of pores (c) Diameter of pores

In food packaging, the presence of pores is mostly considered a defect as they would allow mass transfer between the packaged commodity and the environment. More importantly, microbes might also invade through pores reaching 0.4 µm (400 nm) causing microbial contamination. In addition, the presence of pores affect the package atmosphere, as gases would transfer easily by diffusion through small pores272. It is desirable to have the atmosphere within the packaging free of oxygen to avoid the oxidatie effects that are caused due to the chemical reactions that can take place between oxygen and packaged food components273.

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It was thus of importance to investigate the porosity of the prepared films, especially because they are made of a biopolymer like starch which might be incompletely gelatinized leading to the formation of pores and ZnO NPs were added as well which might are immiscible in the starch matrix and might have led to the formation of a less compact structure274. This was reported in previous studies were the incorpotation of lemon essential oil increased the porosity of corn and wheat starch films275.

4.3.2. Antioxidant Activity Assessment using the DPPH Assay

The antioxidant activity of the 6 films was investigated using the DPPH assay to check whether

ZnO NPs would retain its previously exerted antioxidant activity or not. The results are presented in Figure 83.

The results show that the S/ZnO/BP3 film retained its antioxidant activity, showing a 20.91%

DPPH scavenging activity. The remaining films showed no significant activity which conforms to the previous results of their NPs counterparts. No previous study was found to report the antioxidant activity of ZnO NPs when incorporated in films.

145

Figure 83: DPPH scavenging activity of the prepared films

4.3.3. Antibacterial Activity Assessment using the Disk Diffusion Method (Kirby-

Bauer Method)

The antibacterial activity of the films was investigated using the disk diffusion method. The results are shown in Figures 84 and 85. The inhibition zones when present were calculated by measuring their diameters (Table 7). The results showed that both the commercial film and starch films had no antibacterial activities as expected and thus no inhibition zones were observed. The remaining samples all showed inhibition zones, which were much more apparent in the E. coli and this correlates with the previous results in the previous section where the NPs were reported to show similar pattern.

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Table 6: Disk diffusion results of the films

Film Zone of Inhibition Zone of Inhibition %Inhibition %Inhibition Diameter (S. Diameter (E. coli) (S. aureus) (E. coli) aureus) (cm) (cm) C film 0 0 0 0 S film 0 0 0 0 S/ZnOC film 4.1 4.3 20.5 27.9 S/ZnOW film 4.1 4.7 20.5 46.8 S/ZnO/PEG film 4.0 4.6 17.6 45.3 S/ZnO/BP3 film 4.0 4.8 17.6 50.0

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Figure 84: Disk diffusion results using S.aureus (a) C film (b) S film (c) S/ZnOC film (d) S/ZnOW film (e) S/ZnO/PEG film (f) S/ZnO/BP3 film

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Figure 85: Disk diffusion results using E.coli (a) C film (b) S film (c) S/ZnOC film (d) S/ZnOW film (e) S/ZnO/PEG film (f) S/ZnO/BP3 film

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Chapter 5

Figure5.5: MicroscopicConclusion images and Future Perspectives

150

Chapter 5: Conclusion and Future perspectives

In this study, ZnO NPs were chemically synthesized using zinc acetate and sodium hydroxide with the aid of capping agents to adjust the size distribution and morphology. The NPs prepared were grouped in three classes; those prepared without using capping agents, those prepared using nonionic and anionic surfactants as capping agents and those prepared using BPE with different amounts as capping agent. The ZnO samples were characterized using TEM, SEM, UV/vis spectroscopy, FTIR, XRD, DLS, DPPH and colony counting method together with commercial bulk ZnO particles. It was found that the ZnO NPs prepared using BPE showed the smallest TEM size (reaching 6 nm in ZnO/BP3), uniform spherical morphology under the SEM, stable size and limited particle aggregation, as well as significant DPPH scavenging activity (76.91%). The antibacterial activity of all ZnO samples were found similar, with higher activity observed against

E. coli when compared to S. aureus.

ZnO NPs were then incorporated in biodegradable starch films and the films were characterized with regards to their antioxidant and antibacterial properties and were compared to a commercial nylon/evoh film that is commonly used in Egyptian households and restaurants. All films, except the commercial film, exhibited comparable antibacterial activities when characterized using the disk diffusion assay. In addition, the film prepared using the ZnO/BP3 NPs was the only film that exhibited antioxidant activity as well with a DPPH scavenging activity of 20.91% which makes this film a potential candidate for antimicrobial food packaging that would have an additional antioxidant activity and would be of benefit in lipid rich food that are prone to lipid oxidation.

Further studies would be needed to investigate the antioxidant and antibacterial activities of the films in real food commodities and against more strains of bacteria that are known to cause foodborne infection such as L. monocytogenes and C. albicans. 151

Chapter 6

Figure5.5: Microscopic images Appendices

152

Chapter 6: Appendices

Appendix I: Synthesis of ZnO NPs using fruit extracts

ZnO NPs were intended to be synthesized using the extracts of the pulp and the peel of three fruits: dragon fruit, rambutan and banana (Figure 86).

Figure 86: Photos of the pulps and peels of fruits used (a)banana (b)dragon fruit (c)rambutan

The extracts were prepared from the peels and pulps separately and were then added to a 0.1 M zinc acetate solution for the synthesis of ZnO NPs. Precipitates formed were centrifuged, dried in hot oven and characterized using SEM (Figure 87).

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Figure 87: SEM image of particles prepared using (a) dragon fruit's pulp extract (b) dragon fruit's peel extract (c) rambutan's peel extract (d) banana pulp extract (e)banana peel extract

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Appendix II: Nanocellulose fibers synthesis

Nanocellulose fibers were prepared using wet disk milling for incorporation in the final film together with ZnO to enhance the mechanical properties of the film. 20 grams of sigmacell cellulose type 101 were suspended in water that was adjusted to two liters. This slurry was then left overnight in 4°C. Wet disk milling was then performed using the Supermass colloider

MKZA10 (Masuko Saangyo Co., Ltd., Saitama, Japan). The ceramic nonporous disk grinders were adjusted so that distance between the upper and the lower grinder was 50 µm and they were revolved at 1800 rpm. 20 cycles were performed on the prepared slurry to produce the desired size of nanofibers. The prepared nanocellulose fibers were characterized using SEM (Figure 88).

Figure 88: SEM images of nanocellulose fibers at different magnifications

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Appendix III: Film Synthesis

Biodegradable starch films were synthesized using the prepared nanocellulose fibers at different concentrations (0.4%, 0.8%, 1.6% w/w of starch) to investigate the mechanical properties of the films and and select the optimum nanocellulose concentration to achieve the best mechanical properties. Mechanical testing was performed using the TA.XTplus Texture Analyzer and the film having a nanocellulose concentration of 0.8% was found to be superior among the remaining ones as it showed the highest extensibility and tensile strength (Table 8).

Table 7: Mechanical Testing results of nanocellulose films

Sample Tensile strength Extensibility (N/m2) (mm) Starch 1 3.97 12.71 Starch 2 3.93 10.6 0.4% NC1 2.59 23.37 0.4% NC2 2.26 23.5 0.4% NC3 2.39 22.92 0.8% NC1 4.29 36.79 0.8% NC2 4.14 31.64 0.8% NC3 5.47 27.65 1.6% NC1 4.04 21.64 1.6% NC3 4.61 13.6

The particles prepared from the green synthesis using the BB extract were added to the film together with nanocellulose fibers to render the film antimicrobial. Homogenized commercial ZnO

NPs were also used for the synthesis of the film instead of the green synthesized particles. SEM images of the films can be seen in Figure 89.

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Figure 89: SEM image of (a)starch film (b) starch + homogenized ZnO film (c) starch + nanocellulose + homogenized ZnO film (d) starch + green synthesized particles film

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Appendix IV: Folin Test Calibration Curves

To characterize the banana peel extracts prepared (E1 and E2) prepared, the Folin test was used and the calibration curves (shown in figure 90) were used to calculate the GAE value for both extracts.

Figure 90: Calibration curves of gallic acid solutions used in folin test (a) gallic acid dissolved in water for E1 analysis (b) gallic acid dissolved in acetone:water (1:1) for E2 analysis

158

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