SURFACES TO INACTIVATE PATHOGENIC MICRORGANISMS

EFFICACY OF PHOTOCATALYTIC NANOCOATINGS ON FOOD CONTACT

SURFACES TO INACTIVATE PATHOGENIC MICROORGANISMS

by

VEERACHANDRA K. YEMMIREDDY

(Under the Direction of Yen-Con Hung)

ABSTRACT

TiO2 is a promising photocatalyst for use in food processing environments as an

antimicrobial coating. The overall goal of this research was to develop physically stable

TiO2 nanocoatings with strong bactericidal property on food contact surfaces. A testing

protocol was developed to determine the photocatalytic bactericidal activity of TiO2

nanoparticles (NPs) in suspension. Among the tested TiO2 NPs, Aeroxide®

P 25 was

found to be the most efficient and achieved a 5 log reduction of bacteria in 3h. Type and

source of TiO2, bacterial cell harvesting conditions, volume of suspension, and intensity

of UV-A light had significant effect on the log reduction. Further, the effect of food

organic matter on bactericidal property of TiO2 NPs was investigated. Increasing the

concentration of organic matter decreased the bactericidal efficacy of TiO2. A linear

correlation was observed between chemical oxygen demand (COD) and total phenolics as

well as COD and protein contents. An empirical equation in the form of “Y=me-nX

”

(where Y is log reduction, X is COD and m, n are reaction rate constants) was able to

successfully predict the disinfection kinetics of TiO2 in the presence of organic matter (R2

= 0.944). In the next study, TiO2 coatings having a thickness of 50-100 µm were

developed on stainless steel substrates either by dip-coating or painting. Among several

tested coating formulations using different binders; TiO2 coatings containing shellac,

polyurethane, and polycrylic as binders at 4 to 16 weight percent were physically stable

when subjected to adhesion strength, scratch, and wear resistance tests. An indented

coupon technique was found to be the most appropriate method to determine the

bactericidal property of TiO2 nanocoatings. TiO2 coating with polycrylic showed the

greatest reduction followed by TiO2 coating with polyurethane, and shellac. On repeated

use of coatings for 1, 3, 5, and 10 times, TiO2 coating with polycrylic was found to be

physically more stable and able to retain its original bactericidal property. The results of

this research show promise to development of durable photocatalytic antimicrobial

nanocoatings on food contact surfaces to help ensure a safe food processing environment.

INDEX WORDS: Titanium dioxide, Nanoparticles, Food contact surface, Antimicrobial

coating, Physical stability, Photocatalytic activity, Organic matter, E.coli O157: H7.


SURFACES TO INACTIVATE PATHOGENIC MICRORGANISMS

by


B. Tech., Osmania University, India, 2004

M. S., University of Georgia, 2011

A Dissertation Submitted to the Graduate Faculty of The University of Georgia in partial

Fulfillment of the Requirements for the Degree

DOCOTOR OF PHILOSOPHY

ATHENS, GEORGIA

2015

© 2015

Veerachandra K. Yemmireddy

All Rights Reserved


SURFACES TO INACTIVATE PATHOGENIC MICRORGANISMS

by


Major Professor: Yen-Con Hung

Committee: Yiping Zhao

Joseph F. Frank

Jennifer L. Cannon

Alexander M. Stelzleni

Electronic Version Approved:

Julie Coffield

Interim Dean of the Graduate School

The University of Georgia

May 2015

iv

DEDICATION

To my parents and my teachers who all have made me what I am today

v

ACKNOWLEDGEMENTS

I would like to thank everyone who has been part of this project. I want to

especially thank my major professor, Dr. Yen-Con Hung, who not only supported me in

successful completion of this research but also encouraged and challenged me throughout

my academic program towards the best realization of my goals. The knowledge and the

training that I obtained under his tutelage is invaluable in shaping me up as a researcher.

Dr. Hung, it was a pleasure to work with you and an honor to have you as my mentor

during my graduate studies at UGA.

I would like to thank Dr. Yiping Zhao for being part of my committee and always

challenging me with questions that helped me to better understand the subject of

photocatalytic nanomaterials. I also would like to thank Dr. Frank, Dr. Cannon, and Dr.

Stelzleni for serving on my Ph.D. committee and guide me through this process. I am

deeply thankful to Mr. Glenn Farrell for all his technical help throughout this research. I

acknowledge the support of all my labmates in Dr. Hung’s research group without whom

this could not have been done. In addition, I would like to extend my thanks to all the

personnel in the Melton building for their friendship and support throughout my research

work at UGA-Griffin campus.

Finally, I want to thank my family and friends, especially my father Mr. Linga

murthy, my mother Mrs. Vijaya lakshmi, my brother Mr. Ramesh kumar, and my wife

Srividya for their love, encouragement, and support.

vi

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS……………………………………………………...…........v

LIST OF TABLES ………………………………………………………………………vii

LIST OF FIGURES …………………………………………………………………….viii

APPENDICES……………………………………………………………………………x

CHAPTER

1 INTRODUCTION…………………………………………………………………….1

2 LITERATURE REVIEW……………………………………………………………..5

3 SELECTION OF PHOTOCATALYTIC BACTERICIDAL TITANIUM DIOXIDE

(TiO2) NANOPARTICLES FOR FOOD SAFETY APPLICATIONS……..…….....85

4 EFFECT OF FOOD PROCESSING ORGANIC MATTER ON

PHOTOCATALYTIC BACTERICIDAL ACTIVITY OF TITANIUM DIOXIDE

(TiO2) ...……………………………………………..……………………………...109

5 METHOD DEVELOPMENT FOR CREATING TITANIUM DIOXIDE (TiO2)

NANOCOATINGS ON FOOD CONTACT SURFACES AND METHOD TO

EVALUATE THEIR DURABILITY AND PHOTOCATALYTIC BACTERICIDAL

PROPERTY ……………………………………………………………………….139

6 EFFECT OF BINDER ON THE PHYSICAL STABILITY AND BACTERICIDAL

PROPERTY OF TITANIUM DIOXIDE (TiO2) NANOCOATINGS ON FOOD

CONTACT SURFACES ………………………………………………………….172

7 CONCLUSIONS ……………………………………………….............................201

vii

LIST OF TABLES

Table 3.1. Characteristics of commercial TiO2 NPs………………………….………...104

Table 3.2. Effect of light intensity and volume on bactericidal activity of TiO2 NPs….105

Table 4.1. Comparison of kinetic models to predict the TiO2 disinfection efficacy with or

without organic matter………………………….………………………………………131

Table 4.2. Effect of pH of wash solution containing organic matter on the bactericidal

activity of TiO2…………………………………………………………………………132

Table 4.3. Comparison of fitted isotherm parameters of empirical model……………..133

Table 5.1. Composition of different TiO2 nanocoatings………………………………..164

Table 5.2. ASTM D3359-02 classification of adhesion test results……………………165

Table 5.3. Physical stability results of TiO2 nanocoatings with different binders……..166

Table 5.4. Bactericidal activity of TiO2 nanocoatings using different test methods.….167

Table 6.1. Details of the binders and the composition of different TiO2 nanocoatings..195

Table 6.2. Estimated surface coverage of nanocoatings with the binder and the TiO2

nanoparticles……………………………………………………………………………196

Table 6.3. Physical stability of TiO2 coatings before and after repeated use

experiment……………………………………………………………………………...197

Table B1. Summary of photocatalytic bactericidal activity of various types of Fe2O3 in

suspension and as a nanocoating……………………………………………………….220

viii

LIST OF FIGURES

Fig 2.1. Semiconductor photocatalysis…………………………………………………..83

Fig 2.2. TEM of a dispersion of TiO2 Degussa P-25 (1 mg/L) in contact with E. coli K-12

Cells…………………………………………………………...…………………………84

Fig 3.1. Schematic of photocatalytic disinfection set-up……………………..………...106

Fig 3.2. Effect of TiO2 source and bacterial cell harvesting conditions on the log

reduction………………………………………………………………………………..107

Fig 3.3. Comparison of photocatalytic degradation of methylene blue and photocatalytic

disinfection rate of E.coli O157:H7 among different TiO2 NPs……………………….108

Fig 4.1. Effect of different levels of organic matter from produce and meat extract

solutions on the log reduction of E.coli O157:H7 by TiO2 photocatalysis…………….134

Fig 4.2. Effect of turbidity of produce and meat extract solutions on the log reduction

of TiO2 of E.coli O157:H7 by TiO2 photocatalysis for 3h ……………………………135

Fig 4.3. Correlation between total phenolics and COD of produce extract as well as total

phenolics and log reduction of E.coli O157:H7 by TiO2 photocatalysis ……………..136

Fig 4.4. Correlation between protein content and COD of meat extract as well as protein

and log reduction of E.coli O157:H7 by TiO2 photocatalysis ………………………..137

Fig 4.5. Relationship between COD of produce and meat organic matter extracts and

the log reduction of E.coli O157:H7 by TiO2 photocatalysis ……………………..…138

Fig 5.1. Images of TiO2 nanocoatings with shellac (A), polyurethane (B), and polycrylic

(C) binders at different NP to binder concentrations…………………………………168

ix

Fig 5.2. Scanning electron micrographs of the surface of TiO2 coatings with binders

A, B, and C at different NP to binder concentrations………………..…………………169

Fig 5.3. SEM image of TiO2 coating with binder C at 1:8 NP to binder weight ratio (TC8)

showing regions of binder, surface exposed TiO2 NPs, and unexposed TiO2 NPs that are

partly covered by the binder……………………………..………………………..……170

Fig 5.4. In-house fabricated wear resistance tester……………………..………….…...171

Fig 6.1. Effect of type and concentration of binder on the log reduction of E.coli

O157:H7 by TiO2 nanocoatings at 0.5 mW/cm2 UV-A light intensity for 3 h…………198

Fig 6.2. Effect of UVA light intensity on the log reduction of E.coli O157:H7 by TiO2

nanocoatings……………………………………………………..……………………..199

Fig 6.3. Bactericidal activity of different TiO2 nanocoatings against E.coli O157:H7

before and after repeated use experiment…………………………...………………….200

Fig A1. TiO2 nanocoating on stainless steel surface (SS) using (a) Direct coating,

(b) Layer-by-Layer coatings methods……………………………..…………………..210

Fig A2. Effect of coating method on bactericidal activity of TiO2 coatings……….….211

Fig A3. Comparison of TiO2 nanocoatings with binders A (TA8/AT8), B (TB8/BT8),

and C (TC8/CT8) at 1:8 NP to binder weight ratio created by (i) Direct coating method

(TA8, TB8, and TC8), and (ii) Layer-by-Layer coating method (AT8, BT8, and

CT8)………………………………………………………………………………….212

x

APPENDICES

A. STRATAGIES TO IMPROVE PHOTOCATALYTIC BACTERICIDAL

PROPERTY OF TiO2 NANOCOATINGS………………………………………204

B. STUDIES ON BACTERICIDAL ACTIVITY OF VISIBLE LIGHT ACTIVATED

IRON OXIDE (Fe2O3) NANOPARTICLES AND NANOCOATINGS….……..213

1

CHAPTER 1

INTRODUCTION

Surface cross-contamination of foodborne pathogens to food products during

processing or preparation is a major concern to both consumers and food manufacturers

alike. Goddard (2011) stated that “As food production becomes increasingly automated,

the number of surfaces with which foods comes into contact and the subsequent potential

for contamination increases”. Several studies in the past have demonstrated that both

food contact and non-food contact surfaces are major source of microbial cross-

contamination. In general, hygienic processing is assured by the implementation of

cleaning and disinfection operations adapted to the process using different physical,

chemical, and mechanical procedures. Sanitation of food processing equipment is a

regular practice, but in some cases, conventional cleaning and disinfection operations

may be insufficient to achieve satisfactory decontamination (Meylheuc et al, 2006). It is

reported that bacteria may develop resistance to some disinfectants, and it has been

suggested that different types of disinfectants should be used alternately to prevent

establishment of resistant house strains (Doyle, 2005). In addition, several chemical

disinfection methods are well known for generating toxic disinfection by-products. In this

context, advanced oxidation processes involving photocatalytic nanomaterials have

shown great promise as effective non-targeted disinfectants for wide range of

microorganisms and chemical contaminants.

2

Photocatalysis is a versatile and effective process that can be adapted for use in

many disinfection applications (Gamage et al, 2010). Over the last decade, there is an

increased interest in the application of photocatalytic semiconductor nanoparticles (NPs)

for the purpose of food safety and quality enhancement. Of the various photocatalytic

NPs tested to date, Titanium dioxide (TiO2) has been recognized as the most promising

photocatalyst. Heterogeneous photocatalysis using TiO2 is a safe, non-hazardous, and

ecofriendly process which does not produce any harmful by-products (Lan et al, 2013).

TiO2 NP embedded coatings have shown great promise as effective disinfectants over a

range of microorganisms. However, majority of the past research did not fully address the

problem of durability of these coatings on usage. Application of this technology on food

contact surfaces is required to address crucial aspects of stability of these coatings and

migration or release of NPs into the food systems. However, with appropriate binding

agents, stable and permanent TiO2 nanocoatings with strong bactericidal property can be

developed. Hence, the overall goal of this project was to create physically stable and

durable TiO2 nanocoatings on food contact surfaces and evaluate their photocatalytic

bactericidal property. Specific objectives include:

1. To identify most efficient TiO2 NPs with strong bactericidal property and

determine the optimum conditions for their photocatalytic activity.

2. To determine the effect of food processing organic matter on the

photocatalytic bactericidal activity of TiO2 NPs identified from Objective 1.

3. To develop a method to create TiO2 nanocoatings on stainless steel surfaces

using different binding agents.

3

4. To determine the physical stability and photocatalytic bactericidal property of

TiO2 nanocoatings developed from Objective 3.

This dissertation is divided into total seven chapters. The first chapter presents an

introduction and rationale on which the dissertation is based, including specific

objectives. The second chapter presents the literature review on topics such as microbial

food safety concerns, nanotechnology based intervention strategies, synthesis and

characterization of nanocoatings, antimicrobial activity of TiO2 nanomaterials, safety and

toxicity issues of NPs. The third chapter investigates the effect of different variables on

the photocatalytic bactericidal property of several commercial TiO2 NPs in suspension

and identifies the most efficient TiO2 NPs to create nanocoatings. The fourth chapter

presents the effect of food processing organic matter on bactericidal property of TiO2

NPs that are identified from the study in chapter three. The fifth chapter is based on a

study which developed a method to create TiO2 nanocoatings using different binding

agents on stainless steel substrates. In addition, this study also presents the methods to

evaluate physical stability and bactericidal property of TiO2 nanocoatings. The sixth

chapter describes the effect of different binding agents on durability and bactericidal

property of TiO2 nanocoatings. Finally, the seventh chapter outlines overall conclusions

of the research carried out in this project.

4

References:

Doyle, E. M. (2005). Food antimicrobials, cleaners, and sanitizers, Food Research

Institute, UW-Madison.

http://fri.wisc.edu/docs/pdf/Antimicrob_Clean_Sanit_05.pdf. (Accessed on

November 2012).

Gamage, J., & Zhang, Z. (2010). Applications of Photocatalytic Disinfection.

International Journal of Photoenergy. Article ID 764870,

doi:10.1155/2010/764870, 1-11.

Goddard, J. M. (2011). Improving the Sanitation of Food Processing Surfaces. Food

Technology, 65(10), 40-45.

Lan, Y., Lu, Y., & Ren, Z. (2013). Mini review on photocatalysis of titanium dioxide

nanoparticles and their solar applications. Nano Energy, 2(5), 1031-1045.

Meylheuc, T., Renault, M., & Bellon-Fontaine, M. N. (2006). Adsorption of a

biosurfactant on surfaces to enhance the disinfection of surfaces contaminated

with Listeria monocytogenes. International Journal of Food Microbiology, 109(1-

2), 71-78.

http://fri.wisc.edu/docs/pdf/Antimicrob_Clean_Sanit_05.pdf

5

CHAPTER-2

LITERATURE REVIEW

PHOTOCATALYTIC ANTIMICROBIAL NANOCOATINGS IN FOOD SAFETY-

PAST RESEARCH, PRESENT STATUS AND FUTURE PROSPECTS

6

INDEX

I. Microbial Food Safety Concerns Page #

1 Microbial cross-contamination and its impact on food

safety

7

2 Major sources of microbial cross-contamination 7

3 Role of shiga toxin-producing Escherichia coli in cross-

contamination

9

4 Methods of disinfection and sanitation 10

II. Nanotechnology Based Intervention Strategies

1 Nanotechnology & its applications in food safety 11

2 Antimicrobial nanoparticles 13

3 Photocatalytic nanoparticles 22

4 Principle and mechanism of photocatalysis 23

5 Applications of TiO2 photocatalysis 26

6 Mechanism of TiO2 antimicrobial activity 28

III. Synthesis and Characterization of Nanocoatings

1 Methods to synthesize nanostructured materials 31

2 Methods to develop antimicrobial nanocoatings 32

3 Methods to evaluate surface characteristics and physical

stability of nanocoatings

46

IV. Antimicrobial Activity of TiO2 Nanomaterials 1 Bactericidal activity of TiO2 in suspension vs coating 48

2 Studies related to bactericidal activity of TiO2 for food

safety applications

51

3 Considerations for testing antimicrobial activity of

photocatalytic nanomaterials

53

V. Safety concerns on use of NPs

1 Toxicity issues 58

2 Regulatory framework 60

VI. Knowledge Gap 1 Research scope 62

7

I. MICROBIAL FOOD SAFETY CONCERNS

1. Microbial cross-contamination and its impact on food safety

Microbial cross-contamination is a general term which refers to the direct or

indirect transfer of bacteria or virus from a contaminated product to a non-contaminated

product through various routes. A survey conducted by the World Health Organization in

Europe indicated that almost one quarter of the total foodborne outbreaks are closely

associated with microbial cross-contamination events involving contaminated equipment,

unhygienic processing, contamination through food handlers and inadequate storage

conditions (WHO, 1995). The US Centers for Disease Control and Prevention (CDC)

reported that 19% of foodborne diseases caused by bacteria in the years between 1993 to

1997 in the United States were associated with contaminated equipment and poor hygiene

practices, respectively (IFT, 2004). Similarly, the UK outbreak surveillance system

reported that cross-contamination was the main contributing factor (32%) in the

outbreaks investigated in the period 1999-2000 (WHO, 2003). In addition, several

unaccounted cases of microbial cross-contamination go unnoticed severely impacting the

safety of global food supply chain.

2. Major sources of microbial cross-contamination

Several studies in the past have demonstrated that the microorganisms on the

surfaces in food processing plants are an important source of product contamination and

may lead to food spoilage as well as transmission of disease (Meylheuc et al., 2006;

Chasseignaux et al., 2002; and Salvat et al., 1995). In addition, food residues that

accumulate on inert structural surfaces, such as floor drains, conveyors, and product tote

boxes can act as continuous culture systems in which microorganisms reside and multiply

8

to form mature biofilms that are hard to remove by regular sanitation protocols (Bower et

al, 1996). Surface contamination, colonization, and subsequent biofilm formation events

have been implicated in several foodborne diseases and outbreaks (de Valk et al, 2001).

De Boer and Hahne (1990) showed the ease with which Salmonella can be

transferred from chicken to utensils, a variety of kitchen surfaces, hands, and other foods;

from those surfaces Salmonella cells were recovered up to 6 h after contamination.

Another study by Cogan et al (1999) reported that in kitchens where chicken had been

prepared, the prevalence of Salmonella was 60 % for cutting boards and 10 % for door

handles, cupboards, ovens, sink-rims and refrigerators. On the other hand, minimally

processed foods satisfy the growing consumer demand for more natural, fresh, and highly

nutritious foods with a lower amount of preservatives. However, minimally processed

foods may suffer a high risk of cross-contamination from the processing environment and

equipment, cutting boards, knifes or the working surfaces (Carrasco et al, 2012).

Similarly, microbial cross-contamination is a serious risk in ready-to-eat (RTE) foods as

well. Studies conducted on RTE products revealed that factors such as food handlers,

aprons, utensils, and work surfaces are potential sites for bacterial contamination (Pal et

al, 2008; Christison et al, 2007; Lues and Van Tonder, 2007; Lunden et al, 2002).

Several multi-state foodborne outbreaks in the United States such as an ice-cream

premix contaminated with Salmonella enteritidis (Hennessy et al, 1996), peanut butter

contaminated with Salmonella tennessee (Chang et al, 2013), and cantaloupes

contaminated with Listeria monocytogenes (McCollum et al, 2013) were ultimately

traced back to either contaminated equipment surfaces or unhygienic processing and

preparation, packaging, and transportation conditions. This shows that both food contact

9

and non-food contact surfaces pose high risk of microbial cross-contamination seriously

affecting the public health.

3. Role of shiga toxin-producing Escherichia coli in cross-contamination

Shiga toxin-producing Escherichia coli (STEC) strains of various serotypes are

important foodborne pathogens that pose a serious public health concern, resulting in

significant financial burden. It has been estimated that E.coli O157:H7 is responsible for

over 73,000 cases of illness each year in the United States (Wang et al, 2012). It is

reported that various STEC serotypes have the ability to attach, colonize, and form

biofilms on a wide variety of food contact surfaces commonly used in meat processing

plants as well as on vegetables and meat products (Silagyi et al, 2009). A wide variety of

materials commonly used for food processing equipment such as stainless steel,

aluminum, nylon, Teflon, rubber, plastic, glass, and polyurethane were found to become

ideal hosts for STEC biofilms (Silagyi et al, 2009). It is also reported that STEC may

form biofilms in different areas of food processing environments, such as floors, walls,

pipes, and drains, etc. (Marouani-Gadri et al, 2010). In particular, O157:H7 strains were

found to form biofilms most efficiently on stainless steel and on high density

polyethylene surfaces; and O157:H7 biofilms on stainless steel were able to transfer the

bacteria to meat, poultry, and other food products (Silagyi et al, 2009; Stopforth et al,

2003).

Dourou et al (2011) reported that the contamination of beef carcasses with E. coli

0157:H7 may occur during the slaughtering, dressing, chilling or cutting stages of

processing. As a consequence, there is a potential for E.coli O157:H7 population to be

distributed to the surface of equipment associated with slaughter and fabrication and the

10

environment via aerosols or direct contact, and potentially contaminate unadulterated

carcasses and fresh meat products. For several of the outbreaks the cause is believed to be

lack of or insufficient cleaning and disinfection of equipment and surfaces contaminated

with pathogens (Moretro et al, 2012). For example, contaminated onions due to poor

cleaning and sanitizing of equipment were likely cause of a pathogenic E. coli 0157:H7

outbreak at a fast–food restaurant in Canada that sickened 235 people (NCCE, 2009).

Strong attachment of the STEC biofilms on the food surfaces may also affect the

efficiency of antimicrobial interventions applied to food products for reducing

contamination. Thus, the contamination of STEC strains and subsequent biofilm

formation pose serious threat in process hygiene and may become a source of cross-

contamination in the food processing environment.

4. Methods of disinfection and sanitation

Normally, hygienic processing is assured by implementation of cleaning and

disinfection operations adapted to the process using different physical, chemical, and

mechanical procedures. Most commonly, several alkaline detergents, chlorinated

compounds, iodophors, encapsulated lysozyme, peroxyacetic acid, quarternary

ammonium compounds, electrolyzed water, and numerous other commercial disinfectants

are used for this purpose (Doyle, 2005). Alternative methods, such as ozonation,

irradiation, high pressure water washing, and fumigation were also found to be effective.

Sanitation of food processing equipment is a regular practice, but in some cases,

conventional cleaning and disinfection operations may be insufficient to achieve

satisfactory decontamination (Meylheuc et al, 2006). In addition, several chemical

disinfection methods are well known for generating toxic disinfection byproducts. Other

11

treatment methods such as irradiation have their own problems and limitations, such as

lack of residual effect and generating small colony variants (Doyle, 2005). This indicates

that merely relying upon certain established sanitation and disinfection techniques may

not be sufficient to address emerging problems. Modification of surfaces with

antimicrobial agents to prevent the growth of harmful microorganisms has received much

attention for application in biomedical devices and health as well as in the food and

personal hygiene industries (Rai et al, 2010). Such antimicrobial coatings are required to

have long lasting efficacy, ease of fabrication, and no toxicity for effective use in food

safety applications. In this context, nanotechnology based advanced oxidation processes

involving photocatalytic nanoparticles (NPs) have shown great promise as an effective

non-targeted disinfectants for a wide range of microorganisms and chemical

contaminants.

II. NANOTECHNOLOGY BASED INTERVENTION STRATEGIES

1. Nanotechnology & its applications in food safety

The concept of nanotechnology was first introduced by Richard Feymann in 1959

at a meeting in American Physical Society (Khademhosseini and Langer, 2006). Since

then, nanotechnology has developed into a multidisciplinary field of applied science and

technology. Nanotechnology is the ability to work on a scale of about 1 to 100 nm (1m =

109 nm) in order to understand, create, characterize, and use material structures, devices,

and systems with new properties derived from their nanostructures (Roco, 2003).

Nanomaterials are characterized by having at least one dimension in this size range (i.e. 1

to 100 nm), although the upper limit of 100 nm is used by general consensus in many

cases the nano-properties still exist beyond this size limit (Rossi et al, 2014). At this

12

nanoscale, the surface-to-volume ratios of materials become large and their electronic

energy states become discrete, leading to unique physic-chemical, electronic, optical,

magnetic, mechanical, and biological properties which can be manipulated suitably for

desired applications (Rai and Bai, 2012).

The phenomenon that takes place at the nanometer scale offers lots of

opportunities for innovation that have the potential to impact global food supply.

Nanotechnologies can be applied in the entire food chain, from production to processing,

product safety, packaging, transportation, storage, and delivery (Cushen et al, 2012;

Silvestre et al, 2011; Weiss et al, 2006). While nanotechnology has revolutionized the

fields of medicine, electronics, energy, and defense, its application in food sector is

relatively new, as most of the research in this area is in its infancy. In the last one decade,

several excellent reviews have been written discussing the potential benefits of

nanotechnology in food sector (Rossi et al., 2014; Sastry et al., 2013; Cushen et al., 2012;

Morris., 2011; Chen and Yada, 2011; Duncan and Timothy, 2011; Brody., 2006; Chen et

al., 2006; Moraru et al., 2003). One promising area of such application is the use of

nanotechnology based intervention strategies for food safety and quality.

Many applications, including food production and storage might benefit from the

incorporation of safe, economical, and wide spectrum long-lasting biocides into

polymers, paints, or working surfaces (Fernandez et al, 2008). For the last one decade,

there is an increased interest in the application of photocatalytic disinfection techniques

for the purpose of food safety and quality enhancement. Certain metal ions, such as

copper, silver, zinc, palladium, titanium, or iron, occur naturally and can be used for

novel food safety measures. These materials are recognized to have no adverse effects on

13

eukaryotic cells below certain concentrations (Ibhadon et al, 2013), thus being excellent

candidates for the implementation of novel strategies in food safety by incorporating in

food contact substances. As per the US FD&C Act (Section 409, US FDA,1998a) a food-

contact substance can be defined as "any substance that is intended for use as a

component of materials used in manufacturing, packing, packaging, transporting, or

holding food if such use is not intended to have a technical effect in such food". Common

types of food contact substances include coatings, plastics, paper, adhesives, as well as

colorants, antimicrobials, and antioxidants found in packaging. Hence, NPs can also be

used to develop antimicrobial coatings on food contact and non-food contact surfaces to

provide additional layer of protection along with the existing sanitizers and disinfectants

to ensure safe food processing environment.

2. Antimicrobial nanoparticles

The antimicrobial agents currently used in the food industry can be classified into

two categories: i) organic, and ii) inorganic. The key advantages of inorganic

antimicrobial agents, when compared to their organic counterparts, are improved safety

and stability at high temperature and pressures (Fu et al., 2005; Sawai, 2003). Similarly,

nanomaterials can be grouped into two main categories: i) Organic which include carbon

NPs such as fullerenes, and (ii) inorganic NPs such as those of nobel metals (gold, silver,

and platinum), magnetic NPs (iron, cobalt, and nickel), and those of semiconductor NPs

(oxides of titanium, zinc, and cadmium etc.). Inorganic NPs have gained significant

importance due to their ability to withstand adverse processing conditions without losing

their original characteristics. Currently, a variety of NPs have been explored for their

14

potential antimicrobial properties. These include NPs of silver, gold, zinc, silica,

aluminum, copper, magnesium, titanium, iron and their respective oxide forms.

The antimicrobial activity of the NPs is known to be a function of the surface area

in contact with the microorganisms. The small size and large surface area (i.e. high

surface to volume ratio) of the NPs enhances their interaction with the microbes to carry

out a broad range of probable antimicrobial activities (Rai and Bai, 2012). Inorganic NPs

with antimicrobial activity when embedded and coated onto the surface can find immense

applications in water treatment, food processing, and packaging. Therefore, the use of

inorganic antimicrobial agents as a coating on food processing equipment and other food

contact and non-food contact surfaces to reduce the chances of microbial cross-

contamination has attracted much attention. The reported antimicrobial properties of

some of these NPs were briefly described here:

2.1. Silver

Among inorganic antibacterial agents, silver (Ag) has been used extensively for

very long time to control spoilage and fight infection (Chou et al., 2005). However, the

mechanism of inhibitory action of silver ions and silver NPs on microorganisms is not

well established until now. Several possible mechanisms have been proposed for

antimicrobial property of silver.

It is assumed that the high affinity of silver towards sulfur and phosphorus is the key

element of the antimicrobial effect. Due to the abundance of sulfur-containing proteins on

the bacterial cell membrane, silver NPs can react with sulfur-containing amino acids

inside or outside the cell membrane, which in turn affects bacterial cell viability. It was

also proposed that the Ag+ ions released from Ag NPs can interact with phosphorus

15

moieties in DNA, resulting in the obstruction of DNA replication and inhibition of

enzyme functions in the bacterial cell (Matsumura et al, 2003; Gupta and Silver, 1998).

Another proposed phenomena is that the Ag NPs adhere to the cell surface degrade the

lipopolysaccharides and eventually form pits in the membranes, largely increasing the

cell permeability and eventual death (Sondi and Salopek-sondi, 2004). Rai et al (2009)

provided more detailed review on antimicrobial mechanism of silver NPs.

The antibacterial and antiviral activity of silver, silver ion, and silver compounds

have been thoroughly investigated. Physico-chemical properties such as size, shape, and

concentration of NPs play an important role in the antimicrobial activity of silver. Gogoi

et al (2006) reported that the Ag NPs with size less than 10 nm are more toxic to bacteria

such as E.coli and P. aeruginosa. Pal et al (2007) reported that triangular silver

nanoplates containing more reactive planes were found to be more toxic than silver

nanorods, spheres, or Ag+

ions. Thus, the silver NPs exhibit a shape-dependent

interaction with the bacterial cells. Araujo et al (2012) found that increasing the

concentration of Ag NPs from 6 to 60 µg/mL in the suspension increased the

antimicrobial activity. Kim et al (2008) found that silver ions were also photoactive in the

presence of UV-A and UV-C irradiation, leading to enhanced UV inactivation of bacteria

and viruses.

The most common nanocomposites used as antimicrobial films for food

packaging are based on silver, which is well known for its strong toxicity to a wide range

of microorganisms (Liau et al, 1997) with high temperature stability and low volatility

(Kumar and Munstedt, 2005). Though, silver has been reported as an excellent

antimicrobial agent, its mode of action, dose required for killing the microorganisms in

16

food systems and reported toxic effects limits its usage in food applications (Fernandez et

al 2010). It has been demonstrated that silver is non-toxic to humans cells at lower

concentration. However, high concentrations of silver are required to exert antimicrobial

activity in food systems limiting the feasibility of using silver widely in food safety

applications. For example, Llorens et al (2012) reported that a concentration of 60 mg

Ag+/kg was necessary to reduce the microbial load of 1-log CFU/mL in absorbent pads in

contact with beef meat. They also reported that the natural chelating agents, especially

proteins, counteract the antimicrobial activity of silver ions.

2.2. Gold

Gold (Au) NPs are known to be the most stable NPs and can be engineered to

possess excellent chemical or photo-thermal properties (Rai and Bai, 2012). A detailed

review on chemistry, properties, catalytic and biological applications of Au NPs was

provided by Daniel and Astruc (2004). Photocatalytic gold NPs conjugated with specific

antibodies and antibiotics was found to exhibit excellent antibacterial activity over a

range of Gram-positive and Gram-negative bacteria. Rai et al (2010) reported that

antibiotic cefaclor reduced gold NPs have potential bactericidal activity against S. aureus

and E. coli. They reported that the antibiotic inhibits the synthesis of peptidoglycan layer,

making cell walls more porous and the gold NPs generate holes in the cell wall, resulting

in the leakage of cell contents and eventual cell death. It may also be possible that gold

NPs bind to the DNA of bacteria and inhibit the uncoiling and transcription of DNA thus

promoting the death of bacteria. In another study by Perni et al (2009), polymers

containing methylene blue and gold NPs showed a reduction of up to 3.5 log for

methicillin-resistant S. aureus and E. coli in 5 min when exposed to low power laser light

17

(660 nm). It is believed that the bactericidal activity is due to the light-induced

production of singlet oxygen and other reactive oxygen species (ROS) generated by

methylene blue in presence of gold NPs. They further reported that the presence of gold

NPs enhanced the hydrophobic properties of the polymer as well as its bactericidal

activity. This shows that gold NPs can be used to develop antimicrobial coatings on

different surfaces. However, the feasibility of Au NPs for use in practical applications is

very limited due to cost.

2.3. Copper oxide

Copper oxide (CuO) is another semiconductor metal oxide with a photocatalytic

property. Compared to other NPs, copper oxide is cheap, stable and mixes well with

polymers making it an attractive nanomaterial for a wide range of applications (Rai and

Bai, 2012). CuO was found to be effective in killing a range of bacterial pathogens

involved in hospital-acquired infections. However, like silver NPs, a high concentration

of CuO is required to achieve significant bactericidal effect (Ren et al, 2009). Several

mechanisms have been proposed for antimicrobial activity of Cu/CuO. However, the

exact mechanism behind bactericidal effect of copper NPs is still unclear. One possible

mechanism is that the metallic and ionic forms of copper produce hydroxyl radicals that

damage essential proteins and disrupt the mechanism of DNA replication in bacterial

cells. Studies reported that due to greater abundance of amines and carboxyl groups on

the cell surface of B. subtilis, it has high affinity towards CuO NPs and more susceptible

to inactivation by CuO. Also, presence of copper ions inside bacterial cells disrupts their

biochemical processes (Rai and Bai, 2012). The antimicrobial activity of copper NPs

18

depends on the combination of several factors such as shape, concentration, pH,

temperature, and the concentration of bacteria.

In a study conducted on antibacterial activity of different NPs, CuO was found to be the

most toxic against E. coli, B. subtilis, and S. aureus followed by ZnO, NiO, and Sb2O3

(Baek and An, 2011).

Studies have been conducted to assess the potential of CuO NPs embedded in a

range of polymer materials. A lower contact-killing ability was observed in comparison

with release killing ability against MRSA strains. This suggests that a release of Cu ions

into the local environment is required for optimal antimicrobial activity (Ren et al, 2009;

Cioffi et al, 2005). Noyce et al (2006) reported a diminished risk associated with E. coli

O157:H7, when food processing work surfaces were coated with copper cast alloys.

However, the presence of beef residues found to be a limiting factor for the required

growth inhibition.

2.4. Zinc oxide

Zinc oxide (ZnO) NPs have been used in sunscreens, coatings, and paints due to

their high UV absorption efficiency and transparency to visible light (Franklin et al,

2007). ZnO NPs exhibit strong antibacterial activities on a broad spectrum of bacteria

(Sawai, 2003; Adams et al, 2006; Jones et al, 2008). Even though the antibacterial

mechanism of ZnO is not well understood, the photocatalytic generation of hydrogen

peroxide was proposed to be one of the primary mechanisms (Sawai, 2003). In addition,

penetration of the cell envelope and disorganization of bacterial membranes upon contact

with ZnO NPs were also found to inhibit bacterial growth (Huang et al, 2009).

Contrasting results have been reported regarding the effect of particle size on the

19

antibacterial activity of ZnO. Jones et al (2008) observed that smaller ZnO particles were

more toxic than bigger particles, but no size related effect was found in another study by

Franklin et al (2007). Li et al (2011) studied the potential use of nano-packaging

containing ZnO NPs during the storage of Fuji apple cuts. They observed a better

retention of quality indicators such as ascorbic acid and polyphenol content, and lower

counts of typical altering microorganisms. However, reports suggest that ZnO suffers

from photo-corrosion problems upon excitation in the solution.

2.5. Magnesium oxide

Stoimenocv et al (2002) reported that reactive magnesium oxide (MgO) NPs and

halogen (Cl2, Br2) adducts of these NPs both in the form of dry powder and in water

slurries are very effective against Gram-negative (E.coli) and Gram-positive (B.

megaterium) bacteria as well as spores (B. subtilis). However, spores were found to be

less susceptible to the action of MgO compared to vegetative cells. MgO NPs with the

crystal size less than 10 nm exhibit high bactericidal activities since their high surface

area, presence of defective sites and positive charges on surface exhibit strong affinity

towards electronegative bacteria and spores. Further, the extremely small size of MgO

allows many particles to cover bacterial cells to a high extent and bring high

concentration of halogen in an active form in proximity to the cell. Similarly, Lin et al

(2005) demonstrated that γ -Al2O3 with highly dispersed MgO on the surface is efficient

bactericide, and the one with the 20% load amount of MgO can kill more than 99%

bacteria and spore cells. Similarly, Huang et al (2005) reported a high bactericidal

efficacy of MgO NPs both when used directly and as an additive in an interior wall paint.

20

2.6. Iron oxide

Magnetic NPs have aroused increased interest for their potential applications in

various fields, such as advanced materials, biomedicine, diagnostics, energy, and the food

sector (Cao et al, 2012). Iron oxide (Fe2O3) exists in different polymorphs such as alpha,

beta, gamma and epsilon. Chirita et al (2009) discussed the physical, photochemical and

photoelectrochemical properties and applications of iron oxide. Iron oxide is

particularly interesting because of its stability against photo/chemical corrosion at neutral

or basic pH and has band gap energy of about 2.0 to 2.2 eV corresponding to the

absorption of 564 to 620 nm light (Basnet et al, 2013). Especially, α- Fe2O3 or hematite

form of iron oxide is known for its useful photocatalytic properties for solar energy

conversion and water splitting. In addition, hematite has shown promise as a disinfectant

under visible light (λ< 552 nm) photocatalysis. Fe2O3 biocidal applications are not widely

reported. Sultana et al (2012) reported 90% reduction of E.coli and S. aureus with a bio-

ceramic material with 7% loading of iron and titanium metal oxide incorporation. Prucek

et al (2011) identified minimum inhibitory concentration for ten different bacterial and

four different fungal strains using composite silver, α- Fe2O3 and Fe3O4 (maghamite)

NPs. Zhang et al (2011) studied adsorption kinetics of α- Fe2O3 at different sizes for

inhibition of E.coli and reported faster inhibition rates at smaller particle sizes. Tran et al

(2010) reported inhibition of S. aurues growth using PVA coated iron oxide NPs. Basnet

et al (2013) reported that sputter deposited Fe2O3 thin films and nanorod coatings on

glass exhibited around 1.5 and 4.5 log reductions of E.coli O157:H7, respectively.

However, photocatalytic disinfection studies conducted by our group with commercial

and chemical/physically synthesized Fe2O3 NPs in suspension has shown less than 0.5 log

21

CFU/mL of E.coli O157:H7 even after prolonged treatment time of about 3 to 4 h.

Though, α-Fe2O3 is low cost, abundant, and has narrow bandgap for harnessing solar

energy, it suffers from rapid charge recombination and a short charge carrier diffusion

length. As a result of these draw backs, renewed interest in this material has focused on

its modification with cationic dopants such as Cr and Mo to improve its charge transport

properties or doping with Si to reduce the charge diffusion path length (Tran et al, 2005).

2.7. Tungsten oxide

Tungsten oxide (WO3) has received renewed interest in the photocatalytic

applications due to its narrow bandgap (2.7 eV). However, like Fe2O3 NPs, Tungsten

oxide, has the disadvantage of a low electron conduction band and rapid charge pair

recombination. Studies reported that coupling WO3 with platinum co-catalyst help to

achieve much lower reduction potentials. This has increased the use of WO3 as one of the

very few highly visible-light-active single-phase oxide photocatalysts (Ibhadon et al,

2013).

2.8. Titanium dioxide

Of the various semiconductor nanomaterials tested to date, titanium dioxide

(TiO2) has been recognized as the most promising photocatalyst because of its unique

electronic band structure, photostability, chemical inertness, low cost, non-toxicity,

commercial availability, and capability of repeated use without substantial loss of

catalytic activity (Lan et al, 2013). Titanium dioxide is the oxide of titanium with a

chemical formula of TiO2 and was first discovered in 1791 from ilmenite. It is also

known as titanium (IV) oxide, titania, titanium white or pigment white 6 in building

paints, and E171 in food coloring (Fujishima and Zhang, 2006). TiO2 mainly exists in

22

three polymorphs namely, anatase, rutile, and brookite. Among these, only anatase and

rutile form of TiO2 were found to show high photocatalytic activity. Anatase possesses an

energy band gap of 3.2 eV with an absorption edge at 386 nm which lies in the near UV

range. Anatase is the most stable form of TiO2 and can be converted to rutile by heating

to temperatures above 700 °C. Rutile has a narrow band gap of 3.02 eV, with excitation

wavelengths that extend into the visible light range (410 nm). However, anatase is

considered as the most photochemically active phase of titania. The reason for this higher

activity can be attributed to the combined effect of the higher surface adsorptive capacity

of anatase and its higher rate of hole trapping. Afterwards, several studies have shown

that mixtures of anatase-rutile or brookite-anatase were more active than anatase alone

(Visai et al, 2011). Degussa P-25 is one commercially available form of TiO2 which

consists of both rutile and anatase phase at around 1:3 weight ratios. Several studies

showed that Degussa P-25 has excellent photocatalytic properties and is used as a

standard to compare the photocatalytic activity of other nanomaterials (Mills et al, 1997).

A more detailed review on synthesis, properties, principles, and applications of TiO2

photocatalysts were reported by Chen et al (2007) and Linsebigler et al (1995). The

bactericidal mechanism and the major applications of TiO2 were further discussed in the

next sections.

3. Photocatalytic nanoparticles

Among inorganic antimicrobial NPs, photo-activated antimicrobial nanostructures

are especially interesting. These photocatalysts include various oxide semiconducting

materials, their metal hybrid nanocomposites, and doped structures such as TiO2, ZnO,

CuO, MgO, CdS, ZnS, SnO2, WO3, SiO2, ZrO2, Fe2O3, Nb2O3, Ag/TiO2, TiO2/CuO,

23

TiO2/Pt, Au/TiO2, Fe2O3/TiO2, and N-, C-, S- doped TiO2 (Fu et al., 2005; Sawai, 2003).

Especially, semiconductor nanomaterials like TiO2, Fe2O3, WO3, and ZnO have enough

band-gap energies to carry-out more efficient photocatalytic reactions. Among these

photocatalytic NPs, the unique electronic band structure of TiO2 makes it as a stand-alone

photocatalyst, and an ideal choice for the photocatalysis. TiO2 is the most frequently used

photocatalyst owing to its photostability and low cost, combined with its biological and

chemical inertness and resistant to chemical corrosion. On the other hand, binary metal

sulfide semiconductors such as CdS and PbS are regarded as insufficiently stable for

catalysis and are toxic. ZnO is also unstable in illuminated aqueous solutions while WO3

and Fe2O3 have been investigated as a potential photocatalysts, but these are generally

less active catalytically compared with TiO2.

4. Principle and mechanism of TiO2 photocatalysis

Semiconductor NPs such as TiO2, ZnO, Fe2O3, CdS, and ZnS can act as

sensitizers for light-induced redox reactions due to their electronic structure, which is

characterized by a filled valence band (VB) and an empty conduction band (CB). Each

semiconductor used as a photocatalyst corresponds to a range of light wavelengths with

which electron hole-pairs may be induced. The size of the band gap of the electron hole-

pairs varies between the semiconductors and band gap is the amount of energy the

semiconductor requires to absorb in order to produce an electron-hole pair. As shown in

the Fig.1, when a photon with energy of hʋ matches or exceeds the band gap energy (Eg)

of the semiconductor, an electron (ecb-), is promoted from the VB, into the CB, leaving a

hole (hvb+) behind. Excited state CB electrons and VB holes can recombine in

picoseconds and dissipate the input energy as heat, get trapped in metastable surface

24

states or react with electron donars and electron acceptors adsorbed on the semiconductor

surface such as molecular oxygen and water. This mechanism generates several types of

reactive oxygen species (ROS) which can be effectively utilized to completely mineralize

organic compounds into CO2 and H2O (Hoffmann et al., 1995).

The band gap energy of anatase form of TiO2 is approximately 3.2 eV, which

effectively means that photocatalysis can be activated by photons with a wavelength of

below 385 nm which falls in UVA region. The absorption of photons with sufficient

energy by TiO2 NPs results in generation of electron (e-) - hole (h

+) pairs as described

before. These conduction band electrons and valence band holes on the surface of TiO2

can react with surface bound O2 or H2O molecules to initiate redox reactions. This

process generates ROS such as hydroxide radicals (OH-), superoxide radicals (O2

-), and

hydrogen peroxide (H2O2) in various chain reactions. The major steps involved in the

photocatalytic oxidation of organic compounds by TiO2 were shown below:

Steps:

1. Photo excitation: TiO2 + hʋ ecb- + hvb

+

2. Electron trapping : ecb- e tr

-

3. Hole trapping : h vb +

h tr +

4. Electron-hole recombination : e-

tr + h+ vb (h

+ tr) e

- cb + heat (non-productive)

5. Oxidation of hydroxyls: H2O + h+ vb

•OH + H

+aq

6. Reduction of oxygen: O2(ads) + ecb - O2

.-

7. Formation of hydrogen peroxide: •OH +

•OH H2O2

8. Chain reaction: O2.- + H2O2

•OH + OH

- + O2

9. Hydroperoxyl radical formation: O2.- + H

+ •OOH

25

10. Mineralization of organic compounds: •OH + Organics+ O2 CO2 + H2O

Furube et al (2001) reported that the trapped charge carriers in steps 1, 2, and 3

are usually TiO2 surface bound and do not recombine immediately after photon

excitation. In the absence of electron scavengers, the photoexcited electron recombines

with the valence band hole in nanoseconds with simultaneous dissipation of heat energy

as shown in step 4. Thus, the presence of electron scavengers is vital for prolonging the

recombination rate and efficient photocatalysis. Presence of an electron scavenger such as

molecular oxygen prevents the recombination of electron-hole pair, and allows the

formation of superoxide radical by following reaction step 6. This superoxide radical can

be further protonated to form the hydroperoxyl radical (Step 9). The hydroperoxyl radical

formed was also reported to have scavenging property and thus, the co-existence of these

radical species can doubly prolong the recombination time of holes in the entire

photocatalytic reaction. However, it should be noted that all these occurrences in

photocatalysis were attributed to the presence of both dissolved oxygen and water

molecules. Without the presence of water molecules, the highly reactive hydroxyl

radicals (Step 5) could not be formed and impede the photodegradation of organic

substances. The h+ tr are powerful oxidants (+1.0 to +3.5 V against NHE), while e tr

- are

good reductant (+0.5 to -1.5 V against NHE), depending on the type of catalysts and

oxidation conditions. The hydroxyl radical is a powerful oxidizing agent, and attacks

organic pollutants present at or near the surface of TiO2. It results in complete

decomposition of toxic and bio-resistant compounds into harmless species such as CO2

and H2O (Step 10).

26

5. Applications of TiO2 photocatalysis

After the initial discovery of photocatalytic water-splitting of TiO2 by Fujishima

and Honda (1972), TiO2 has been widely studied for use in several other applications.

Lan et al (2013) has briefly summarized the photocatalytic applications of TiO2 as

follows:

i. Photocatalytic water-splitting

Fujishima and Honda (1972) first reported the ability of TiO2 to split water into

H2 and O2 in the presence of light having a wavelength shorter than 410 nm. Later,

TiO2 photocatalysis has attracted much attention as one promising method to produce

hydrogen for energy requirements.

ii. Environmental decontamination

Several reviews have been published on environmental decontamination of TiO2

photocatalysts (Ibhadon et al, 2013; McCullagh, 2007). TiO2 photocatalysis can

degrade and mineralize a large variety of environmental contaminants, including

organic and inorganic materials into CO2, H2O, and harmless inorganic anions (Lan et

al, 2013). Photocatalytic properties of TiO2 were effectively used in order to

decompose organic pollutants and purify soil, air, and water (Yu and Brouwers, 2009;

Chaleshtori et al, 2008; McCullagh, 2007).

iii. Photocatalytic disinfection

Since Matasunga et al (1985) first reported the photocatalytic disinfection efficacy

of TiO2 to inactivate bacteria, several studies have been reported the use of TiO2

photocatalysis to kill a wide range of microorganisms. More recently, Foster et al

(2011) reported a comprehensive review on photocatalytic disinfection using TiO2.

27

iv. Photocatalytic self-cleaning surfaces

A TiO2 coated surface becomes superhydrophilic upon irradiation with UV light due

to formation of metastable hydroxyl groups which results in the formation of very thin

closed liquid layer that is characterized by a small contact angle (Watanabe et al, 1999).

This phenomenon supports the cleaning process and enables fast evaporation of the water

film. However, this functionality of TiO2 is reversible and depends on the light exposure

(Wolfrum et al 2002). This photo-induced self-cleaning property of TiO2 has been

extensively used in several applications such as exterior and interior construction

materials, road-construction materials, and household goods etc. (Fujishima and Zhang,

2006). In addition, these properties of TiO2 help to reduce the usage of cleaning agents

and to shorten the cleaning cycles. Especially in the food industry where frequent

cleaning cycles are the norm, this phenomenon could help reduce costs in the long run

(Muranyi et al, 2013).

Heterogeneous photocatalysis using TiO2 is a safe, nonhazardous, and ecofriendly

process which does not produce any harmful by-products. Based on the unique properties

of the photo-induced electron-hole pairs, inertness to chemical environment and long-

term photostability has made TiO2 an attractive material for many commercial

applications, ranging from drugs to foods, cosmetics to catalysts, paints to

pharmaceuticals, and sunscreens to solar cells in which TiO2 is used as a desiccant,

brightener, or reactive mediator (Kamat, 2012). In addition, due to the antibacterial

applications of TiO2-mediated photooxidation, this process shows promise for the

elimination of microorganisms in areas where the use of chemical cleaning agents or

biocides is ineffective or is restricted by regulations, for example in the pharmaceutical

28

and food industries (Skorb et al 2008). Antimicrobial surface coatings based on the

semiconductor TiO2 could provide a positive contribution to maintain the process

hygiene.

6. Mechanism of TiO2 antimicrobial activity

First known bactericidal activity of TiO2 photocatalytic reactions was reported by

Matasunga et al (1985). Since then several important photo-killing mechanisms have

been proposed for TiO2. It is believed that the bactericidal effect of TiO2 is initiated by

the photochemical oxidation of intracellular coenzyme A, which alters the respiratory

activities and leads to eventual death of bacterial cell (Matsunaga et al., 1985, 1988).

Later, Saito et al (1992) reported that a rapid leakage of K+ ions and slow leakage of

RNA and proteins from treated bacterial cells due to TiO2 photocatalytic reaction is the

possible cause for the bactericidal property. A similar mechanism has been suggested by

Hu et al (2007). Another study by Zheng et al (2000) investigated the mechanism of cell

death with a focus on the gross features of cell wall and cytoplasmic membrane damages

caused by TiO2 photocatalytic reactions. Their study measured the hydrolytic rate of

permeability of marker probe (ONPG, a chromogenic substrate) upon reaction with

intracellular β- glycosidase enzyme. This reaction is only possible if there is damage to

the outer membrane and inner membrane through permeation of ONPG towards inside of

the cell or leakage of β- glycosidase towards outside of the cell. Their results suggested

that the initial oxidative damage takes place on the cell wall, where TiO2 photocatalytic

surface makes first contact with intact cell. Cells with damaged cell wall are still viable.

After degradation of outer membrane, oxidative damage proceeds to the underlying

cytoplasmic membrane. This condition results in free efflux of intracellular contents that

29

eventually leads to cell death. Several spectroscopic studies supported this mechanism

and confirm the order of destruction being outer membrane followed by inner membrane

and peptidoglycan layer. Sunada et al (1998), reported photocatalytic degradation of E.

coli endotoxin which is an integral part of the outer membrane. However, there is also

more direct evidence that the lethal action is due to outer membrane and cell wall damage

(Fig. 2). This is mainly due to the production of ROS like hydroxyl radicals (•OH) and

hydrogen peroxide (H2O2) by the photocatalysts under illumination, which can lead to

phospholipid peroxidation and ultimately cell death (Sunada et al., 2003; Cho et al.,

2005). Pigeot-Remy (2012) and Maness et al (1999) demonstrated that lipid peroxidation

can be initiated on cell membrane polyunsaturated phospholipids through TiO2

photocatalytic reaction. Hydroxyl radicals generated by the TiO2 photocatalyst are very

potent oxidants and are nonselective in reactivity. Their findings suggested that OH·, O2-,

and H2O2 generated on the irradiated TiO2 surface resulted in breakdown of cell

membrane structure of E. coli. In contrast, Goginat et al (2006) reported that aggregation

of bacteria onto TiO2 particles is a driving force for the bactericidal effect in suspension.

They observed cytoplasmic membrane started to disintegrate even before illumination.

This NP adsorption alone alters membrane integrity and greatly amplified under

illumination.

It is clear that the first contact between microorganism and ROS occurs on the cell

surface. Therefore, it is the primary target of initial oxidative attack (Maness et al., 1999).

It is assumed that ROS act at different distances. For instance, H2O2 can diffuse into the

solution in contrast to OH radicals which are bound on the surface or react close to it due

to high and unselective oxidation (short lifetime) (Schwegmann et al, 2012). There is a

30

greater consensus that the OH radicals are the primary ROS responsible for the

inactivation of cells (Cho et al, 2005) and hence the physical contact between

photocatalyst and cell have a high impact on the disinfection rate (Goginat al, 2006;

Marugan et al, 2010). The surface interaction of microorganisms with the photocatalyst

during the photo-disinfection is essential for enhancing the inactivation rate. Hence the

transfer of bacterial cell to the close vicinity of the surface generated ROS site remains as

the rate-limiting step in the photocatalytic disinfection.

Induction of oxidative stress due to formation of ROS triggers the NP toxicity and

it depends on various factors such as composition, surface modification, intrinsic

properties of NPs and the bacterial species (Hajipour et al, 2012). In particular, many

previous studies have explored the photogeneration of ROS on the surfaces of metal-

oxide NPs. Hydroxyl radical (·OH) is a strong and nonselective oxidant that can damage

virtually all types of organic biomolecules, including carbohydrates, nucleic acids, lipids,

proteins, DNA, and amino acids. Singlet oxygen (1O2) is the main mediator of the

phototoxicity and can irreversibly damage the treated tissues causing membrane

oxidation and degradation. Although, superoxide anion is not a strong oxidant, it acts as a

precursor for .OH and

1O2. Consequently, these three types of ROS (

.OH, O2

.- and

1O2 )

contribute to the major oxidative stress in biological systems (Li et al, 2012).

Li et al (2012) compared the ROS generation potential of seven types of metal

oxide NPs (nTiO2, nCeO2, nZnO, nCuO, nSiO2, nAl2O3 and nFe2O3) and their bulk

counterparts based on their band energy structures and subsequently analyzed their

antibacterial activity. Bulk particles other than bTiO2 and bZnO did not produce

measurable ROS, whereas all the NPs other than nCuO generated ROS. The average

31

concentration of total ROS (i.e. .OH, O2

.- and

1O2) are in the order of nTiO2 > nZnO >

nAl2O3 > nSiO2 > nFe2O3 > nCeO2 > nCuO and bZnO > bTiO2. Among NPs, nZnO

generated the most O2.-, followed by nFe2O3, nTiO2, and nCeO2, whereas for bulk

materials, only bZnO favors O2.- generation. nTiO2 generated the most

.OH, which was

approximately 2-fold and 6-fold more than that generated by nZnO and nFe2O3. nTiO2

generated the most 1O2, followed by nAl2O3, nZnO, and nSiO2. The enhanced ROS

generation potential of NPs compared to their bulk counterpart is likely due to their large

surface areas, which provide more available reaction sites for light absorption. Other

potentially size dependent properties such as light absorption or scattering, defective

sites, and structural disorder may also lead to the difference in photoactivity.

III. SYNTHESIS AND CHARACTERIZATION OF NANOCOATINGS

1. Methods to synthesize nanostructured materials

Two building strategies are currently used in nanotechnology: a ‘top-down”

approach and the “bottom-up” approach. The commercial scale production of

nanomaterials basically involves the “top-down” approach, in which nanometric

structures are obtained by size reduction of bulk materials, by using milling,

nanolithography, or precision engineering (de Azeredo et al, 2009). The newer “bottom-

up” approach, on the other hand allows nanostructures to be built from individual atoms

or molecules capable of self-assembling (Moraru et al, 2003). Based on these two

approaches, synthesis of NPs or nanomaterials can be broadly classified into two main

categories: 1. Physical synthesis methods and 2. Chemical synthesis methods. Several

physical and chemical synthesis methods have been reported to design, fabricate, and

manipulate nanostructured materials by innovative approaches (Hu et al, 2009). In

32

addition, several reviews and book chapters are available on the synthesis and properties

of different types of NPs such as metal NPs, semiconductor NPs, carbon-based NPs, and

NPs in general. Burda et al (2005) provided a more comprehensive list of methods to

synthesize NPs. Chen and Mao (2007) as well as Linsebigler et al (1995) reported various

approaches of TiO2 NP synthesis, their properties, modifications and applications.

Nanostructured TiO2 materials in the form of NPs, nanorods, nanowires, nanotubes,

films or coatings, and nanoporous structures can be prepared by using various physical

and chemical synthesis methods. Physical synthesis methods are based on subdivision of

bulk materials (top-down approach) and the NPs thus produced are usually large in size

and have wide size distribution. Most commonly used physical synthesis methods include

but not limited to ball milling, thermal decomposition, laser ablation, arc-ion plating,

spray pyrolysis, flame pyrolysis, magnetron sputtering, ion-beam sputtering, and physical

vapor deposition etc. On the other hand, chemical synthesis methods are based on the

reduction of metal ions or decomposition of precursor solutions to form atoms followed

by aggregation into nano-sized particles (bottom-up approach). The NPs thus prepared by

chemical synthesis methods usually have a narrow size distribution and good control over

composition. Sol-gel synthesis, hydrothermal treatment, sonochemical method, co-

precipitation, anodic oxidation, and electrophoretic deposition are the most common

examples of chemical synthesis methods.

2. Methods to develop antimicrobial nanocoatings

Goddard (2011) stated that the antimicrobial agents commonly used for coating

on food contact and non-food contact surfaces can be classified into two categories. 1.

Migratory and 2. Non-migratory antimicrobial agents. Migratory antimicrobial agents are

33

blended throughout the bulk of the material. These antimicrobials exhibit activity through

migratory effect and do not need close contact of bacterial cell to the antimicrobial agent.

However, their antimicrobial activity diminishes over a time, limiting its long time

functionality (Goddard, 2011; Page et al, 2009). In contrast, non-migratory antimicrobial

agents are strongly attached to the food processing surface by the strongest possible

chemical bond and therefore less likely to migrate from the surface (Goddard, 2011).

Such non-migratory antimicrobial materials therefore have the potential for long-lasting

antimicrobial activity and have the added benefit of being unlikely to migrate to the food

product. Further, non-migratory antimicrobial materials require direct contact with

microorganisms to be effective (Goddard, 2011).

Several methods have been proposed to develop inorganic and organic nanocoatings

on different materials. Visai et al (2011) presented an overview of available surface

technologies allowing the deposition and/or structuring of TiO2 films with antibacterial

properties. Bastarrachea et al (2015) reported various methods used to develop

antimicrobial coatings on food equipment surfaces. In the following section most

commonly used approaches for coating were briefly summarized largely based on the

above mentioned two review papers.

2.1. Graft polymerization: Graft polymerization is a widely used method for coating

on polymeric substrates. However, steel and other inorganic metals can also undergo

graft polymerization (Bastarrachea et al, 2015). The surface chemistry of a solid support

will be changed by grafting polymeric chains with desirable characteristics (Kato et al,

2003). Two different approaches are followed in graft polymerization (Bastarrachea et al,

2015). In the first approach, the solid surface will be pre-treated with either

34

gamma/UV/high-energy electron beam irradiation or by treatment with ozone, plasma,

corona, or flame to create reactive group in order to initiate grafting of antimicrobial

monomers. In the second approach a preformed polymers are immobilized onto a

functionalized solid support.

2.2. Cross-linkable coatings: Cross-linkable coatings are polymers that can bond

with each other after deposition by inclusion of chemical cross-linkers or subsequent

exposure to irradiation or heat curing. UV curing and chemical treatments are the most

often used methods of cross-linking polymers (Bastarrachea et al, 2015).

2.3. Self-assembled monolayers: Self-assembled monolayers form when molecules

spontaneously form into a single layer of relatively ordered groups on a material surface

via strong interaction between anchoring group and the surface (Bastarrachea et al, 2015).

N-alkyl silanes on hydroxylated inorganic surfaces such as silica, glass, steel and thiols

on gold are some common examples of self-assembled monolayers (Raynor et al, 2009).

Self-assembled monolayers have a thickness of one to several nanometers and relatively

simple to deposit over large surface areas. However, their stability under aqueous

conditions has not been well established and remains challenge to commercial adaptation

(Bastarrachea et al, 2015).

2.4. Langmuir-Blodgett films: Langmuir-Blodgett films are comprised of single or

multiple layers of highly organized surfactants that form when a solid support is removed

from a solution containing the material to be deposited (Zasadzinski et al, 1994). Unlike

self-assembled monolayers, these films are rely on physisorption rather than

chemisorption and covalent bond formation and are therefore not as stable as self-

assembled monolayers.

35

2.5. Layer-by-Layer: Layer-by-Layer (LbL) self-assembly is a method by which a

multiple layer coating/film of nanometer-thick layers can be made by sequential

adsorption of oppositely charged polyelectrolytes on a solid support (Bastarrachea et al,

2015). Various antimicrobial components can be incorporated into the bilayers, such as

silver NPs (Dubas et al, 2006), N-halamines (Bastarrachea and Goddard 2013),

quaternary ammonium compounds (Grunlan et al, 2005), and chitosans (Gomes et al,

2013). LbL assembly is rapid, cost-effective, conformal coating technique that can create

durable layers on many surfaces, from polymers to stainless steel (Bastarrachea et al,

2015).

2.6. Electroplating or Electroless plating: Electroplating is a solution-based process

that creates a thin coating of metal on another metal by applying an electrical current. In

contrast, electroless plating is an autocatalytic plating method that uses a chemical

reducing agent in the bath instead of electricity (Mallory and Hajdu, 1990). The thickness

of electroless-plated coatings is typically limited to a few micrometers, and thorough

cleaning of the surface to be plated is critical for optimal adhesion (Ghodssi and Lin

2011).

2.7. Electrophoretic deposition: Electrophoretic deposition is a technique that

exploits the movement of charged particles in suspension in the presence of an

appropriate electric field. In electroplating the coatings are built from metallic ions

converted into atoms when discharged at cathode, whereas in electrophoretic process the

coatings are formed by a deposition of relatively large powder particles. Electrophoretic

deposition allows the deposition of coating from almost any material class, including

metals, polymers, and ceramics. (Visai et al, 2011).

36

2.8. Anodic oxidation: In this method an electrical field driven metal and oxygen ion

diffusion lead to formation of an oxide film at the anodic surface. The anodic oxide film

growth is a two-stage process that results in either a thin or thick TiO2 film. A linear

growth in the nanometric range of the TiO2 film is achieved up to 160 V of applied

voltage drop in the electro chemical cell. However, when anodization carried out at

higher voltages, an increased gas evolution and often sparkling are obtained, resulting in

TiO2 films of higher thickness. This process is generally called micro-arc oxidation or

anodic spark deposition. Doping of metal ions in TiO2 films is also possible with anodic

oxidation process. The films show controlled porosity, morphology, chemical

composition, and allotropic structure (Visai et al, 2011).

2.9. Chemical vapor deposition: In chemical vapor deposition (CVD), a thin film is

formed on a heated solid support from a gaseous phase in a closed chamber, followed by

removal of unreacted gas and chemical by-products from the chamber. A commercial

limitation to CVD coating of food processing equipment is that the apparatus required for

CVD processing is complex, and the size and throughput of the materials to be coated is

limited. Nevertheless, the robust, high purity coatings imparted by CVD may be useful in

certain applications in food processing equipment such as small and irregularly shaped

sanitary valve components (Bastarrachea et al, 2015). Sobczyk-Guzenda et al (2013)

found that the radiofrequency plasma enhanced chemical vapor deposited TiO2 coatings

has exhibited better mechanical properties and bactericidal activity compared to sol-gel

synthesized TiO2 films.

2.10. Plasma spraying: Plasma spray is also a widely used technique to form ceramic

and oxide coatings on wide range of inorganic substrates. The process is based on the

37

action of an electric arc that melts and sprays materials onto a solid surface. Generally,

the material to be deposited is injected in powder form using an inert gas such as argon as

powder carrier. Ctibor et al (2012) created TiO2 powder coatings on stainless steel

substrate using water stabilized plasma gun with argon or nitrogen as carrier gases. They

found that argon assisted TiO2 coatings found to be slightly more stable than nitrogen

assisted TiO2 coatings. However, nitrogen assisted coatings were found to be

photocatalytically more active in decomposing acetone.

2.11. Sol-gel synthesis: Sol-gel synthesis method is a wet chemical technology based

on a sequence of synthesis steps. This method involves hydrolysis and condensation of

metalloorganic alkoxide precursors. Sol-gel technique allows the possibility of

incorporating metal ions, nanometric clusters, and bactericidal molecules (Visai et al,

2011). Sol-gel coatings can be deposited mainly either via dip, spin, or spray coating.

This technique is particularly useful in producing advanced antibacterial coatings due to

the simple industrial scale-up and the esthetic quality of the resulting film. In addition,

sol-gel synthesis is the most widely reported method in the literature. A typical sol-gel

process consists of three major steps:

i) Synthesis of NPs

ii) Coating

iii) Heat treatment

i) Synthesis of NPs

In the first step of sol-gel synthesis, a colloidal suspension, or a sol, is formed

from the hydrolysis and polymerization reactions of the precursor solutions. Complete

polymerization and subsequent loss of solvent leads to the transition from the liquid

38

solution (sol) into solid (gel) phase (Chen et al, 2007). TiO2 NP synthesis and coating by

sol-gel approach is the most popular technique used in several studies. Different types of

precursor solutions, organic solvents along with various surfactants, stabilizers, and

binding agents will be used for this purpose. Process conditions during hydrolysis and

polymerization reactions in sol-gel synthesis are crucial to achieve NPs of desired

characteristics. Sugimoto et al (2003) conducted extensive studies on sol-gel method for

the formation of TiO2 NPs of different sizes and shapes by tuning the reaction mixtures

and by use of different shape controllers. Oskam et al (2003) reported the time-

temperature dependence of NP formation during sol-gel synthesis. They found that the

rate constant for coarsening increases with temperature due to temperature dependence of

the viscosity of the solution and the equilibrium solubility of TiO2. They also reported the

formation of secondary particles at longer times and higher temperatures. In another

study by Barati et al (2009) investigated the effect of pH on homogeneity and particle

size of the precursor solution. They found that the TiO2 crystal size decreases by

decreasing the pH of precursor solution.

ii) Coating

In the second stage, the synthesized sol-gel suspension was coated on a substrate

using different approaches. Dip coating and spin coating are the most widely reported

approaches in the literature. In dip coating method the substrate will be dipped in a

precursor solution for a specified time and later slowly pulled out from the precursor

solution at a specified withdrawal speed. As the substrate pulled up, the film dries due to

convectional air movement. Later, it will be subjected to heat treatment in an oven at

higher temperatures for a specified duration. By repeating this process a thin film of

39

desired thickness will be formed on the substrate. Whereas in spin coating, the substrate

will be mounted on a spin coater and the precursor solution of certain volume will be

poured on the surface. Later, the substrate will be rotated at a certain speed (rpm) for a

specific period of time. The centrifugal force generated during this process spreads the

sol as a thin film on the substrate by expelling excess solution outside. After drying the

film as described before, the process will be repeated to generate a film of desired

thickness.

The above methods are the most simple and efficient. However, as such they are

not practically feasible methods of application in commercial scale and have some

limitations. Alternatively, different coating techniques were used in several studies.

Marcos et al (2008) developed TiO2 coatings on glazed ceramic tiles using screen-

printing method. Anatase TiO2 powder was printed through different sieved screens (55

and 136 µm) on glazed tiles. MacFarlane et al (2011) developed physically stable and

robust TiO2 surfaces on aluminum substrates by using jet spray screen printing method

adopted from Marcos et al (2008). Cerna et al (2011) followed a novel method of coating

using an inkjet printer. In this method the prepared TiO2 solution was deposited on glass

substrate of different film thickness by adjusting printer cartridge and print settings.

Parthasarathy et al (2009) used a hand held spray gun for coating TiO2 on textile fabrics.

A dispersion of NP was filled in a hand spray gun and the fabric substrate was fixed on a

vertical board. The suspension was evenly sprayed over the fabric by maintaining a

constant distance between the fabric and spray gun nozzle. In another study by Ricon et

al (2001) used a spray gun to paint the TiO2 emulsion on to stainless steel substrates.

Film thickness was controlled by the number of spray cycles. The thickness of the 15-

40

cycle spray-painted coating, after sintering at 450°C in air, is roughly around 5-6 µm.

Shinde et al (2008) used a pneumatic spray system to coat TiO2 thin film on glass. The

coating parameters were optimized to obtain uniform, homogeneous and adherent thin

films up to 800 nm thickness. Witanachchi et al (2006) used laser-assisted spray pyrolysis

for the growth of TiO2 and Fe2O3 NP coatings on silicone substrates. Coatings with much

smaller and well-defined grains have been grown by laser heating the droplets. Tomeszek

et al (2006) reported plasma sprayed TiO2 functional coating on SS substrate with a

thickness range of 30 to 50 µm. Structural characterization of these films revealed better

properties than powder deposition techniques. Taniguchi et al (2003) reported successful

fabrication of La1-x Srx Co1-y Fey O3 thin films by electro static spray deposition and

studied the effect of various deposition temperatures, deposition times and liquid flow

rates on the film structure. Ctibor et al (2012) analyzed structural, mechanical and

photocatalytic activities of TiO2 thin films created by using water-stabilized plasma gun.

iii) Heat treatment

In the third step of sol-gel synthesis, the coated substrates will be first preheated

to evaporate the solvent followed by calcination or sintering at higher temperatures to

create stable thin films. Several studies have reported that drying at around 100°C for 30-

60 min followed by calcination in the range of 400 to 600°C is ideal for obtaining anatase

form of crystalline TiO2 thin films with good photocatalytic activity. Mathews et al

(2009) studied the effect of annealing temperature on structural, optical and

photocatalytic properties of TiO2 thin films on soda lime glass slides. The dip coated

films were dried in air at room temperature and later heated for 2 min in oxygen at

100°C. Subsequently, the coatings were calcinated at 200-600°C under oxygen flow.

41

Their results showed that the structure of the film changes from amorphous to

polycrystalline after annealing at 400°C and the band gap of the created film decreased

from 3.4 to 3.32 eV after annealing at 600°C. Barati et al (2009) reported that the TiO2

thin films created on stainless steel showed anatase structures when calcinated at 350-

550°C followed by solvent bath drying. Vigil et al (2009), used microwave assisted

chemical bath for deposition of TiO2 thin films on stainless steel using different precursor

solutions. The thin film coated coupons were microwave irradiated (2.45 x 109 Hz. 0.6

kW) and subsequently calcinated at temperatures lower than 700°C. Results showed that

amorphous TiO2 was deposited on stainless steel from the TiOSO4 precursor solution

while a disordered anatase phase was deposited from the (NH4)2TiF6 precursor solution.

Ilmenite (FeTiO3), as well as, hematite (Fe2O3) appeared with heat treatment, indicating

that Fe ions diffuse into the TiO2 film. Yang et al (2007) subjected the dip coated TiO2

thin film to heating for 5 min using a domestic microwave oven. The resultant thin film is

well characterized with smooth morphology and spherical shape with grain size of 68.2

nm and strong absorption band in the range of 300-387 nm with band gap energy of 3.4

eV. Alrousan et al (2009) dried TiO2 thin films using IR lamp in between the coats.

Machida et al (2005) studied the antibacterial efficacy of TiO2 film photo-deposited silver

ions on tiles which are first air-dried and later calcinated at 880-980ºC for 1 hr. Results

showed that when calcination temperature was < 900ºC, the antibacterial activity was

100%, irrespective of TiO2 thickness. They also found that the calcination temperature,

film thickness has significant impact on anatase to rutile composition, and photo

deposited silver.

42

The above mentioned studies indicate that the synthesis of NPs beginning with a

precursor solution is a complex process and need to control several variables to obtain

desired characteristics. In addition, aggregations of NPs, crack formation on films are

some common problems that are widely reported in the literature. Alternatively, few

studies reported the use of commercially available ready-made TiO2 NPs dispersed in an

organic solvent to form sol-gel for direct coating. This method of approach limits the

complexity of choosing suitable chemical ingredients for precursor solutions and control

over the reaction parameters. Marcos et al (2008) suspended TiO2 powder with 0.29 µm

size and 12.94 cm2/g specific surface area in an organic media and the resultant

suspension was used for coating on glazed ceramic tiles. Similar technique was used by

MacFarlane et al (2011) on aluminum substrates and reported the formation of uniform

coatings with high surface area and physical stability. Kim et al (2008) used aqueous

solution of commercial Degussa P25 powder mixed with carbowax binder (PEG) to

deposit thin films on glass substrate and tested their antimicrobial activity. Their results

showed that number of coatings on glass showed no difference in the antimicrobial

activity. Similarly, Alrousan et al (2009) used a suspension of Degussa P25 in methanol

to develop TiO2 thin films for bacterial inactivation in surface water. Machida et al

(2005) sprayed commercial TiO2 solution on ceramic tiles (3 g/m2) and studied the

antibacterial efficacy of resultant TiO2 thin film.

2.12. Wet chemical methods using different binding agents: Wet chemical methods

involve use of an organic/inorganic binder and a suitable solvent along with an

antimicrobial agent in order to prepare a suspension for coating on a substrate. Depending

upon the nature of the constituents used in the coating, a subsequent heat treatment may

43

be required to evaporate the solvent and to remove the organic binder from the coating to

achieve high bactericidal property and physical stability. Three different mechanisms

namely oxidation, solvent evaporation, and polymerization or chemical cross-linking will

be involved while creating coatings using a binding agent. Oxidation of binder in the

coating lead to thermosets which remain hard on exposure to heat. Usually this type of

coatings contains drying oils which take time to dry and achieve good moisture as well as

chemical resistance. However, over a time these coatings lead to cracking, embrittlement,

and deterioration. Solvent evaporation of coatings leads to thermoplastics which deform

or soften by exposure to heat. In general, water or organic solvents are used in these types

of coatings. Whereas, polymerization or chemical cross-linking mechanism leads to

thermosets.

Many studies have reported the use of organic and inorganic binding agents for

developing nanocoatings on different substrates. Kasanen et al (2011) studied the UV

stability of polyurethane binding agent on multilayer photocatalytic TiO2 coating on glass

substrate. They found that the optimal dilution of polyurethane binder in water is 1:4 for

better photocatalytic activity and binding of TiO2 to the substrate. Similarly, Bhargava et

al (2012) investigated the effect of TiO2 concentration (pigment-to-binder ratio) and

dispersing agent percentage on the peel strength of high reflectivity waterborne

polyurethane based coatings on aluminum substrate. Dhoke et al (2012) developed

polyurethane based ZnO coatings on stainless steel. Their results showed 0.1% ZnO with

polyurethane showed better UV, scratch, and abrasion resistance. Qiao et al (2012)

fabricated boron carbide green sheets using a suspension with polyvinyl butyral as a

binding agent, di-n-butyl phthalate as a plasticizer, castor oil as dispersant and single

44

oleic acid glycerol as wetting agent. Their study found that shear thinning behavior of the

slurry was found to be the most suitable for coating. In addition, optimal contents of

binder and plasticizer were found to be 5 wt %, wetting and dispersing agents were found

to be 2 wt%. Addition of diethylene glycol (DEG) while preparing TiO2 precursor

solution using titanium isopropoxide and ethanol enhanced the thin film physical

characteristics such as adherence, robustness, and surface smoothness on stainless steel

surface (Kajitvichyanukul et al, 2005). Also, the photocatalytic activity of resultant TiO2

coating remained unchanged when compared with the control. Similar results were also

observed by Cerna et al (2010) when using polyethylene glycol (PEG) in precursor

solution for thin film formation on glass substrate. In another study by Fretwell et al

(2001) added PEG and DEG in a precursor solution along with ethanol as solvent to

avoid aggregation of titania particles. Addition of PEG and DEG helped to form a

network with NPs and avoid aggregation. Tsoukleris et al (2007) and Chorianopoulos et

al (2011) prepared TiO2 pastes by using different solvents, rheology agents, and surface

modifiers along with PEG for coating on glass and stainless steel substrates. In addition,

Chorianopoulous et al (2011) tested TiO2 coated glass and steel substrates prepared with

Triton X-100 surfactant to disinfect Listeria monocytogenes biofilms in a food processing

environment.

Shi et al (2008) used an epoxy resin (Bisphenol-A) based coating embedded with

TiO2 NPs. Their study found that epoxy resin showed good chemical (acid/alkali), heat,

and wear resistance but has high rigidity, weak photostability due to oxidation of

methylene chains. Cheema et al (2012) fabricated optically transparent nanocomposites

with enhanced mechanical properties using stable dispersions of ZrO2 NPs mixed with a

45

commercially available bisphenol-A-based epoxy resin and cured with a mixture of two

amine-based curing agents. Wagner et al (1998) developed novel corrosion resistant hard-

coatings for metal surfaces. A sol-gel precursor solution is prepared by adding inorganic

fillers such as GPTS (ɤ- glycidyloxi propyl trimethoxy- silane) and PTMS (propyl tri-

methoxy-silane). They found that the epoxy group of GPTS generates a polyethylene

oxide network beside the inorganic backbone by organic polymerization. PTMS has been

used as a network modifier to increase the relaxation behavior of the material to achieve a

more hydrophobic behavior of the material. Bhave (2007) reported a good adhesion of

GPTS and MTMS based TiO2 (brookite phase) coatings on glass substrate when

compared to coating consisting of Degussa P 25 TiO2. The differences in the adhesion

strength of TiO2 coatings might be attributed to the poor wetting and dispersion

properties of Degussa P 25 compared brookite phase TiO2 NPs. Schmidt et al (1997)

created a corrosion resistant nanocomposite coating using GPTS, and SiO2 NPs by wet

coating method. These coatings showed excellent abrasion resistance and the physical

stability of these coatings was found to be in the order of anodizing processes. Dhoke et

al (2009) created ZnO nanocoatings on stainless steel substrates using waterborne

silicone modified alkyd resin, a cross-linking agent and a neutralizing medium. They

found that by increasing the concentration of ZnO, scratch resistance and abrasion

resistance of the coatings increased. Similarly, Allen et al (2004) used TiO2 NPs to mix

with alkyd paint for coating on stainless steel substrates. Faure et al (2011) used silicone

co-polymers along with an antimicrobial agent nisin for dip coating on stainless steel.

They found that the coatings are durable and exhibited good microbicidal activity. Li et al

46

(2009) created ZnO coating on plastics using acrylic resin mixed with a curing agent and

a thinner.

Gergely et al (2011) developed corrosion resistant zinc rich alumina coatings on

stainless steel surface using a two component mixture consisting of epoxy resin and a

cross-linking agent. Caballero et al (2010) suspended TiO2 NPs in acrylic paints along

with extenders such as CaCO3, silica, and talc at different TiO2 concentrations. The paint

formulations were then coated on polyester sheets and tested for photocatalytic

bactericidal activity. Kumar et al (2012) mixed TiO2 NPs that are functionalized with

silanes with epoxy paint and cured with cycloaliphatic amine. The suspensions were then

spray coated on carbon steel. They found that scratch hardness, adhesion strength,

abrasion resistance, flexibility, and corrosion resistance were improved with silane

treated TiO2 in the coating when compared to untreated TiO2. Marolt et al (2011) studied

the photocatalytic activity of TiO2 coating on card board using acrylic binder.

Han et al (2012) prepared TiO2 coated polyester fibers using colloidal silica as binding

agent. They reported that the spray-coated samples of TiO2 showed higher photocatalytic

activity and physical stability when compared to dip coated samples.

3. Methods to evaluate surface characteristics and physical stability of

nanocoatings

Understanding the structural characteristics NPs and nanocoatings such as size,

shape, chemical composition, and orientation etc, are important for estimating their

photocatalytic properties. Several techniques such as X-ray diffraction (XRD), X-ray

photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), transmission

electron microscopy (TEM), atomic force microscopy (AFM), optical, and Raman

47

spectroscopy were widely used techniques for this purpose. In addition, evaluating the

physical stability of the nanocoatings is vital for estimating their long-term durability.

Several methods can be used for this purpose. Scratch hardness of the coating can be

determined based on ASTM G171-03 standard method. This method characterizes the

resistance of a solid surface to penetration by a moving stylus of given tip radius under a

constant normal force and speed. The hardness of coated surface can be expressed as

scratch hardness number in GPa. Adhesion strength of the coating on the substrate can be

evaluated by ASTM D3359-02 method. In addition, ASTM D4060-14 is the standard test

method for measuring abrasion resistance of a coating which is also referred as the Taber

test. The test specimen is mounted to the Taber abrader and rotated at a fixed speed under

a weighted CS-10 or CS-17 abrading wheel. The weight loss per specified number of

revolutions under specified load is expressed as wear index. Simunkova et al (2003)

discussed the tests used for evaluating the mechanical properties such as adhesive

strength, cohesive behavior, wear resistance, micro hardness and fracture hardness of thin

film substrate systems. A reciprocating test to simulate food processing cleaning

operations was mentioned in a report by European Commission which evaluated the

effect cleaning on the antibacterial activity of coated surfaces. In addition, prolonged

water exposure (one week immersion), thermal cycling (5 cycles: 2 min 70°C water + 30

min -18°C), acid (pH 3.1) and caustic (pH 10) exposure (1 hour immersion), manual

brush cleaning and sand falling test were also discussed to measure the physical stability

and antimicrobial performance of the coatings.

48

IV. ANTIMICROBIAL ACTIVITY OF TIO2 NANOMATERIALS

1. Bactericidal activity of TiO2 in suspension vs coating

Since the early work of Matasunga et al (1985), many research groups have

reported the application of semiconductor photocatalysis for the inactivation of different

types of pathogenic microorganisms, such as bacteria, viruses, algae, fungi or protozoa.

As described earlier, the bactericidal properties of TiO2 are attributed to the high redox

potential of the surface species formed by photo-excitation. The type and the source of

TiO2 has shown to exhibit significant effect of bactericidal activity. This is mainly

attributed to the rate of formation of ROS depends on the particle size, crystalline phase,

the isoelectric point, and the specific surface area of the NPs. All these surface properties

can be controlled by the method of synthesis and various with the type of NPs. On the

other hand, the biological parameters of the microorganisms such as microbial species,

growth phase, initial cell density etc., were also found to have significant effect of

photocatalytic disinfection. In addition, the experimental conditions such as the

concentration of NPs, the light intensity, the wavelengths, and the treatment time were

also important (Hitkova et al, 2012). Most recently, our studies on TiO2 bactericidal

activity in suspension revealed that the type and source of NPs, bacterial cell harvesting

conditions, volume of reaction mixture, and the intensity of UV light has showed

significant effect on the bactericidal property of TiO2 NPs (Yemmireddy and Hung,

2015).

Hitkova et al (2012) studied the antibacterial activity of sol-gel synthesized TiO2

NPs in suspension at 1 mg/mL concentration. Their results showed that E.coli were more

susceptible to photocatalytic disinfection when compared to P. aeruginosa, and S.

49

aureus. Faure et al (2011) studied the photocatalytic inactivation of TiO2 on five different

photocatalytic supports, in terms of TiO2 type and source (Degussa P25 vs Millennium

PC500) and configurations (catalyst was impregnated on supports, alone or with binder,

or suspended in water). They found that for the same type of TiO2, inactivation efficiency

was better in suspension (up to 4 log in 2 h) followed by TiO2 impregnated without

binder (up to 2 log in 2 h) and finally TiO2 with binder (only 0.5 log after 2 h). Although,

several studies have found that TiO2 in suspension has high microbicidal property, its

practical application in suspension is limited due to difficulties in post-reaction catalyst

separation. Many efforts have been devoted to immobilize NPs on inert supports for more

practical applications.

Kuhn et al (2003) studied the photocatalytic bactericidal activity TiO2 coatings on

glass substrate using different bacterial strains. They reported that the order of bacterial

susceptibility to photocataytic disinfection is as follows: E.coli > P. aeruginosa > S.

aureus > E. faecium > C. albicans. Marugan et al (2008) found that TiO2 supported on

silica show a significant decrease in the bactericidal activity when compared with

bacterial activity of TiO2 (Degussa P-25 ) in suspension. They also reported that in a

coated supports, the contact between TiO2 and the microorganisms is limited to the TiO2

crystal located in the external surface of the particles. This area represents only a small

fraction of the semiconductor loading that is available for actual photo-killing effect.

Chorianopoulos et al (2011) developed TiO2 (Degussa P-25) coatings on stainless steel

and glass substrates using acetyl acetone as a solvent and Triton X-100. These coatings

reduced Listeria monocytogenes biofilms by 3 log CFU/cm2 after 90 min UVA light

illumination. Kim et al (2008) created TiO2 thin films on glass substrate by using

50

Degussa P-25 and carbowax binder. They reported that the number of coatings had no

effect on the antimicrobial property. Evans et al (2007) developed TiO2 thin films on the

surface of stainless steel using a combination of flame-assisted chemical vapor deposition

(FACVD) and thermal atmospheric pressure –assisted chemical vapor deposition

(APCVD). They reported a 6 log reduction of E.coli in less than 3 h when irradiated the

TiO2 coatings at 2.2 mW/cm2 UVA light intensity. However, the intensity of UV light

alone used in this study is high enough to show significant bactericidal activity. In

another study by Ditta et al (2008) reported the antibacterial activity of APCVD coated

TiO2, CuO, and TiO2/CuO dual layers on E. coli and bacteriophage T4. Their results

suggested that the bacteriophage T4 was more susceptible to photocatalysis than E.coli.

Among the tested coatings, TiO2/CuO composite coatings showed higher efficacy which

might be attributed to the additional toxicity of Cu ions. George et al (2010) studied the

bactericidal performance of flame-sprayed TiO2 and TiO2-Cu composite coatings against

Pseudomonas aeruginosa. Under the same conditions, the TiO2-Cu composite coatings

had the same bactericidal capability as pure Cu surfaces. The change of TiO2 from

anatase to rutile phase during high temperature flame spraying was found to be one

possible reason for the low bactericidal property. However, the composite coatings

showed improved bactericidal performance under light irradiation. MacFarlane et al

(2011) studied the photocatalytic microbicidal activity of jet spray formed TiO2 surfaces.

The microbial inactivation rates were highest for P. aeruginosa (Gram-negative)

followed by S. aureus (Gram-positive) and C.albicans (yeast). Xiao et al (2014) reported

antibacterial and antifungal activity of Fe-doped TiO2 coating with chitosan under visible

light irradiation. A slurry of Fe- doped TiO2 mixed with chitosan using a crosslinking

51

agent (epichlorohydrin) was spread coated on glass slides. They reported that the

composite coatings were very efficient in reducing the levels of the three tested strains

E.coli, C. albicans, and A. niger.

2. Studies related to bactericidal activity of TiO2 for food safety applications

TiO2 has been approved by the American Food and Drug Administration (FDA)

for use in human food, drugs, cosmetics, and food contact materials (FDA, 2014) and it

permits up to 1% of TiO2 as an inactive ingredient in food products. Also, TiO2 has been

evaluated positively as a food additive by European Union (EU Directive 94/36/EC,

1994). It is widely used as an additive in various products such as cosmetics (e.g.

lipsticks), food (e.g. salami, chewing gum, cookies) and pharmaceuticals as a white

pigment. Malato et al (2009) reported that TiO2 is the most active photocatalyst under the

photon energy of 300 nm< l < 390 nm and remains stable after the repeated catalytic

cycles. TiO2 can kill both Gram-negative and Gram-positive bacteria, although Gram-

positive bacteria are less sensitive due to their ability to form spores (Wei et al, 1994).

However, contrasting information is available regarding sensitivity of Gram-negative and

Gram-positive bacteria towards TiO2 photocatalysis.

TiO2 photocatalytic activity has been found particularly useful to decontaminate

wash water used for cleaning minimally processed products (Chaleshtori et al, 2008).

TiO2 coatings on food contact surfaces was found to diminish the growth of Listeria

biofilms (Chorianopoulos et al, 2011) or to improve cleanability of stainless steel (Verran

et al, 2010). The biocide capacity of TiO2 nanocomposites with typical packaging

materials has also been tested (Cerrada et al, 2008; Diaz-Visurraga et al, 2010). Kim et al

(2009) reported a reduction of up to 2.8 log CFU/g for E.coli, L. monocytogenes, S.

52

aureus and S. typhimurium in inoculated iceberg lettuce when germicidal UV light (254

nm) illuminated through a TiO2 coated quartz glass. In vitro studies using TiO2 coated

polypropylene films were effective in decreasing the counts of E. coli up to 3 log CFU/g

(Chawengkiwanich and Hayata, 2008). In the same study, a reduction of over 1 log

CFU/g was observed during extended storage. Their results also showed that the

antimicrobial effect of TiO2 coated film is dependent on the UVA light intensity, but it is

independent of the particle size of TiO2 used for coating. Similarly, Maneerat and Hayata

(2006) reported antifungal activity of TiO2 powder and TiO2 coated plastic film to

prevent Pencillium fruit rot on apples, tomatoes, and lemons. Their study revealed that

the concentration of TiO2 and the growth of Penicillium expansum were inversely

correlated. Chorianopoulos et al (2011) reported photocatalytic disinfection potential of

TiO2 coatings on stainless steel and glass substrates against Listeria monocytogens

biofilms. Wang et al (2010) found that a nano-packaging containing Ag and TiO2 in

combination with a hot air treatment were efficient in improving green mold control and

ethylene production in Chinese bayberries. TiO2 photocatalysis has also shown to be

effective for the inactivation of foodborne pathogens such as Salmonellas spp, Vibrio

parahaemolyticus, and Listeria monocytogenes (Kim et al, 2008). Bodaghi et al (2013)

developed TiO2 nanocomposite packaging film through melt blending technique using an

extruder. They reported a 4 log and 2 log CFU/mL reduction of Pseudomonas spp and

Rhodotorula mucilaginosa, respectively when using these packaging films. Further, in

vivo tests on fresh pears packaged in TiO2 nanocomposite film showed significant

reduction in mesophilic bacteria and yeast growth when stored at 5 °C for 17 days.

53

Studies with E.coli strains (PHL 1273) synthesizing curli, a type of appendage

that allows the bacteria to stick to surfaces and form biofilms, found that titania and

various types of UV irradiation were able to inactivate this organism. In dark event

studies, following the bacterial inactivation, no bacterial cultivability was recovered even

after 48 h, indicating that the durability of the TiO2 disinfection was adequate (Gamage et

al, 2010). In a study by Gelover et al (2006), found that a complete inactivation of fecal

coliforms was achieved in 15 min by exposing water in TiO2 coated plastic containers to

sunlight whereas the same extent of inactivation required 60 min with uncoated

containers. This study also found that the bacteria exposed to TiO2 photocatalytic

disinfection do not self-repair. Nano-sized TiO2 was also reported to kill viruses

including poliovirus-1 (Watts et al, 1995), hepatitis B virus (Zan et al, 2007), herpes

simplex virus (Hajkova et al, 2007), and MS2 bacteriophage (Cho et al, 2005). Nakano et

al (2012) reported antiviral properties of TiO2 thin film coatings against influenza virus.

This study also investigated the effect of UV intensity, irradiation time, and bovine serum

albumin concentration in the viral suspensions on the inactivation kinetics.

3. Considerations for testing antimicrobial activity of photocatalytic nanomaterials

Several studies in the literature have used different approaches to determine the

antimicrobial property of photocatalytic nanocoatings. However, the International

Standard Organization (ISO) suggested a standard method ISO 27447 for evaluating

antibacterial activity of semiconducting photocatalytic materials (ISO, 2009). This

standard specifically applies to evaluate antibacterial activity on photocatalytic ceramic

materials and other materials that are generated through coating or mixing with

photocatalysts. Similarly, ISO 13125 (E) is the standard method for testing antifungal

54

activity of semiconducting photocatalytic materials (ISO, 2013). Though each of these

methods including the standardized methods that are followed in the literature have their

own advantages and limitations, the variety of methods used to determine antimicrobial

activity impedes an accurate comparison of the test results.

The approaches that are used to test the antimicrobial activity of photocatalytic

nanomaterials based on the method of inoculation can be broadly classified into:

i) Direct inoculation method

ii) Glass cover-slip method

iii) Direct immersion method

iv) Indented well method

v) Agar diffusion method

3.1. Direct inoculation method

Direct inoculation method is the most widely reported test method in the literature

(Xiao et al, 2014; Faure et al, 2011; Ditta et al, 2008; Evan et al 2007; Kuhn et al, 2003;

Sunada et al, 2003; Yu et al, 2003; Kikuchi et al, 1997). Briefly, in this method a

specified volume test inoculum is directly pipetted on to the surface of photocatalytic

material to be tested. After the treatment, the samples are enumerated as per standard

microbiological recovery protocols. Even though this method is simple to conduct, the

main disadvantages include: (i) non-uniform coverage of the inoculum on the test

surface, and ii) drying out of the inoculum during the photo-treatment. These conditions

may lead to inaccurate determination of photocatalytic antimicrobial property of test

surface.

55

3.2. Glass cover-slip method

Glass cover-slip method is mainly based on ISO 27447:2009 standard test

method. As per this method an adhesion film or glass cover-slip is placed on top of the

inoculum in order to ensure good contact of the bacterial cells to the photocatalytic test

surface. Also, Kim et al (2008) has suggested using polypropylene adhesion film based

on better light transmittance through the polypropylene film. Later, in a review conducted

by Mills et al (2012) outlined the pros and cons of using film and glass cover-slip

methods. The main issue associated with using the cover slip method is that it is difficult

to avoid leakage of test inoculum from the sides of the photocatalytic test surface. In

addition, this method of testing inhibits the contact of sufficient atmospheric oxygen with

the NPs to catalyze the process of photocatalytic disinfection.

3.3.Direct immersion method

Krysa et al (2011) compared two different test methods to evaluate bactericidal

property of TiO2 coatings with a modified ISO 27447:2009 method using adhesive glass.

In the first method (50 cm3 method), TiO2 coatings were immersed in 50 mL bacterial

suspension, and in the second method (3 cm3

method) 3 mL of bacterial suspension was

poured into a petridish containing TiO2 coated sample. Their studies concluded that the

50 cm3 method is only useful to test pure TiO2 coatings with strong bacterial property and

the 3 cm3 method is not appropriate for testing. They also found that 3 cm

3 method did

not allow a clear distinction between the inhibition effect of TiO2 and UV light itself and

also it created several unreactive dead zones in the test system. They found that modified

ISO method using adhesive glass is most suitable compared to other two methods.

Similarly, Nakano et al (2012) reported antiviral properties of TiO2 thin film coatings

56

against influenza virus. Their study also suggested a minor modification of the ISO test

method for antibacterial effects of TiO2 photocatalysis to evaluate antiviral activity.

3.4. Indented well method

The indented well method is not a widely reported approach to test microbicidal

activity of photocatalytic materials in the literature. However, this method has several

advantages in solving some issues discussed in previous methods. Cushnie et al (2010)

used an aseptically sealed glass cell ring on the surface of photocatalytic glass slides.

Later, a 300 µL bacterial suspension was carefully pipetted into the ring cells and the

material subsequently irradiated. Another study by George et al (2010) used pyrex wells

mounted on thermal sprayed TiO2 coatings on stainless steel using a silicone rubber in

order to contain bacterial solutions on to the coated substrate. Similarly, Bonetta et al

(2013) used a glass ring fixed with inert cement on TiO2 coated ceramic tiles to inoculate

bacterial culture and determine the photocatalytic bactericidal property. The advantage of

this type of approach is that it allows the bacterial suspension to be accurately deployed

to a known area of the surface under investigation. Also, this method of testing offers the

advantage of uniform coverage and good contact of bacterial cells with the photocatalytic

surface. However, the relative complexity of mounting a well with good seal limits wide

spread application of this test procedure.

3.5. Agar diffusion method

The agar diffusion method is another approach which is widely used to determine

the bactericidal activity of NPs in general. In this method, the NPs is immersed in

bacterial growth media which is already inoculated with test bacterium. After the

treatment, the size of the growth inhibition zone is a measure of bactericidal activity of

57

NPs. For example, Azam et al (2012) compared the antimicrobial activity of metal oxide

NPs such as ZnO, CuO, and Fe2O3 using agar well-diffusion method and minimal

bacterial concentration. They found that ZnO had the highest antibacterial activity

followed by CuO and Fe2O3 when tested against Gram-positive (S. aureus and B.

subtilis) and Gram-negative (P. aeruginosa and E.coli) bacteria. They also found that

Gram –negative bacteria showed more resistance to NPs compared to Gram-positive

bacteria. However, the agar diffusion method is not suitable for photocatalytic NPs such

as TiO2 since these NPs require light penetration and activation in order to exhibit

significant bactericidal property.

Effect of other test conditions

Suspension medium: The chemical nature of the suspension medium was found to have

significant effect on the photo-killing rate of TiO2 NPs. Cushnie et al (2009) reported that

the greatest antibacterial activity was observed when aqueous sodium chloride solution

was used when compared to aqueous tryptone solution. In contrast, Ditta et al (2008)

reported a similar rate of killing by using either saline or water as re-suspension media.

Growth media: Rincon and Pulgarian (2004) reported that the choice of growth media

for enumerating bacterial recovery following photocatalytic treatment is an important

factor that may affect the experimental results. Their study revealed that the non-selective

medium plate count agar showed 1000-times higher response than that of the selective

media CHROM agar following photocatalytic disinfection of E.coli. Similarly, Faure et al

(2011) compared the bacterial re-growth on two different culture media, such as tryptic

soy agar (TSA) and eosin methylene blue (EMB) agar after photocatalytic treatment.

They found that for the same sample, the number of colonies counted on EMB was

58

always lower than on TSA after photo-treatment. They concluded that the lactose

metabolism of bacteria was severely affected due to photocatalytic treatment and hence

showed variable recovery on different media. McCullagh et al (2007) have suggested that

under certain circumstances where it is considered necessary to use a selective media

then it is also important to include a non-selective agar to substantiate and compare the

results.

Incubation time: Many studies have used different incubation times for bacterial

enumeration following photocatalytic treatment with NPs. Cushnie et al (2009) found that

the factors such as osmatic pressure, and incubation time has significant effect on the

outcome of TiO2 photocatalytic disinfection results. Bacteria treated with only UV light

grew more slowly than those treated with TiO2 and UV, often taking in excess of 24 h to

produce visible colonies.

V. SAFETY CONCERNS ON USE OF NPS

1. Toxicity issues

Despite projected benefits, nanotechnology is raising regulatory issues and public

concern regarding safety and environmental effects. Rossi et al (2014) in their review on

scientific basis of nanotechnology, implications for the food sector and future trends

discussed the major issues associated with toxicity of NPs when used in food

applications. The main issues raised by Rossi et al (2014) include:

i. Knowledge gap on how altered physico-chemical properties of NPs may

influence their toxicological properties when ingested into human body via

food.

59

ii. Current toxicity testing approaches appear to be inadequate to detect all the

toxicity aspects of nano-sized materials.

iii. Various dose metrics such as size and other physicochemical parameters of

engineered nanomaterials has to be explored since mass concentration alone is

not sufficient.

iv. Effect of factors such as dissolution rate, agglomeration, aggregation,

adsorption or binding with other food components, and reactions with acid

and digestive enzymes may affect the fate of engineered nanomaterials in the

gut and more understanding on these aspects is needed.

Dutta and Waldman (2012) studied the effect of Si, ZnO, TiO2, and Ag NPs on

the human epithelial cells in a stomach model as well as in a stomach and intestinal

model and measured the uptake, cell death, cell proliferation, and mitochondrial activity.

Their results showed that the ZnO NPs dissolve in acidic medium in the stomach and

showed modest toxicity while TiO2 and silica had almost no cell toxicity; whereas Ag

NPs are found to be very toxic. In another study, Lin and Mustapha (2012) spiked

engineered NPs such as Ag, ZnO, and TiO2 from a commercial source into various food

samples such as wheat flour, yam, corn starch, and pears and used a combination of

techniques, including Scanning Electron Microscopy (SEM), Energy Dispersive

Spectroscopy (EDS), and Inductive Couple Plasma-Optical Emission Spectroscopy (ICP-

OES) to detect the NPs. In addition, they also exposed the NPs to natural gut microflora

such as E.coli, Lactobacillus acidophilus, and Bifidobacterium and human intestinal

epithelial cells to different concentrations of ZnO and Ag NPs. Their results showed that

the tested NPs have some antibacterial properties that inhibit growth of bacteria and

60

higher concentrations of NPs showed toxicity to human epithelial cells. Ammendolia et al

(2014) studied the L. monocytogenes behavior in presence of non-UV-irradiated TiO2

NPs with a special focus on biofilm formation and intestinal cell interaction. Their study

revealed that the TiO2 NPs influenced both production and structural architecture of

listerial biofilm in addition to their interaction with intestinal cells.

The International Agency for Research on Cancer (IARC), after extensive studies

has rated TiO2 nanoparticles as carcinogenic (Group 2b) for humans (Baan et al, 2006).

In vivo studies on the ability of TiO2 NPs to penetrate the GI tract has revealed that the

NPs can be found in systemic organs after an oral exposure of 10 days (Jani et al, 1994).

Liu et al (2010) reported that TiO2 NPs showed intracellular accumulation of ROS

leading to apoptosis in PC12 cells. In addition, Tassinari et al (2013) demonstrated that

reproductive and endocrine effects of short term oral exposure to low doses 0-2 mg/kg

body weight per day of TiO2 NPs. However, due to lack of sufficient data supporting the

toxicity of engineered NPs and the scientific consensus on this matter, further studies

need to be warranted to evaluate potential toxicity of engineered NPs on human health

and the environment.

2. Regulatory framework

For regulatory purposes related to use of nanotechnology in food sector, various

countries have adopted different and sometimes diverging approaches (Cushan et al,

2012). The National Nanotechnology Initiative (NNI) is a U. S. Government’s

multiagency, multidisciplinary research and development program on nanotechnology. It

brings together the expertise of 25 federal agencies and supports collaborative research

and development in academic, government, and industry laboratories (National

61

Nanotechnology Initiative, 2010). The U.S. Food and Drug Administration (FDA) did not

issue strict definitions of NPs and considers food manufacturing processes that involve

nanotechnology in the same manner as any other food manufacturing technology (FDA,

2011). However, the FDA clearly states that, as with any studies to support the safety of

food substances, studies to establish the safety of food substances manufactured using

nanotechnology should have been appropriately validated (FDA, 2012). The Center for

Food Safety and Applied Nutrition (CFSAN) at the U.S. FDA has a research program to

explore the safety assessment of nanomaterials in food and cosmetic products. It focuses

on examining the possibility of NPs leaching from food packaging materials and

determining whether there is a safety concern, and investigating approaches to study the

potential toxicity of NPs (Mermelstein, 2013). Further, the FDA’s National Center for

Toxicological Research is developing analytical tools and procedures to quantify

nanomaterials in complex matrices and conducting toxicity studies on NMs. In addition,

The U.S. National Toxicology Program (NTP) through its Nanotechnology Safety

Initiative, conducting research on several classes of nanoscale materials, including metal

oxides, fluorescent crystalline semiconductors, carbon based fullerenes, carbon

nanotubes, nanoscale silver, and nanoscale gold (Mermelstein, 2013).

The European Food Safety Authority (EFSA), in its scientific opinion on the potential

risks arising from nanoscience and nanotechnologies on food and feed safety, affirmed

that the risk assessment paradigm (i.e. hazard identification, hazard characterization,

exposure assessment and risk characterization) is applicable for engineered nanomaterials

(EFSA, 2009). However, since different types of engineered nanomaterials even with the

same chemical composition may vary as to their toxicological properties, the risk

62

assessment of engineered nanomaterials has to be performed on a case-by-case basis. The

EFSA stated that appropriate data for risk assessment of an engineered nanomaterial in

the food and feed area include comprehensive characterization of the engineered

nanomaterial, information on whether it is likely to be ingested in nanoform, and, if

absorbed, whether it remains in nanoform at absorption or not.

VI. KNOWLEDGE GAP

1. Research scope

Foster et al (2011) reported that about 11,000 publications are available on

photocatalysis and out of which about 800 publications reported photocatalytic

disinfection. Henderson (2011) reported that approximately 2400 heterogeneous

photochemistry papers were published in 2008 and out of which 80 % involved TiO2

based materials. This shows the extent of research activity carried out in the area of TiO2

photocatalysis. Over the last decade, the antimicrobial property of photocatalytic TiO2

NPs has aroused much interest in the food sector. However, our knowledge is very

limited on the use of TiO2 photocatalysts for food safety applications. As reported in the

literature, microbial cross-contamination is a major issue in the entire food chain.

Nanotechnology based intervention strategies using photocatalytic TiO2 NPs has great

potential to help reduce the risk of microbial cross-contamination in the food processing

environment. The potential areas of research in this direction may include but not limited

to:

Developing antimicrobial nanocoatings on food contact and non-food contact

surfaces using TiO2 NPs.

63

Developing testing methods to determine the antimicrobial activity of TiO2

nanomaterials.

Determining the bactericidal efficacy of TiO2 nanomaterials in food processing

environmental conditions.

Determining the durability of the nancoatings for use in food safety applications.

64

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Fig 2.1. Semiconductor photocatalysis

Ref: http://dev.nsta.org/evwebs/1952/photocatalysis.html

http://dev.nsta.org/evwebs/1952/photocatalysis.htm

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Fig 2.2. TEM of a dispersion of TiO2 Degussa P-25 (1 mg/L) in contact

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CHAPTER 3

SELECTION OF PHOTOCATALYTIC BACTERICIDAL TITANIUM DIOXIDE

(TIO2) NANOPARTICLES FOR FOOD SAFETY APPLICATIONS1

1Veerachandra K. Yemmireddy and Yen-con Hung. LWT- Food Science and Technology.

61 (2015) 1-6. Reprinted here with permission of the publisher.

86

Abstract

The main objective of this study was to develop a systematic testing protocol for

selecting bactericidal TiO2 nanoparticles (NPs). Photocatalytic bactericidal activity of

TiO2 NPs at 1 mg/mL concentration was tested against E.coli O157:H7. The effect of

source of NPs (three different commercial samples referred as T1, T2 and T3), bacterial

cell wash conditions (1 vs 3 wash), volume of reaction mixture (10, 20 and 30 mL) and

intensity of UVA light (1 vs 2 mW/cm2) on bactericidal activity has been determined.

Sample T3 was found to be the most effective among the tested TiO2 samples. Increasing

the number of cell washes from 1 to 3 increased the log reduction (2.91 vs 4.57).

Increasing the light intensity increased the overall log reduction (3.27 vs 4.22).

Decreasing the volume of reaction mixture increased the log reduction. This study has

identified the best testing protocol for evaluating TiO2 NP bactericidal efficacy as single

wash of bacterial cells with a reaction mixture volume of 20 mL and UVA light intensity

of 2 mW/ cm2. In addition, it was found that photocatalytic oxidation of organic dyes can

be used as a quick and easier way to screen bactericidal TiO2 NPs prior to actual

microbiological tests.

Keywords: TiO2; Nanoparticles; Photocatalyst; Bactericidal activity; E.coli O157:H7.

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1. Introduction

Advanced oxidation processes based on heterogeneous photocatalysis using

photocatalytic nanoparticles (NPs) is gaining popularity in food safety applications.

Heterogeneous photocatalysis utilizes light along with a semiconductor NPs to produce

reactive oxygen species (ROS) which can inactivate bacteria and degrade a wide range of

chemical contaminants (Mills & Le Hunte, 1997). Of the available semiconductor NPs

which can be used as photocatalysts, TiO2 is generally considered to be the best

semiconductor photocatalyst available at present (Mills & Lee, 2002) due to its strong

oxidizing power at ambient temperature and pressure, stable, non-toxic, cheap and readily

available. TiO2 has been approved by the Food and Drug Administration (FDA) for use in

human food, drugs, cosmetics, and food contact materials (Chorianopoulos et al., 2011).

TiO2 photocatalysts generate strong oxidizing power when illuminated with UV light of

wavelength less than 385 nm. TiO2-mediated photooxidation shows promise for the

elimination of microorganisms in areas where the use of chemical cleaning agents or

biocides is ineffective or is restricted by regulations such as pharmaceutical and food

industries (Skorb et al., 2008). In addition, TiO2 becomes superhydrophilic upon

irradiation with UV light and this functionality is reversible and depends on the light

exposure (Chen & Mao, 2007). These properties of TiO2 help to reduce the usage of

cleaning agents and to create shorter cleaning cycles in the food industry.

Since the photochemical sterilization of E. coli using Pt-TiO2 was reported by

Matsunga et al. (1985), TiO2 photocatalysts have extensively studied to disinfect a broad

spectrum of microorganisms including viruses, bacteria, fungi, algae, and cancer cells

(Kim et al., 2003). The bactericidal properties of TiO2 are attributed to the high redox

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potential of the surface species also known as ROS, such as hydroxyl radical (.OH),

superoxide radical (O2.-), hydrogen peroxide (H2O2) formed by photo-excitation. The type

and the source of TiO2 plays an important role during bacterial inactivation because the

rate of formation of ROS is a function of the particle size, crystalline phase, the

isoelectric point, and specific surface area of the nanostructure (Hitkova et al., 2012). On

the other hand, the biological parameters of the microorganisms such as microbial

species, growth phase, and initial cell density etc., are also important and photocatalytic

disinfection process may vary depending on the light intensity, the wavelengths and

experimental conditions (Hitkova et al., 2012). Several commercial photocatalytic TiO2

products are available on the market and notably, Degussa P25 TiO2, which is considered

a standard is often used for comparison in scientific experimentation for determining

photocatalytic activity (Mills & Le Hunte, 1997). Hoffmann et al (1995) have suggested

that the anatase/rutile structure of P25 promotes charge-pair separation and inhibits

recombination. The different electron-hole pair recombination lifetimes and interfacial

electron-transfer rate constants may be due to the different preparation methods of the

samples that result in different crystal defect structures and surface morphologies (Chen

& Mao, 2007).

Several studies claimed high bactericidal activity of synthesized and commercial

TiO2 NPs that are equivalent to Degussa P-25 (Kim et al., 2003; Hitkova et al., 2012).

However, none of these studies have considered important factors like bacterial cell

harvesting conditions and reaction mixture volume that may negatively influence the

results of photocatalytic disinfection. Hence developing a systematic testing protocol to

evaluate bactericidal efficacy of NPs helps proper selection of TiO2 NPs for food safety

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related applications. The overall objective of this study was to develop a systematic

testing protocol to determine the bactericidal efficacy of different TiO2 nanoparticles in

suspension. Specific objectives include: i) to determine the effect of type and source of

TiO2 NPs on bactericidal activity, ii) to determine the effect of cell harvesting conditions,

light intensity and volume of suspension on bactericidal activity.

2. Materials and Methods

2.1. Nanoparticles

A total three different types of commercial TiO2 NPs (referred as T1, T2 and T3) of

known characteristics (Table 3.1) were used in this study. Samples T1 and T2 were of

anatase crystal phase with 10-25 nm size and > 99 % purity. While sample T3 is mixture

of anatase/rutile phase (~80:20 wt %) with ~ 21 nm size and > 99.5 % purity. The details

of the nanoparticle characteristics as provided by the individual manufacturer are listed in

Table 3.1. Aqueous suspensions of TiO2 NPs at 1 mg/mL concentration were prepared by

sonication in water-bath (Model # FS30, Fisher Scientific, Waltham, MA, USA) for

about 1 h at 23°C using sterile deionized water. The suspensions were prepared fresh

every time prior to each photocatalytic disinfection experiment.

2.2. Bacterial strains and inoculum preparation

Five strains of E. coli O157: H7 isolated from different sources: E009 (beef),

EO932 (cattle), O157-1 (beef), O157-4 (human), and O157-5 (human) were used in this

study. All bacterial strains were stored at -70 °C in tryptic soy broth (TSB) (Difco,

Becton Dickinson, Sparks, MD, USA) containing 20 % glycerol. Prior to the experiment,

cultures were activated at least twice by growing them overnight in 10 mL of TSB at 37

°C. Later each bacterial stain was cultured separately in 10 mL of TSB and kept on a

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shaking incubator at 230 rpm and 37°C for 16 h. Following the incubation, cells were

harvested by sedimentation either once (4000 x g for 12 min) or resuspended and

sedimented three times (3200 x g for 10 min) in sterile phosphate-buffered saline (PBS,

pH 7.2) in order to determine the effect of cell harvesting conditions on the photocatalytic

disinfection efficacy. The harvested cells were re-suspended in 10 mL PBS and an equal

volume of each strain suspension was combined to obtain 10 ml of a five strain cocktail

containing approximately 108 CFU/mL. Cell concentration was adjusted by measuring

the absorbance of bacterial suspension at 600 nm using a UV/Vis spectrophotometer

(Beckman DU520, Beckman Coulter Inc., Brea, CA, USA) and confirmed by plating 100

µL portions of the appropriate serial dilution on tryptic soy agar (TSA) (Difco

Laboratories) plates incubated at 37 °C for 24 h.

2.3. Photocatalytic experiments

The photocatalytic disinfection experiments were carried out in a sterile glass petri-

dish (90 x18 mm2; diameter x depth) mounted on a magnetic stirrer (Model# H1190M,

Hanna Instruments, Smithfield, RI, USA) which were together placed in a photocatalytic

disinfection chamber (Fig. 3.1). Aqueous suspensions of TiO2 NPs of 9, 18 and 27 mL

volumes were added into the petri-dish along with 1, 2 and 3 mL of bacterial cultures,

respectively. In this way, the effect of volume of suspension (10, 20 and 30 mL) on the

photocatalytic bactericidal activity was investigated for each commercial TiO2 sample

individually. The initial concentration of the bacterial culture in the suspension was fixed

at approximately 107 CFU/mL. The petri-dish with bacteria-NP suspension was

illuminated with a UVA light system fitted with four 40 W lamps (American DJ®, Model

UV Panel HPTM

, LL-UV P40, Los Angeles, CA , USA) from the top under continuous

91

stirring at medium speed with a magnetic stirbar (3.8 cm length x 1.25 cm diameter) (Fig.

3.1). The intensity of the light was measured using a UV radiometer (Peak sensitivity 365

nm, UVP®, Upland, CA, USA). The light intensity reaching the surface of the sample

was adjusted to either 1 or 2 mW/cm2 (±0.15) by changing the distance between the light

source and the sample. A positive (photocatalyst in dark) and a negative (without

photocatalyst under UVA light) control samples were also included. All the experiments

were conducted at room temperature using indoor air as oxidant. A sample of 1 mL was

withdrawn from the treatment solution at every 30 min for 3 h and added into 9 ml sterile

PBS. Appropriate serial dilutions of the samples were prepared and the surviving bacteria

from the control and treatments were enumerated by spiral plating 50 µl of each dilution

on TSA. The plates were incubated at 37 °C for 24 h, and colonies were counted and

recorded as log CFU per mL. It was noticed that under tested conditions, positive and

negative controls had negligible effect on log reduction.

Further, the photocatalytic activity of TiO2 samples was evaluated by degradation of

methylene blue (MB) solution. The photodecay rate was measured by using 20 mL of

TiO2 aqueous solution (1 mg/mL) saturated with MB dye (20 mg/L) in a petridish

illuminated with UVA light at 2 mW/cm2 under continuous stirring as described earlier.

A 3 mL sample was collected at every 60 min for 3 h and the TiO2 NPs were separated

by centrifuging the suspension at 4000 rpm for 15 min at 4°C. The photodecay rate of

MB was determined by measuring the absorbance of supernatant at 664 nm using UV-

Vis spectrophotometer. Residual concentrations of MB (mg/L) due to TiO2 photocatalytic

activity was calculated by using standard MB curve.

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2.4. Data analysis

All the experiments were replicated three times. Data were subjected to an analysis of

variance with a completely randomized factorial design. Statistical analysis was

performed using SAS (2008) General Linear Model procedure performed with SAS

Software Release 9.3 (SAS Institute). T-tests were used for pairwise comparisons. Least

significant difference of means tests were done for multiple comparisons, and all tests

were performed with a level of significance 0.05.

3. Results and discussion

3.1. Effect of type and source of TiO2

The reduction trend of TiO2 (T1, T2 and T3) samples using single wash of

bacterial cells, 20 mL bacteria-NP suspension and 2 mW/cm2 UVA light intensity over a

3 h photocatalytic disinfection treatment is shown in Fig 3.2. Under tested conditions,

Sample T3 exhibited the highest log reduction (5.78 CFU/mL) followed by T2 (2.59

CFU/mL) and T1 (1.81 CFU/mL), respectively. This indicates that the type and source of

TiO2 has played important role on the log reduction. In particular, the difference in the

log reduction among the tested samples might be attributed to the differences in the

surface characteristics of the individual TiO2 NPs (Table 3.1). Sample T1 and T2 used in

this study have wide particle size distributions of 10-25 nm and sample T3 has an average

particle size of 21 nm. Size of the particle is an important factor which influences the

quantum yield of ROS responsible for the photocatalytic disinfection. Changes in particle

size influence photoactivity through changes in surface area, light scattering and light

absorptivity. Gerischer (1995) demonstrated that the quantum yield increases with

decreasing illumination intensity and size of the particle during photocatalysis. Bui et al.

93

(2008) pointed out that the differences in particle size, surface area and charge among

different varieties of TiO2 in powder affect the photocatalytic efficiency. In the current

study, though samples T1 and T2 have similar size distribution (10-25 nm), T2 showed

more bactericidal activity compared to sample T1 under similar test conditions. This

difference could be attributed to the high particle specific surface area (SSA) of T2 (200-

240 m2/g) compared to T1 (50-150 m

2/g). Increasing the particle surface area provides

high relative OH- ion coverages for hydroxyl radical (OH

·) formation which is an

important ROS responsible for photocatalytic disinfection. In contrast, sample T3 with

mixed phase of anatase (~80%) and rutile (~20%), an average particle size of ~21 nm and

relatively low SSA (35-65 m2/g) showed higher bactericidal activity than T1 and T2.

As reported in several studies, high bactericidal activity of mixed phase of

anatase-rutile TiO2 powder, particularly Degussa P-25 (sample T3) could be attributed to

the generation of high amounts of ROS such as OH·, O2- and H2O2. This could be due to

effective charge separation and by avoiding electron-hole pair recombination during

photocatalytic disinfection process. Gumy et al (2006) demonstrated that neither a high

surface area nor a high aggregate size can be the sole properties of a TiO2 photocatalyst

leading to optimal E.coli inactivation. Another study reported that Degussa P-25 obtained

by flame pyrolysis (Degussa, 1997) is an effective photocatalyst for bacterial

inactivation. Bickley et al (1991) reported that the dynamic process of Degussa P-25

preparation would create a complex variety of multiphased particles characterized by a

juxtaposition of anatase with rutile phases. This condition results in increased charge

separation and slows down electron (e-) - hole (h

+) pair recombination rate which in-turn

results in high photocatalytic activity. Nguyen et al (2005) reported for the same mass of

94

NPs, dispersed particle sizes of a commercial TiO2 PC500 are approximately three times

larger than TiO2 P-25. As a result P-25 provides a larger surface area per unit weight for

contact with bacteria than PC 500 and exhibits improved efficiency. This indicates that

the preparation method of the TiO2, NP surface structure, size, and crystallographic

structure all played important roles during the interfacial charge transfer between TiO2

and E.coli leading to disinfection (Gumy et al, 2006). The results of current study further

support the importance of type of TiO2 on bactericidal activity. Degussa P-25 was found

to be most effective among tested commercial TiO2 NPs.

However, several studies that reported high bactericidal activity of other

synthesized or commercial TiO2 NPs did not accounted for the effect of other influencing

factors such as bacterial culture harvesting method and volume of suspension used during

photocatalytic disinfection. These factors are important and need to be considered when

selecting TiO2 NPs for food safety applications.

3.2. Effect of cell harvesting conditions

The reduction trend of samples T1, T2 and T3 with respect to number of cell

washes at 2 mW/cm2 UVA intensity and 20 mL reaction mixture volume was shown in

Fig 3.2. Using single wash of bacterial cells, sample T1 and T2 showed reductions of

only 0.14, 0.62 log CFU/mL after 120 min and 1.81, 2.51 log CFU/mL after 180 min

treatment, respectively. Both samples required at least 90 min for initiating

photocatalytic disinfection. Whereas, sample T3 being more reactive right after 30 min

treatment showed a reductions of 3.12 and 5.78 log CFU/mL after 120 and 180 min,

respectively. It should be noted that depending on the reactivity of NP there will be a lag

in bacterial cell killing. This is in part attributed to the time required for the ROS to react

95

with bacterial cell membrane and facilitate damage and destruction of intracellular

components and eventual death of cell. It is clearly visible that sample T3 is the most

reactive among the tested TiO2 samples.

However, increasing the number of cell washes from 1 to 3 showed significant

change in the reduction efficacy of all three TiO2 samples. Sample T1 and T2 started

killing bacterial cells from around 30 min (a decrease of initial reactivity time at 90 min

for 1 wash) and showed a reduction of 3.28 and 4.48 log CFU/mL in 3h respectively

which is about 81 and 78% respective increase from the reduction at 1 wash treatment.

Sample T3 is least benefited by increasing number of cell washes as it showed no

difference in the reduction potential at the end of 3 h treatment. However, sample T3

showed better reduction between the treatment times 90 (98% increase) and 120 (44%

increase) min when compared with 1 wash treatment. These results indicate possible

damage to the bacterial cell membrane during additional centrifugation/sedimentation

prior to the photocatalytic disinfection treatment might have enhanced reduction efficacy

of TiO2 NPs.

Peterson et al. (2012) stated that centrifugation in essence involves compacting

bacteria into a pellet, causing collisions against each other that result in shear forces on

the bacterial cell surface, which may lead to cell surface damage with a potential effect

on the outcome of surface-sensitive experiments. Gilbert et al. (1991) reported a decrease

of 25 and 40% in the viability of exponential-phase Pseudomonas aeruginosa following

centrifugation at 5,000 x g and 10,000 x g respectively. Similar experiments with

stationary and exponential phase E. coli cells greatly altered biocide sensitivity (Gilbert et

al., 1990). Pembery et al. (1999) reported loss of viability and modification of

96

physicochemical cell surface properties of E. coli or S. epidermis by high-speed

centrifugation (15,000 x g) when compared to harvesting at (5000 x g). Similarly,

subjecting the bacterial cells to multiple washing steps in current study might have

damaged the outer cellular membrane and makes it more susceptible to ROS attack

during photocatalytic disinfection treatment. Especially, increasing the number of cell

washes resulted higher bactericidal efficacies by less effective commercial samples T1

and T2 (Fig 3.2). This situation may leads to false prediction of bactericidal efficacy of

TiO2 NPs while selecting NPs for food safety applications such as coating on food

contact and non-food contact surfaces. The results of this study suggest that bacterial

harvesting conditions are at-most important to accurately determine bactericidal activity

of photocatalytic NPs. It is recommended to use less severe harvesting conditions

depending on the type of test organism.

3.3 Effect of light intensity

Effect of UVA light (365 nm peak wave length) intensity on the log reduction of

E.coli O157:H7 due to TiO2 samples (T1, T2 and T3) is shown in Table 3.2. Increasing

the light intensity from 1 mW/cm2 to 2 mW/cm

2 increased the bacterial cell reduction

from 3.27 to 4.22 log CFU/mL. Although the trend of bactericidal efficacy was

unchanged (i.e. T3>T2>T1), the intensity of UVA light was shown to have a significant

effect on efficacy of individual TiO2 samples. Increasing the UVA intensity from 1 to 2

mW/cm2 significantly increased the log reduction of bacterial cells by T1 (2.04 to 3.04

CFU/mL), T2 (2.99 to 3.94 CFU/mL) and T3 (4.77 to 5.67 CFU/mL). Benabbou et al.

(2007) reported a decrease in light intensity from 3.85 to 0.48 mW/cm2

increased the time

necessary to totally inactivate E. coli (3 log) from 90 to 180 min. Increasing the light

97

intensity increases the amount of photon generation which results in more electron-hole

pair formation, eventually leading to the formation of more OH radicals (Marugan et al.,

2010). On the other hand, Cho et al. (2004) reported the existence of linear negative

correlation between inactivation of E.coli and OH concentration. This linear dependence

of reaction rate with the photon flux is only found at low intensities of irradiation,

because at high intensities the concentration of charge carriers is so high that

recombination is more favored, limiting the efficiency of the process (Hermann, 1999).

However, it is believed that the tested intensity range (1-2 mW/cm2) of the current study

operated in the linear region corresponding with optimal light utilization. This implies

that the saturation of TiO2 acting as the photosensitizer was not reached when increasing

the light intensity from 1 to 2 mW/cm2. Benabbou et al. (2007) noticed an induction

period in the first 10 min for lower light intensities, which suggests that self-defense and

auto-repair mechanisms for protecting the bacteria were more efficient at a low intensity.

However, self-defense mechanism unable to protect bacterial cells over a long treatment

time under the light. Similar results were also observed in the current study with an initial

lag period of ~30 min at the lower intensity (i.e. 1 mW/cm2) for photocatalytic

disinfection (data not shown). To further evaluate the photocatalytic disinfection efficacy

of TiO2 NPs, the effect of suspension volume was also investigated.

3.4. Effect of volume of suspension

Effect of volume of TiO2 NP-bacterial suspension (10, 20 and 30 mL) on

photocatalytic bactericidal activity for 3 h was shown in Table 3.2. Statistical analysis of

the data revealed that the overall log reduction of TiO2 is higher (4.32 CFU/mL) at lower

volume (10 mL) when compared to higher volumes (20 and 30 mL). No significant

98

difference in the reduction was observed between 20 and 30 mL volume of suspensions at

the end of 3 h treatment time. As expected, all the tested TiO2 samples (T1, T2 and T3)

exhibited high bactericidal activity at lower volume of suspension (10 mL). However,

volume does not showed any effect on overall reduction potential of least effective TiO2

sample T1 at the end of 3 h (avg ~ 2.5 log CFU/mL). Whereas, sample T2 showed

significantly higher reduction (4.30 log CFU/mL) at 10 mL and almost similar reductions

at 20 and 30 mL (~ 3 log CFU/mL). The most effective sample, T3 only showed

significant difference in the reduction between 10 mL (5.64 log CFU/mL) and 30 mL (4.7

log CFU/mL).

These results indicate that volume has significant effect on the bactericidal

activity of individual TiO2 NPs. We expected the improved efficacy of the least effective

TiO2 sample (T1) by optimal light utilization at lower volume of 10 mL. However, as per

the current study, no significant difference in the log reduction was observed by

decreasing the suspension volume from 30 mL to 10 mL for least effective sample T1.

Even though it is beyond the scope of our study to understand the agglomerate size of

NPs in suspension, its effect on ROS generation potential of individual TiO2 samples

should not be ruled out. If agglomerates were formed even providing more light flux by

decreasing the volume of suspension it will not improve photocatalytic disinfection

efficacy. To support this hypothesis, Gumy et al. (2006) reported that out of several

surface properties, aggregate sizes of several commercial NPs in suspension played an

important role during the interfacial charge transfer between TiO2 and E. coli leading to

bacterial abatement. Agglomerated condition reduces effective surface area of NPs

available for bacteria to come in-contact with while the suspension is stirred during

99

photocatalytic disinfection. Further we observed, a minimum of a 20 mL volume of

suspension is necessary for effective mixing of NP and bacteria while conducting

bactericidal efficacy tests in suspension.

3.5. Comparison of photocatalytic oxidation and photocatalytic disinfection rates of TiO2

The photocatalytic degradation rate of MB by three commercial TiO2 samples is

shown in Fig 3.3. Under tested conditions, sample T3 was most efficient (26% decay)

followed by sample T2 (10%) and T1(1%) in the degradation of MB solution by

photocatalytic oxidation. Similar photocatalytic disinfection trends in TiO2 samples (T1,

T2 and T3) were observed for E.coli inactivation (Fig 3.3). This shows that the

photocatalytic degradation potential of organic compounds such as MB can be used as a

prior screening test to indirectly predict bactericidal efficacy of TiO2 NPs since the

mechanism of both the processes depend on ROS generation rate. Chen et al. (2009)

reported apparent correlation between the photocatalytic processes of decomposing

formaldehyde and inactivating E.coli. They noticed a similar trend with respect to key

parameters such as light intensity, initial concentration, and type of nanomaterial on the

effect of photodegradation and disinfection. This study concluded analogously that this is

potential method to evaluate the antimicrobial effect based on organic compound

degradation. Similarly, Marugan et al. (2010) reported that similarities between

photocatalytic degradation of chemicals and microorganism inactivation which were

agreeable only when analyzing the effect of operational variables such as catalyst

concentration or light intensity. However, different microbiological aspects (osmotic

stress, repairing mechanism, regrowth, bacterial adhesion to TiO2 surface, etc) makes

disinfection kinetics significantly much more complex than the oxidation of chemical

100

compounds (Marugan et al., 2010). Hence certain similarities and differences exist

between photocatalytic oxidation and photocatalytic disinfection. However,

photocatalytic degradation of organic dyes such as MB can be used a quick and easier

test to screen bactericidal TiO2 NPs for food safety applications prior to actual

microbiological tests.

4. Conclusions

Photocatalytic disinfection efficacy of three different commercial TiO2 NPs to

inactivate E.coli O157: H7 has been systematically investigated. Type and source of TiO2

has showed significantly effect bacterial log reduction. Among the tested commercial

TiO2 samples, T3 (Degussa P-25) was found to be the most efficient photocatalyst

followed by T2 and T1. The same trend has been observed for photocatalytic degradation

of MB solution. Increasing the number of bacterial cell washes from 1 wash to 3 washes

prior to photocatalytic disinfection treatment increased the log reduction of even the least

effective TiO2 samples. It is preferred to use less severe cell harvesting conditions for

accurate determination of bactericidal efficacy of TiO2 NPs. As expected, increasing the

light intensity increased the bactericidal efficacy all TiO2 samples. Volume of suspension

showed variable effect on the efficacy of tested TiO2 NPs. As per the current study, using

20 mL of suspension with single wash of cells at less severe harvest conditions and 2

mW/cm2 UVA intensity was found to be best testing protocol for evaluating bactericidal

efficacy of TiO2 NPs.

Acknowledgements

Funding for this study was provided by the Agriculture and Food Research

Initiative grant no 2011-68003-30012 from the USDA National Institute of Food and

101

Agriculture, Food Safety: food Processing Technologies to Destroy Food-borne

Pathogens Program- (A4131).

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Table 3.1. Characteristics of commercial TiO2 NPs

a Commercial TiO2 samples T1: Sky Spring Nanomaterial’s; T2: US Research

Nanomaterials; T3: Degussa P-25 from Aldrich

TiO2

samplea

Crystal phase Purity

(%)

Size

(nm)

Specific Surface area

(m2/g)

True density

(g/cm3)

T1 Anatase 99.5 10-25 50-150 NA

T2 Anatase >99 10-25 200-240 3.9

T3 Anatase- Rutile ≥99.5 ~21 35-65 NA

105

Table 3.2. Effect of light intensity and volume on bactericidal activity of TiO2 NPs

Variable Log reductiona (CFU/mL) after 3 h treatment

Intensity (mW/cm2) Over all T1 T2 T3

1 3.27B

2.04B

2.99B

4.77B

2 4.22A

3.04A

3.94A

5.67A

Volume (mL)

10 4.32A 3.02

A 4.30

A 5.64

A

20 3.55B 2.32

A 3.09

B 5.32

AB

30 3.36B

2.28A

3.01B

4.70B

a Mean values with the same superscript in the same column within the same

variable combination are not significantly different (p >0.05)

106

Fig 3.1. Schematic of photocatalytic disinfection set-up; 1. Wooden chamber; 2. UVA

light bulbs (40 W each); 3. Magnetic stirrers; 4. Glass petridish with stirbar; 5. Height to

adjust light intensity.

2

4

3

1

5

107

Fig 3.2. Effect of TiO2 source and bacterial cell harvesting conditions on the log

reduction.

UVA: Without NP UVA alone; T1: Sky Spring Nanomaterial’s; T2: US Research

Nanomaterials; T3: Degussa P-25.

0

1

2

3

4

5

6

7

0 30 60 90 120 150 180

UVA(1 and 3 washes)

T1: 1 wash

T2: 1 wash

T3: 1 wash

T1: 3 wash

T2: 3 wash

T3: 3 wash

Log

red

uct

ion

(C

FU

/mL

)

Treatment time (min)

108

Fig 3.3. Comparison of photocatalytic degradation of methylene blue and photocatalytic

disinfection rate of E.coli O157:H7 among different TiO2 NPs.

T1: Sky Spring Nanomaterial’s; T2: US Research Nanomaterials; T3: Degussa P-25

0

1

2

3

4

5

60.7

0.75

0.8

0.85

0.9

0.95

1

0 30 60 90 120 150 180

Log r

edu

ctio

n,

CF

U/m

L

Met

hyle

ne

blu

e d

ecay, C

/C 0

Treatment time (min)

T1

T2

T3

T1

T2

T3

Photo disinfection

Photo oxidation

109

CHAPTER 4

EFFECT OF FOOD PROCESSING ORGANIC MATTER ON PHOTOCATALYTIC

BACTERICIDAL ACTIVITY OF TITANIUM DIOXIDE (TIO2)2

2Veerachandra K. Yemmireddy and Yen-con Hung. International Journal of Food

Microbiology 204 (2015) 75-80. Reprinted here with permission of the publisher.

110

Abstract

The purpose of this study was to determine the effect of food processing organic matter

on photocatalytic bactericidal activity of titanium dioxide (TiO2) nanoparticles (NPs).

Produce and meat processing wash solutions were prepared using romaine lettuce and

ground beef samples. Physico-chemical properties such as pH, turbidity, chemical

oxygen demand (COD), total phenolics (for produce) and protein (for meat) content of

the extracts were determined using standard procedures. The photocatalytic bactericidal

activity of TiO2 (1 mg/mL) in suspension with or without organic matter against

Escherichia coli O157:H7 (5-strain) was determined over a period of 3 h. Increasing the

concentration of organic matter (either produce or meat) from 0% to 100% resulted in

85% decrease in TiO2 microbicidal efficacy. Turbidity, total phenolics, and protein

contents in wash solutions had significant effect on the log reduction. Increasing the total

phenolics content in produce washes from 20 to 114 mg/L decreased the log reduction

from 2.7 to 0.38 CFU/mL, whereas increasing the protein content in meat washes from

0.12 to 1.61 mg/L decreased the log reduction from and 5.74 to 0.87 CFU/mL. Also, a

linear correlation was observed between COD and total phenolics as well as COD and

protein contents. While classical disinfection kinetic models failed to predict, an

empirical equation in the form of “Y=menX

” (where Y is log reduction, X is COD and m

and n are reaction rate constants) predicted the disinfection kinetics of TiO2 in the

presence of organic matter (R2=94.4). This study successfully identified an empirical

model with COD as a predictor variable to predict the bactericidal efficacy of TiO2 when

used in food processing environment.

Keywords: TiO2; Bactericidal activity; Organic matter; Kinetics; E.coli O157:H7

111

1. Introduction

More than two thirds of all fresh water abstraction worldwide goes toward food

production (Kirby et al., 2003). From the primary production of food to subsequent

processing requires copious amounts of water. One challenge for the food industry is to

minimize water consumption and waste water discharge rates (Olmez and Kretzschmar,

2009). Current trends toward sustainable production practices necessitate food industry

to reuse the water after proper treatment. However, it should be noted that water serves as

a source of cross-contamination as reusing processing water may result in the buildup of

microbial loads, including undesirable pathogens from the crop (Gil et al., 2009). Several

recent outbreaks related to foods can be traced back to contaminated process wash water

and irrigation water with pathogens. This shows inadequacy of existing physical and

chemical disinfection technologies.

Among several water disinfection technologies, chlorination is the most

extensively used for the last three decades (Pigeot-Remy et al., 2012). However, studies

show that in many cases chlorinated water is not fully effective in reducing pathogens

(Zhang et al., 2009) and has potential to generate harmful chlorinated disinfection by-

products like trihalomethanes, haloacetic acids, haloketones, and chloropicrin in presence

of organic matter (Gil et al., 2009; Lopez-Galvez et al., 2010). Moreover, pathogens such

as viruses, protozoa, or helminthes are generally more resistant to chlorine than bacteria

by varying degrees (Kirby et al., 2003). Other commonly used treatments such as

ozonation and filtration also have certain inherent limitations. In this context, advanced

oxidation processes (AOPs) involving photocatalytic nanoparticles (NPs) are gaining

popularity as a viable alternative to existing disinfection technologies.

112

Among various photocatalysts, titanium dioxide (TiO2) has been extensively

studied in the last 25 years for its photocatalytic disinfection properties (Hitkova et al.,

2012). TiO2 photocatalysts generate strong reactive oxygen species (ROS) such as the

hydroxyl radical (·OH), superoxide radical (O2.-), and hydrogen peroxide (H2 O2) when

illuminated with UV light with a wavelength of less than 385 nm. The photogenerated

ROS has proven to exhibit excellent microbicidal activity and is responsible for

mineralization of organic compounds. TiO2 is non-toxic and has been approved by the

American Food and Drug Administration (FDA) for use in human food, drugs, cosmetics,

and food contact materials (Chawengkijwanich and Hayata, 2008). Bactericidal and

fungicidal effects of TiO2 on Escherichia coli, Salmonella choleraesuis, Vibrio

parahaemolyticus, Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus

aureus, Diaporthe actinidiae, and Pencillium expansum have been discussed by Foster et

al. (2011). Among several commercial and synthesized TiO2 NPs, Degussa P-25 is

considered as a standard for determining photocatalytic activity (Mills and Le Hunte,

1997).

Several studies in the past have explained the disinfection mechanism of TiO2

(Foster et al., 2011) and explored the effect of nanoparticle size, concentration, UV light

intensity, pH, bacterial cell concentration, inorganic salts, and model organic matter on

the disinfection properties of TiO2 (Rincon and Pulgarian, 2004). However, the effect of

food processing organic matter on the bactericidal activity of TiO2 NPs is not well

reported. In general, the majority of the research studies concerning the evaluation of

sanitizing agents on the reduction of pathogenic microorganisms during washing do not

take into account the presence of organic matter (Stopforth et al., 2008). When potable

113

water is used to evaluate different sanitizing agents, it might lead to unrealistic results

with no practical application (Gil et al., 2009). Meat and produce wash operations in food

processing industries release abundant phenolic, protein, and lipid rich organic matter

along with several viable or nonviable pathogenic and spoilage microorganisms. Any

study exploring the optimum conditions for inactivation of pathogens and the effect of

organic matter on photocatalytic disinfection properties of UV activated TiO2 would help

to apply these novel technologies in still unexplored sectors like food processing waste

water treatment. Also, identifying the disinfection mechanism in suspension consisting

organic matter would help to develop effective strategies while coating these NPs on

abiotic surfaces and packaging materials.

Hence, the overall objective of this study was to determine the effect of organic matter on

bactericidal activity of TiO2 NPs. Specific objectives include the following:

i) To determine the bactericidal efficacy of TiO2 in wash water rich in phenolic and

protein contents

ii) To identify the factors those are most useful to predict the disinfection potential

of TiO2 NPs in real food processing environment

2. Materials and Methods

2.1.Preparation of wash water containing organic matter

Wash waters rich in organic matter representing produce and meat processing

operations were used in this study. Romaine lettuce was purchased from a local

supermarket (Griffin, GA, USA) and stored at 4°C until use. Any wilted and damaged

outer leaves of the lettuce were removed and discarded while internal leaves were cut into

about 2.5 cm2 pieces using clean and sterile scissors. Subsequently, 50 g of lettuce were

114

placed into stomacher bags (Whirl Pak®) containing 200 mL sterile deionized water, and

the mixture was homogenized for 2 min in a stomacher (Seward Stomacher®, 80

biomaster, Worthing, UK). Ground beef samples were prepared by separating lean and

visible fat portions from primal cuts of beef chuck (ExcelTM

, Cargill Meat Solutions

Corporation, Wichita, KS, USA). The separated lean meat portions were ground in a

meat grinder (LEMTM

, Size #8, West Chester, OH, USA) to obtain a near 100% lean

ground beef samples. Later, A 10 g sample of ground beef at different lean to fat weight

ratios (100:0, 80:20, 60:40, 40:60, 20:80 and 0:100) were weighed into a stomacher bag

containing 200 mL sterile deionized water and homogenized as described earlier. The

resultant extracts were filtered through a sterile Whatman® filter paper (No. 2, 185 mm

diameter, 8 µm pore size) and further diluted by a 1:2, 1:3, and 1:4 factor of lettuce or

beef extract to deionized water in order to provide different levels of the organic load.

These solution were referred as produce (lettuce extracts), and meat (beef extracts) wash

solutions and kept at 4 °C in darkness prior to use.

2.2. Analysis of wash water properties

The physico-chemical properties such as pH, turbidity, COD, total phenolics (for

produce) and protein (for meat) contents of the wash solutions were determined. The pH

of the samples was determined using a pH meter (Model # AR50, Fischer Scientific,

Pittsburgh, PA, USA). The turbidity was measured using a turbidity meter (Model #

19952, HF Scientific, Fort Myers, FL, USA) and expressed as nephelometric turbidity

units (NTU). The COD was determined by following reactor digestion method (Jirka and

Carter, 1975). Briefly, 1 mL of an appropriate dilution of the sample was added to the

COD reagent vial (P/N# TT20711, Orbeco, Sarasota, FL, USA) and the contents were

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mixed thoroughly. The samples were digested for 2 h on a heating block preheated to 150

°C. The digested samples in the vials were cooled down to room temperature, and the

COD values were read on a colorimeter (Model # DR/890, HACH®, Loveland, CO,

USA) and expressed as mg/L.

Total phenolic content of produce wash solution was determined using the Folin-

Ciocalteu assay as outlined by Singleton and Rossi (1965). One milliliter of sample was

added to 70 mL deionized water in a 125 mL screw cap bottle then 5 mL Folin-

Ciocalteu’s phenol reagent (Sigma Aldrich Co., St Louis, MO, USA) was added to the

solution. After thorough mixing, 15 mL of 20% (w/v) sodium carbonate solution was

added followed by enough water to bring the total volume to 100 mL. The mixtures were

sealed and incubated for at least 2 h at room temperature. The samples were then read at

750 nm in a 1 cm quartz cuvette using a DU 520 UV/Vis spectrophotometer (Beckman

Coulter Inc., Brea, CA, USA). The total phenolic content of a test sample was calculated

using catechol as a standard and reported as mg/L.

Total protein content of meat wash solution was determined using the Bradford assay

(Bradford, 1976). Briefly, 0.1 mL of sample was mixed with 5 mL Bradford’s reagent

(Sigma- Aldrich Co., St Louis, MO, USA). The samples were then read at 595 nm in a 1

cm quartz cuvette using a DU 520 UV/Vis spectrophotometer mentioned earlier. The

total protein content of a test sample was calculated using bovine serum albumen as a

standard and reported as mg/L.




116


Becton Dickinson, Sparks, MD, USA) containing 20% glycerol. Prior to the experiment,

cultures were activated at least twice by growing them overnight in 10 ml of TSB at 37

°C. Later, each bacterial stain was cultured separately in 10 ml of TSB and kept on a

shaking incubator at 230 rpm and 37°C for 16 h. Following the incubation, bacterial cells

were harvested by sedimentation at 4000 xg for 12 min and re-suspended in a sterile

phosphate-buffered saline (PBS, pH 7.2), and equal volumes of each strain suspension

were combined to obtain 10 mL of a five strain cocktail containing approximately 108

CFU/mL. Cell concentration was adjusted by measuring the absorbance of bacterial

suspension at 600 nm using a UV/Vis spectrophotometer and confirmed by plating 100

µL portions of the appropriate serial dilution on tryptic soy agar (TSA) (Difco

Laboratories) plates incubated at 37 °C for 24 h.

2.4. Photocatalytic disinfection experiments

TiO2 NPs (Aeroxide® P25, Sigma-Aldrich, St. Louis, MO, USA) with a surface

area of 50 m2 g

-1 and a particle size of ~21 nm (as per supplier specifications) were used

in this study. Suspensions of TiO2 (1 mg/mL) in produce and meat wash solutions were

prepared by sonication in water-bath (Model # FS30, Fisher Scientific, Waltham, MA,

USA) for about 1 h at 23°C. Photocatalytic disinfection experiments were carried out by

adding 2 mL bacterial culture in 18 mL NP suspension at 2 mW/cm2 UVA light intensity

by following method of Yemmireddy and Hung (2015). Briefly, the procedure involves,

20 mL bacteria-NP suspension, which was added into a sterile glass petri-dish (90x18

mm2; diameter x depth) mounted on a magnetic stirrer (Model# H1190M, Hanna

Instruments, Smithfield, RI, USA) and illuminated with a UVA light system fitted with

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four 40 W lamps (American DJ®, Model UV Panel HP

TM, LL-UV P40, Los Angeles, CA,

USA) from the top under continuous stirring. The intensity of the light was measured

using UV radiometer (Peak sensitivity 365 nm, UVP®, Upland, CA, USA). A control

sample of bacterial culture suspended in wash water without photocatalyst under UVA

light was also included. All the experiments were conducted at room temperature using

indoor air as oxidant. A 1 mL sample was withdrawn from the treatment solution at every

1h for 3 h and added into 9 mL sterile PBS. Appropriate serial dilutions of the samples

were prepared, and the surviving bacteria from the control and treatments were

enumerated on Sorbitol Macconkey Agar (SMAC). The plates were incubated at 37 °C

for 24 h, and the colonies were counted and recorded as log CFU per mL. All the

experiments with produce wash were replicated five times and meat wash were

duplicated.

2.5. Kinetic models

The kinetics of photocatalytic bacterial inactivation is usually described using

empirical equations. The following five well-known disinfection kinetic models were

considered in order to find a best-fit model for the experimental results involving

photocatalytic bactericidal activity of TiO2 in the presence of organic matter:

The Chick-Watson model (Chick, 1908; Watson, 1908) with a constant

concentration of photocatalyst:

log (N/N0) = -k t (1)

where N/N0 is the reduction in bacterial concentration, k is the kinetic constant of

inactivation, and t is the treatment time.

118

The delayed Chick-Watson model (Cho et al., 2004) to accommodate if there

exists any initial lag time (t0) in the disinfection is computed as follows:

log (C/C0) = 0 for t ≤ t0

- k (t-t0) for t > t0 (2)

The modified Chick-Watson model (Cho et al., 2003) to accommodate either the

existence of a shoulder at the beginning of the reaction or a tail at the end of the reaction:

log (C/C0) = k1[1-exp(-k2t)] (3)

The Homs model (Hom, 1972) when the inactivation rate deviates log-linear

behavior is calculated as follows:

log (C/C0) = - k th (4)

Where h is the second parameter. If h=1, Homs model becomes a Chick-Watson linear

equation, h>1 for existence of a shoulder, h<1 for existence of a tail.

The modified Homs model (Cho et al., 2003) to accommodate shoulder, log-

linear, and tail regions is calculated as follows:

log (C/C0) = k1[1-exp(-k2t)]k3

(5)

To determine which model best described the data, the estimated coefficient of

determination (R2) and the F-value were calculated using the following equations:

Residual sum of squares

R2

= 1 - (6)

Uncorrected total sum of squares

Mean regression sum of squares

F

= (7)

Mean squared error

119


3.1. Kinetics of TiO2 disinfection and the effect of organic matter

Fig. 4.1 shows the results of the photocatalytic disinfection of E. coli O157:H7

using TiO2 aqueous suspensions with different levels of organic matter from meat and

produce extract solutions. TiO2 in suspension without organic matter has showed a

reduction of around 5.78 log CFU/mL after 3 h treatment. While, TiO2 suspended in meat

and produce organic matter extracts at 25% level of incorporation in the reaction mixture

showed a reduction of only 3.7 and 2 log CFU/mL, respectively. Further increasing the

organic matter concentration to 100% in the reaction mixture significantly reduced the

disinfection potential of TiO2 to below 1 log CFU/mL. This shows that increasing the

organic matter content in the reaction mixture has detrimental effect on the TiO2

bactericidal activity. This can be explained based on the premise that the process of

decomposing organic matters and photo-killing of microbes is perceived to follow the

similar mechanisms of the attack by ROS (Chen et al., 2009). However, the organic

matter present in the reaction mixture competes with bacteria for hydroxyl radical (OH.),

which is a major ROS responsible for the killing of bacteria and also hinders the

interaction between the bacteria and the TiO2 catalyst (Grieken et al., 2010). The same

phenomena might be the reason for decreased bactericidal activity of TiO2 in the current

study. However, the effect of specific components of organic matter in the meat and

produce extract solutions on the photocatalytic disinfection efficacy of TiO2 need to be

further investigated.

The photocatalytic disinfection kinetics of TiO2 with or without organic matter

has followed a non-linear trend with a shoulder (Fig. 4.1). The experimental data were

120

fitted with most commonly used empirical models that are described earlier. When the

reaction mixture is free from any organic matter, almost all the tested empirical models

were able to fit the experimental data well with an R2

value greater than 0.94 (Table 4.1).

When considering both R2 and F-statistic values to predict the goodness of fit, only the

modified Chick -Watson and the modified Homs model were able to give the best fit with

an R2

value of 0.98 and F-value of 274.81. However, due to lack of a tail region at the end

of photocatalytic disinfection treatment, modified Homs model is insignificant to fit the

data obtained from the current study. Hence, the modified Chick-Watson model to

accommodate initial lag or shoulder effect was found to be the most appropriate model to

predict the disinfection kinetics of TiO2. With the incorporation of organic matter like

produce or meat extract in the reaction mixture, none of the tested empirical models were

able to fit the TiO2 disinfection data well. Only, the modified Chick-Watson model was

able to predict the disinfection trend of TiO2 for up to 25% level of organic matter from

both meat (R2= 0.993) and produce (R

2 = 0.933) extracts (Table 4.1). However, the

modified Chick-Watson model failed to predict the TiO2 disinfection kinetics when

organic matter concentration was more than 25%. This further supports the hypothesis

that effect of individual components of organic matter need to be accounted to better

predict the disinfection kinetics of TiO2 in the presence of organic matter.

3.2. Effect of pH

The effect of pH of produce and meat extract solutions on the disinfection

potential of TiO2 was shown in Table 4.2. Decreasing the organic load in wash solutions

from 100% to 25% increased the log reduction of TiO2 from 0.54 to 2.07 for produce and

0.87 to 3.7 for meat extract solutions, respectively. Although, the pH values of produce

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and meat extract solutions were significantly different from each other, they are not

different within the same type of extract solutions at different levels of organic load. In

both types of extracts, even at same level of pH, the log reductions are significantly

different from each other. For example, produce extract solution at concentration of

organic load 50% and 75% with pH 6.2 showed significantly different reductions of 1.4

and 0.74 log CFU/mL, respectively. This implies that under tested conditions, pH of the

solutions containing organic matter alone does not have an effect on the disinfection

potential of TiO2. Gumy et al. (2006) reported that the electrostatic attraction between the

E. coli and the Degussa P-25 TiO2 is not a controlling factor in the pH range of 4.5 to 6.0

since E. coli is negatively charged between pH 3 and 9 and TiO2 is positively charged up

to pH 7. In another study by Rincon and Pulgarin (2004), modification of pH of TiO2

suspension did not show any effect on the E.coli inactivation rate in the pH range of 4

and 9. Similarly, the pH range (5 to 6.27) of produce and meat extract solutions used in

the current study may not have an effect on the photocatalytic disnfection efficacy of

TiO2.

3.3. Effect of Turbidity

The effect of turbidity of produce and meat extract solutions on the disinfection

potential of TiO2 was shown in Fig. 4.2. Increasing the turbidity of produce extract from

36 to 148 NTU decreased the log reduction from 2.08 to 0.54 CFU/mL. Similarly,

increasing the turbidity of meat extract solutions from 17 to 50 NTU decreased the log

reduction from 3.7 to 0.38 CFU/mL. This shows that increasing the turbidity of wash

solutions decreased the bactericidal efficacy of TiO2. Turbidity caused by the presence of

components leaching from tissues of the cut produce surface and meat, is a measure of

122

the waters ability to scatter and absorb light, which depends on a number of factors such

as size, number, shape, and refractive index of the particles and the wave length of

incident light (WHO, 1996). The photogenerated ROS, such as hydroxyl radical are

highly active for both the oxidation of organic substances and the inactivation of bacteria

(Kim et al., 2003). Both the bacteria and the organic matter present in the suspension

compete for the ROS generated through photocatalytic disinfection process. This

condition reduces the disinfection potential of TiO2 to inactivate bacteria. In addition,

increasing the turbidity of the reaction mixture decreases the penetration power of UVA

light into the solution and limits the ability of TiO2 NPs to generate ROS. Selma et al.

(2008) studied the turbidity effect of various fresh-cut vegetable wash waters on the

disinfection potential of TiO2. Their study reported that differences in water turbidity

were associated with different bacterial inactivation rate. Onion wash water with highest

turbidity (5040 NTU) has least bacterial inactivation rate and carrot wash water with

lowest turbidity (0.6 NTU) has highest inactivation rate. However, lettuce (87.4 NTU),

escarole (95.7 NTU), chicory (42.4 NTU) and spinach (88.9 NTU) wash waters with

intermediate level of turbidity have showed lower bacterial inactivation. In our study,

upon gradual decrease in the lean to fat ratio of meat extract solution from 100:0 to 0:100

resulted in almost 18% to 40% decrease in the turbidity (results not shown). However, no

significant increase in the bactericidal activity of TiO2 was observed. This clearly shows

that turbidity itself is not a rate limiting factor and the presence of other components of

the organic matter such as fat content may affect the bactericidal efficacy of TiO2. The

presence of components such as protein and fat in the reaction mixture might have

blocked the surface active sites on TiO2 NPs to generate ROS and reduced the efficiency

123

of photocatalytic disinfection process. This implies that the efficacy of the photocatalytic

system will be highly depend on the physicochemical characteristics of the suspension

containing organic matter and increasing the turbidity of the suspension reduced the

photocatalytic inactivation rate of bacteria.

3.4. Effect of total phenolics and its correlation with COD

Fig. 4.3 presents the effect of total phenolics content in the produce extract on

photocatalytic bactericidal activity of TiO2. Total phenolics content in the suspension

showed significant effect on the bacterial inactivation. For example, increasing the total

phenolics content in the suspension from 20.4 to 113.6 mg/L decreased the log reduction

from 2.7 to 0.38 CFU/mL. The reduction trend can be best fitted with an exponential

equation in the form of Y = A eBX

(where Y is the log reduction in CFU/mL, X is the total

phenolics in mg/L, and A and B are constants) with an R2 value of 0.943. Also, a linear

correlation was observed between total phenolics and COD of the produce extract

solution (Fig 4.3). It followed a regression trend of Y=40.22X-220.77 (where Y = COD in

mg/L, X = Total phenolics in mg/L) with a correlation coefficient 0.928. One possible

reason for the decreased photocatalytic activity of TiO2 can be attributed to the increased

concentration of phenolic compounds in the suspension. Phenolic compounds such as

tocopherols, flavonoids, and phenolic acids are well known for their antioxidant activity.

In general, these compounds inhibit or delay the oxidation of other molecules by

inhibiting the initiation or propagation of oxidizing chain reactions. TiO2 photocatalysis,

which involves series of oxidation and reduction reactions, is highly dependent on the

generation of ROS. The phenolic compounds present in the produce extract might have

quenched the generated ROS by irradiated TiO2 NPs, which in turn reduced its efficacy to

124

inactivate bacteria. Rincon and Pulgarin (2004) reported a significant decrease in the

TiO2 photocatalytic inactivation of E. coli in the presence of organic compounds such as

dihydroxybenzenes, hydroquinone, catechol, and resorcinol. They reported that the

formation of an optical screen on TiO2 surface by organic and inorganic components for

light penetration as well as competition of organic compounds for OH radicals are some

reasons for decreased photocatalytic efficacy. Similar phenomena can be attributed to the

decreased bactericidal activity of TiO2 in the presence of phenolic-rich organic matter

used in the current study.

3.5.Effect of protein and its correlation with COD

The effect of protein content in meat extract on the log reduction was shown in

Fig. 4.4. As expected, increasing the protein content from 0.12 to 1.61 mg/L in the

reaction mixture decreased the log reduction from 5.74 to 0.84 in mg/L. The reduction

trend can be represented with an exponential equation in the form of Y = A eBX

(where Y

is the log reduction in CFU/mL, X is the protein content in mg/L, and A and B are

constants) with R2 value of 0.904. Like phenolics, a linear correlation between protein

and COD of the meat extract was noticed with an R2

value of 0.725 (Fig 4.4). Variable

proportions of lean to fat ratios (100:0 to 0:100), and the relative complexity of meat

extract might be one possible reason for the distorted trend and poor correlation of the

protein with log reduction and COD. In general, TiO2 NPs tend to agglomerate in

aqueous solutions in the absence of agitation. The presence of organic matter rich in

protein further enhances the formation of agglomerated NPs in suspension irrespective of

agitation. In addition, it is possible that the fat molecules present in the meat extract

forms an outer layer on the surface of TiO2 NPs, which results in blockage of surface

125

active sites for the photocatalytic reaction to takes place and subsequent generation of

ROS. Gumy et al. (2006) reported that out of several surface properties, the aggregate

size of several commercial NPs in suspension played an important role during the

interfacial charge transfer between TiO2 and E. coli leading to bacterial abatement.

Agglomerated condition reduces the effective surface area of NP available for bacteria to

come incontact with while stirring the suspension during photocatalytic disinfection. The

same might be the reason for the decreased bacterial inactivation rate of TiO2 in the

presence of organic matter rich in protein. However, further studies need to be conducted

to understand the effect of individual components on photocatalytic disinfection

mechanism of TiO2 in complex food systems such as meat extract.

3.6. Effect of COD

Although, total phenolics and protein contents are reasonably good in predicting

the bactericidal efficacy of TiO2 in the presence of organic matter, using a common factor

such as the COD might be practically more beneficial. As discussed before, COD of

produce and meat extract solutions had a linear correlation with phenolics (R2= 0.92) and

protein contents (R2= 0.72), respectively. Hence, COD can be used as a predictor variable

to determine the kinetics of TiO2 bacterial inactivation in the presence of organic matter.

Fig. 4.5 shows the correlation between the COD of the produce or meat extract solutions

and the log reduction of E.coli O157:H7. Increasing the COD values of both meat and

produce extracts decreased the log reduction. Experimental data from both meat and

produce extract solutions were best fitted with an empirical model in the form of Y= menX

(where Y is the log reduction, X is the COD of organic matter, and m and n are reaction

rate constants) (Table 4.3). A study conducted by Selma et al, (2008) on different types of

126

produce wash waters reported that onion wash water with highest COD was associated

with the least bacterial reduction after treatment with the photocatalytic system.

According to these results, it appears that the inactivation data can be better correlated

with the COD of organic matter in the suspension. TiO2 photocatalytic action was

attributed to the promotion of peroxidation of phospholipid components of the lipid

membrane, inducing cell membrane disorder, subsequent loss of essential functions such

as respiratory activity, and cell death (Ibanez et al, 2003). The generation of hydroxyl

radical induced by UV radiation rapidly overcomes the self-protection mechanisms of the

bacterial cell, and as a result microbial counts decrease exponentially. In the last period of

photo-treatment, the rate of microbial inactivation becomes slower because OH radicals

produced by the irradiated TiO2 act against the few active bacteria remaining in the UV-

irradiated water but also against the inactivated bacteria and the metabolites released

during the photocatalytic treatment (Rincon and Pulgarian, 2003). A similar mechanism

can be attributed to the decrease in photocatalytic bactericidal efficacy of TiO2 NPs in the

presence increasing levels of organic matter.

4. Conclusions

The results of this study showed that the presence of organic matter from both

produce and meat extract solutions has a significant effect on the bactericidal efficacy of

TiO2. Under tested conditions, the pH level of the produce and meat wash solutions had

no significant effect on the bactericidal activity of TiO2, whereas turbidity, COD, total

phenolics, and protein content had a significant effect on the bactericidal efficacy of

TiO2. A linear correlation was observed between COD and total phenolics as well as

COD and protein content. While classical disinfection models failed to predict, an

127

empirical equation with COD as predictor variable successfully fit the experimental data.

The empirical equation proposed in this study helped to predict the photocatalytic

disinfection efficacy of TiO2 in the presence of food processing organic matter.

Acknowledgments

Funding for this study was provided by the Agriculture and Food Research

Initiative grant no 2011-68003-30012 from the USDA National Institute of Food and

Agriculture, Food Safety: Food Processing Technologies to Destroy Food-borne

Pathogens Program-(A4131).

128

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Table 4.1. Comparison of kinetic models to predict the TiO2 disinfection efficacy with or

without organic matter

Type Model R2

F-statistic

TiO2 without organic matter Chick-watson 0.947 194.98

TiO2 without organic matter Delayed Chick-Watson 0.948 200.56

TiO2 without organic matter Hom's model 0.985 233.28

TiO2 without organic matter Modified Hom's model 0.982 274.81

TiO2 without organic matter Modified Chick-Watson 0.982 274.81

TiO2 with 25% produce extract Modified Chick-Watson 0.933 125.69

TiO2 with 25% meat extract Modified Chick-Watson 0.993 417.94

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Table 4.2. Effect of pH of wash solution containing organic matter on

the bactericidal activity of TiO2

Type Organic load

(vol%)

pHa

Reductiona at 3 h

(Log CFU/mL)

Produce 100 6.11A

0.54E

75 6.20A 0.74

E

50 6.20A

1.40CD

25 6.27A 2.07

B

Meat 100 5.30B 0.87

DE

75 5.04B 1.04

CDE

50 5.08B 1.60

CB

25 5.07B 3.70

A

a Mean values with the same superscript within the same column are

not significantly different (p >0.05).

133

Table 4.3. Comparison of fitted isotherm parameters of empirical model

Model equation Y= m e nX

; where Y= Log reduction (CFU/mL),

X = COD (mg/L), and m and n are reaction rate constants.

Type of organic matter m n R2 F -statistic

Produce 3.5181 -0.00065 0.972 315.37

Meat 7.7101 -0.00157 0.960 266.64

Combined 6.7116 -0.00134 0.944 353.92

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Fig 4.1. Effect of different levels of organic matter from produce and meat

extract solutions on the log reduction of E.coli O157:H7 by TiO2

photocatalysis.

0

1

2

3

4

5

6

7

0 30 60 90 120 150 180

Log r

educt

ion (

CF

U/m

L)

Time (min)

TiO2 without organic matter TiO2 with 25% produce wash

TiO2 with 100% produce wash TiO2 with 25% meat wash

TiO2 with 100% meat wash UVA only

135

Fig 4.2. Effect of turbidity of produce and meat extract solutions on the

log reduction of E.coli O157:H7 by TiO2 photocatalysis for 3h.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 15 30 45 60 75 90 105 120 135 150

Log r

educt

ion (

CF

U/m

L)

Turbidity (NTU)

Produce extract

Meat extract (100% lean)

136

Fig 4.3. Correlation between total phenolics and COD of produce extract as well

as total phenolics and log reduction of E.coli O157:H7 by TiO2 photocatalysis.

Y = 4.175 e-0.026X

R² = 0.943

y = 40.22x - 220.77

R² = 0.928

0

1000

2000

3000

4000

5000

0

0.5

1

1.5

2

2.5

3

20 30 40 50 60 70 80 90 100 110 120

CO

D (

mg/m

L)

Log r

educt

ion (

CF

U/m

L)

Total Phenolics (mg/L)

137

Fig 4.4. Correlation between protein content and COD of meat extract as well as

protein and log reduction of E.coli O157:H7 by TiO2 photocatalysis.

Y = 6.2625e-2.8211X

R² = 0.904

Y = 1088.5X + 403.1

R² = 0.725

0

500

1000

1500

2000

2500

0

1

2

3

4

5

6

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8

CO

D (

mg/L

)

Log r

educt

ion (

CF

U/m

L)

Protein (mg/L)

138

Fig 4.5. Relationship between COD of produce and meat organic matter

extracts and the log reduction of E.coli O157:H7 by TiO2 photocatalysis.

0

1

2

3

4

5

6

0 500 1000 1500 2000 2500 3000 3500 4000 4500

Log r

educt

ion (

CF

U/m

L)

COD (mg/L)

Produce extract

Meat extract

139

CHAPTER 5

METHOD DEVELOPMENT FOR CREATING TITANIUM DIOXIDE (TIO2)

NANOCOATINGS ON FOOD CONTACT SURFACES AND METHOD TO

EVALUATE THEIR DURABILITY AND PHOTOCATALYTIC BACTERICIDAL

PROPERTY3

3Veerachandra K. Yemmireddy, Glenn D. Farrell and Yen-con Hung. Submitted to

Journal of Food Science, 2/9/2015.

140

ABSTRACT: Titanium dioxide (TiO2) is a well-known photocatalyst for its excellent

bactericidal property under UVA light. The purpose of this study was to develop

physically stable TiO2 coatings on food contact surfaces using different binding agents

and develop methods to evaluate their durability and microbicidal property. Several types

of organic and inorganic binders such as polyvinyl alcohol, poly ethylene glycol,

polyurethane, polycrylic, sodium and potassium silicates, shellac resin and other

commercial binders were used at 1:1 to 1:16 nanoparticle to binder weight ratios to

develop a formulation for TiO2 coating on stainless steel surfaces. Among the tested

binders, polyurethane, polycrylic, and shellac resin were found to be physically more

stable when used in TiO2 coating at 1:4 to 1:16 weight ratio. The physical stability of

TiO2 coatings was determined using adhesion strength and scratch hardness tests by

following standard ASTM procedures. Further, wear resistance of the coatings was

evaluated based on a simulated cleaning procedure used in food processing environments.

TiO2 coating with polyurethane at a 1:8 nanoparticle to binder weight ratio showed the

highest scratch hardness (1.08 GPa) followed by coating with polycrylic (0.68 GPa) and

shellac (0.14 GPa) binders. Three different techniques, namely direct spreading, glass

cover-slip, and indented coupon were compared to determine the photocatalytic

bactericidal property of TiO2 coatings against E.coli 0157:H7 at 2 mW/cm2 UVA light

intensity. Under the tested conditions, the indented coupon technique was found to be the

most appropriate method to determine the bactericidal property of TiO2 coatings.

Keywords: TiO2 coating; Food contact surface; Antimicrobial test; Binding agent;

Physical stability, E.coli.

141

Practical Application: A simple approach to create physically stable and bactericidal

TiO2 nanocoatings was developed on food contact surfaces of stainless steel using

different binding agents. The developed TiO2 nanocoatings might help to minimize

microbial cross-contamination and ensure safe food processing environment.

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Introduction

Microbial-cross contamination is a major issue in the food processing

environment leading to several foodborne outbreaks and illnesses in the recent times.

Cross contamination through both food contact and non-food contact surfaces such as

knifes, cutting boards, working surfaces, equipment and the processing environment is

well reported. Also, increased popularity of fresh and minimally processed foods poses

additional risk of cross-contamination from food contact and non-food contact surfaces

(Lloret and others 2012).

Conventional sanitation and disinfection procedures that are widely followed in

the industry are not sufficient to address the emerging risks of microbial cross-

contamination involving resistant pathogens. In addition, many of the established

sanitizers and disinfectants lack residual effect for extended period of protection and

generate toxic disinfection by-products (Meylheuc and others 2006). Modification of

surfaces with antimicrobial agents to prevent the growth of harmful microorganisms has

received much attention for several industrial applications (Rai and others 2010). In this

context, nanotechnology based advanced oxidation processes involving photocatalytic

titanium dioxide (TiO2) nanoparticles (NPs) have shown great promise as an effective

non-targeted disinfectants for killing a wide range of microorganisms.

Over the last decade, there is an increased interest in the application of TiO2

photocatalytic disinfection technique for the purpose of food safety and quality

enhancement (Manreet and Hayata 2006; Chawengkijwanich and Hayata 2008;

Chorianopoulos and others 2011). TiO2 has been recognized as the most promising

photocatalyst due to its appropriate electronic band structure, photostability, chemical

143

inertness, low cost, ready availability, and capable of repeated use without substantial

loss of catalytic activity (Ibhadon and Fitzpatrick 2013). TiO2 has been approved by the

American Food and Drug Administration (FDA) for use in human food, drugs, cosmetics,

and food contact materials (Chorianopoulos and others 2011). TiO2 photocatalysts

generate strong oxidizing power when illuminated with UV-A light of wavelength less

than 385 nm. The bactericidal properties of TiO2 were attributed to the high redox

potential of the reactive oxygen species (ROS) such as hydroxyl radical (.OH),

superoxide radical (O2.-), and hydrogen peroxide (H2O2) formed by the photo-excitation

(Foster and others 2011). Several techniques have been proposed to immobilize TiO2 NPs

on hard surfaces for the purpose of photocatalytic disinfection (Visai and others 2011).

Sol-gel synthesis of NPs and subsequent dip, spin, or spray coating is the most

widely reported procedure in the literature. Other coating methods such as

electrochemical deposition, electrophoretic coating, chemical vapor deposition,

sputtering, and plasma spraying were complicated and costly for practical application

(Kasanen and others 2011). Alternatively, a simple direct coating of the NPs using wet

chemical approaches were also reported. However, poor adhesion on the substrate and the

lack of physical stability of the developed coatings is the major issue when using direct

coating methods (Mills and Lee 2002; Keshmiri and others 2004; Han et al, 2012). Thus,

binding agents are usually necessary for direct coating in order to form strong adhesion

between the NP and the substrate. Either organic polymer or inorganic binding materials

have been used for photocatalyst immobilization (Du and others 2008; Lim and others

2009; Henderson and others 2011). However, no extensive stability studies have been

conducted in the past to evaluate the durability of the nanocoatings when they are

144

intended to use in food processing environment. In addition, several methods have been

proposed to determine the bactericidal activity of photocatalytic nanocoatings with each

having their own advantages and disadvantages.

For use in food safety applications, the antimicrobial coatings expected to have

characteristics such as long lasting efficacy, ease of fabrication, durability, and no

toxicity. With appropriate binding agents, physically stable and durable TiO2

nanocoatings with strong bactericidal property can be developed on food contact

surfaces. Hence, the main goal of this study was to identify appropriate binders to create

stable TiO2 nanocoating on stainless steel surface and identify methods to evaluate

physical stability and bactericidal property of TiO2 nanocoating. Specific objectives

include:

i) To identify the most promising binders to create TiO2 coatings on stainless steel.

ii) To develop a simple method for coating TiO2 NPs on stainless steel.

iii) To evaluate the physical stability and the durability of the TiO2 coatings.

iv) To identify a suitable testing method to determine the bactericidal property of

TiO2 coatings.

Materials and Methods

Selection of TiO2 NPs and Binders for coating

TiO2 (Aeroxide®

P25, Sigma-Aldrich, St. Louis, MO, USA) NPs with an

approximate particle size of 21 nm and specific surface area of 50 m2 g

-1 as per suppliers

specifications were used for developing nanocoatings in this study. Ten different types of

polymeric, silicate, and resin based organic and inorganic binding agents: (i) Polyvinyl

alcohol (PVA) (TCI America, Portland, OR, USA), (ii) Polyethylene glycol (PEG) (TCI

145

America, Portland, OR, USA), (iii) Ludox AS-40 (LAS-40) (Sigma-Aldrich, Co., St.

Louis, MO, USA), (iv) Two types of potassium silicates (PS1& PS6) (KASIL®

1 &

KASIL®

6, PQ Corporation, Valley Forge, PA, USA), (v) Two types of sodium silicates

(SSO & SSN) (O® & N

®, PQ Corporation, Valley Forge, PA, USA), (vi) Ceramabind 830

(C830) (Aremco Products, Inc., Valley Cottage, NY, USA), (vii) Ceramabind 643-1

(C643-1) (Aremco Products, Inc., Valley Cottage, NY, USA), (viii) Water based oil-

modified polyurethane (B) (Miniwax®, Miniwax company, Upper saddle river, NJ,

USA), (ix) Water based polycrylic (C) (Miniwax®, Miniwax company, Upper saddle

river, NJ, USA), (x) Shellac (A) (Zinsser Co., Inc. Somerset, NJ, USA) were tested to

evaluate their feasibility to incorporate in coating solution to achieve physically stable

TiO2 coatings.

Substrate selection and preparation

Stainless steel (Type 304, finish #2B, 25 mm2) coupons were used as model food

contact surface for coating. Prior to coating, each coupon was slightly roughened to

increase coating adhesion and achieve a high level of bond strength by using an electric

sander fitted with a P100 fine grit sand paper for 1 min on each side of the coupon. Later,

the surface roughened coupons were degreased first by washing in acetone followed by

ethanol and finally rinsed with deionized water. The cleaned stainless steel coupons were

dried in a hot air oven at 60°C for 30 min before used for coating.

Screening of binders for developing stable TiO2 coating

Several preliminary experiments were conducted in order to select binding agents

that are most suitable to develop physically stable TiO2 coatings on stainless steel. In the

first stage of experiments, TiO2 coatings were prepared by using two types of organic

146

binders PVA and PEG. Several suspensions of TiO2 NPs mixed with PVA or PEG

binders at 1:1 to 1:5 NP to binder weight ratios were prepared by using ethanol as

solvent. Stainless steel coupons were dip coated with the resultant suspensions using an

Instron (Model #5544, Instron Corporation, Canton, MA, USA) operated with a dipping

speed of 10 mm/s, residence time of 10 s and a withdrawal speed of 0.5 mm/s. In this

manner single or multiple coatings of TiO2 were applied on each coupon based on the

viscosity of coating suspension and uniformity of the coated film. The coated coupons

were dried in a hot air oven at 60 °C for 1 h. The dried coupons were visually inspected

for coating uniformity and washed under running water for about 5 to 10 min in order to

quickly assess the adherence behavior and physical stability of the coatings. Even though

heat treatment after coating helped to increase the adherence of the coating to stainless

steel surface, heat treatment also resulted in formation of clumps on the TiO2

nanocoating. The results of these experiments showed that the TiO2 nanocoatings with

PVA and PEG as binders are non-uniform in nature and unable to withstand washing

under running water (data not shown).

In the second stage of coating experiments, inorganic binders such as Ludox AS-

40 (LAS-40), potassium (PS1 and PS6) and sodium (SSO and SSN) silicates from a

commercial source as well as two other commercial binders of unknown composition

(C830 and C643-1) were used for TiO2 nanocoating. Twenty different paste formulations

(5 different binders at 4 different NP to binder ratios) were prepared for coating by

mixing TiO2 NPs with each binder at 1:1 to 1:4 NP to binder weight ratios. About 1 g

(±0.15) of the TiO2 paste was weighed and painted on each coupon using a Crayola paint

brush so as to form a layer of uniform TiO2 coating. TiO2 coatings with potassium and

147

sodium silicates (PS1, PS6, SSO & SSN) and C830 were air-dried at room temperature

for about 1 h; while the TiO2 coatings with LAS-40 and C643-1 were air-dried at room

temperature for 2 h and then cured at 93°C for 1 h in a hot-air oven as per the

manufacturer guidelines. Increasing the concentration of binder (i.e. less NPs to binder

ratio), increased the viscosity of TiO2 pastes and physical stability of the resultant

coatings. TiO2 coatings with potassium and sodium silicates at 1: 4 weight ratio was

found to be physically more stable upon scratching with pencil points of 2H hardness.

However, these coatings were not stable upon washing in running water followed by

sonication in water bath for 15 min. Based on these results it was found that the TiO2

coatings with tested inorganic binders helped to achieve uniform and physically stable

coatings (data not shown). However, TiO2 coatings with these binders are not suitable for

application in moist conditions encountered in food processing environment.

In the third stage of coating experiments, polymer based sealers polyurethane (B)

and polycrylic (C), as well as a natural resin, shellac (A), were tested for their feasibility

to incorporate in TiO2 coatings. The composition of different TiO2 coatings with binders

A, B, and C are presented in Table 5.1. Based on the nature of each binder, the viscosity

of coating solution increased with decreasing NP to binder ratio to a point where it is not

feasible for coating. Hence, the reported compositions in Table 5.1 were selected to

achieve feasible viscous suspensions for coating. Suspensions for coating were prepared

by mixing TiO2 NPs with binders A, B, and C at 1:4 to 1:16 (TiO2: Binder) weight ratios

in a mortar for about 15 min. Stainless steel coupons were then dip coated with the

resultant suspensions as described earlier. The coated coupons were air-dried over night

at room temperature. These coated coupons were found to be uniform and physically

148

stable upon scratching with pencil points of 2H hardness. Also, washing under running

water as well as sonication in water bath for about 15 min did not remove the coating

from the substrate significantly. Hence, TiO2 coatings with the binders A, B, and C were

selected for further studies to evaluate their physical stability.

Surface characteristics of TiO2 nanocoatings

In order to maintain consistency in use of samples for evaluating both physical

stability and bactericidal properties of the nanocoatings, an indented stainless steel

coupon having dimensions 46 x 12.5 x 1.25 mm3 and surface area of 540 mm

2 were used

for the TiO2 coatings with the binders A, B, and C, respectively. A sample of 0.25 g of

the coating solution at 1: 4 to 1:16 NP to binder weight ratio were poured into the well of

stainless steel indentation to form a uniform layer of TiO2 coating. The coated coupons

were dried in air overnight at room temperature. This approach of coating is referred as

solution deposition technique. The thickness of the coatings was measured by using a

thickness gauge (Elcometer® 345) at eight different locations on each coupon. Film

morphology and microscopic structure of the coating surface was characterized by a

variable pressure scanning electron microscope (VPSEM, Zeiss 1450 EP) with

accelerating 25 kV. The SEM images were further analyzed using an image processing

software (Paint. NET) to estimate the area ratio of coated surface covered by the NPs vs

the binder.

Measurement of physical stability of TiO2 nanocoatings

The hardness of the coatings was evaluated with the help of a scratch test based

on the ASTM G171-03 method (ASTM, 2009). Briefly, Instron fitted with a

hemispherical diamond tip indenter of 76.5 µm having a conical apex angle of 120°

149

(J&M Diamond Tool, Inc., Rumford, RI, USA) and an anti-vibration table was used for

this test. Three linear scratches of at least 5 mm length at 2 mm apart from each other

were made on each coating with an applied load of 1N to 3N. The width of each scratch

was measured at three different locations equidistance from each other using a digital

microscope pro (Celestron LLC, Model # 44308). Scratch hardness number (HSp) was

calculated as per the standard using following equation:

HSp = kP/w2

Where

HSp is the scratch hardness number (GPa)

K is the geometrical constant (24.98)

P is the applied normal force (grams-force)

W is the scratch width (µm)

In addition, adhesive strength of the coatings was evaluated using a scotch tape

test based on the ASTM D3359-02 test method B (ASTM, 2002). Six parallel cuts of

about 20 mm length at 2 mm apart were made through the coating in one steady motion

using a straight edged metal guide and a sharp razor blade as described in ASTM D3359-

02. Similarly, another six cuts were made through the coating at a 90̊ angle to the

previous cuts to make a lattice pattern of small squares of about 0.5 x 0.5 mm2

dimensions on the coating. Later, about 25 x 50 mm2 pressure-sensitive adhesive tape

(Permacel 99, Permacel, New Brinswick, NJ, USA) was applied over the lattice pattern

and smoothed into the place by using a pencil eraser over the area of the incisions to

ensure good contact with the coating. Adhesive tape was then removed by pulling it off

rapidly with a constant force at close to a 180° angle. The possible crumbling at the edges

150

of the cuts is a measure of the coating adhesion strength and is ranked from 5B to 0B

according to the descriptions and illustrations provided in the ASTM standard (Table

5.2).

A cleaning procedure commonly used in food processing environment was simulated

by means of an in-house developed reciprocating test (Fig 5.4). In this test set-up, a

texture analyzer (TA. XT Plus™

, Texture Technologies Corp, Scarsdale, NY, USA) was

fitted with a moving head consisting of scrubby side of sponge (3M Scotch-Brite™

,

Heavy-Duty scrub sponge, St. Paul, MN, USA) as four individual brushes to simulate

cleaning procedure (Fig 5.4b). Briefly, the weight of each coated coupon was measured

using a calibrated balance before subjecting to cleaning procedure. Later, a 2 mL of

diluted detergent solution (Dawn Ultra™, Procter & Gamble, Cincinnati, OH, USA) was

poured onto each TiO2 coated coupon. The coupons were then subjected to cleaning

procedure using the test set-up described earlier at a moving head speed of 20 mm/s for

up to 500 cycles (i.e. 1000 to-and-fro motions) with an applied load of 1 N on each

coupon. The cleaned coupons then were air-dried overnight at room temperature and the

weight of each coupon was measured again. The difference in the weights of TiO2 coated

coupons before and after cleaning was measured to determine the wear resistance of the

TiO2 coatings.

Measurement of bactericidal property of TiO2 nanocoatings

In order to select most appropriate test method to determine photocatalytic

bactericidal activity TiO2 nanocoatings, three different techniques: (i) Direct spreading,

(ii) Glass cover-slip, and (iii) Indented coupons were investigated. TiO2 coating with

binder A on stainless steel coupons (25 mm2) were used in direct spreading and glass

151

cover-slip techniques; while indented stainless steel coupons (540 mm2) as described

earlier were used for the indented coupon method. Further, E.coli has been widely studied

bacteria in several of the photocatalytic disinfection experiments involving TiO2 NPs.

However, the susceptibility of pathogenic strains of E.coli to photocatalytic disinfection

is not well reported. As a reason, a five strain cocktail of E. coli O157: H7 isolated from

different sources: E009 (beef), EO932 (cattle), O157-1 (beef), O157-4 (human), and

O157-5 (human) was used as a test pathogen in this study. Each bacterial stain was

cultured separately in 10 mL of tryptic soy broth (TSB) (Difco, Becton Dickinson,

Sparks, MD, USA) and kept on a shaking incubator at 230 rpm and 37°C for 16 h.

Following the incubation, bacterial cells were harvested by sedimentation at 4000 x g for

12 min and re-suspended in a sterile phosphate-buffered saline (PBS, pH 7.2). An equal

volume (2 mL) of each strain suspension was combined to obtain a 10 mL of a five strain

cocktail containing approximately 107 CFU/mL bacterial cells. Cell concentration was

adjusted by measuring the absorbance of bacterial suspension at 600 nm using a UV/Vis

spectrophotometer and confirmed by plating 100 µL portions of the appropriate serial

dilution on tryptic soy agar (TSA) (Difco Laboratories) plates incubated at 37 °C for 24

h.

Prior to antibacterial activity tests, TiO2 coated coupons were pre-sterilized under

UVC light for 1 h in a bio-safety cabinet. The sterile coupons were placed in a 90 mm

diameter petri-dish containing moist filter paper to maintain humidity during the

treatment. Bacterial culture was inoculated on each TiO2 coupon as follows: (i) Direct

spreading method: A drop of 100 µL inoculum was spread on the surface of TiO2 coating

using a sterile loop based on the direct spreading technique, (ii) Glass cover-slip method:

152

A drop of 100 µL inoculum was spread on the surface of TiO2 coating as before and then

a glass cover-slip of same size as stainless steel coupon was placed on top of the bacterial

culture, (iii) Indented coupon method: A 300 µL inoculum was pipetted into the well of

indented TiO2 coated coupon to cover entire indented coated surface. The samples were

then illuminated from above with a UV-A light system (American DJ, Model UV Panel

HP ™, LL-UV P40, Los Angeles, CA, USA) at 2 mW/cm2 intensity. The intensity of

light reaching the surface of the coating was measured using a UV radiometer (UVP®,

Upland, CA, USA). Plain stainless and only binder coated stainless steel coupon under

UV-A light were used as negative and positive controls. After 2 h UV treatment, TiO2

coated coupons were immersed in 10 mL or 30 mL (for indented coupon technique) PBS

solution containing 0.1% tween 80 and vortexed for 30 s to re-suspend the bacteria. A

viability count was performed by appropriate dilution and plating on E.coli O157:H7

selective Sorbitol MacConkey agar (SMAC) and incubation at 37°C for 24 h. All the

experiments were conducted in triplicate.

Statistical analysis

Data were analyzed by the analysis of variance (ANOVA) procedure using Statistical

Analysis System (SAS/STAT 9.3, 2011). T-tests were used for pairwise comparisons.

Least significant difference of means tests was done for multiple comparisons, and all

tests were performed with a level of significance 0.05.

Results and Discussion

Effect of binders on TiO2 coatings

Fig. 5.1 shows the images of different TiO2 coatings with binders A, B, and C

prepared by suspension deposition technique. As seen in the figure, all the nanocoatings

153

were uniform and strongly adhered to the stainless steel substrate. We found that

increasing the binder concentration in the coating resulted in smoother surfaces with

fewer visible aggregates as seen in the image of sample TB16 when compared with

sample TB8. Also, no cracks were formed on the TiO2 coating with binders A, B, and C.

However, TiO2 coating with binder A at 1:4 NP to binder weight ratio (i.e., TA4) showed

formation of cracks along the edges of the coating. As the concentration of binder A

further increased (TA8), no visible cracks were noticed on the coating. Thickness of TiO2

coatings with binders A, B, and C at different NP to binder weight ratios were shown in

Table 5.3. Thickness of nanocoatings ranged from 50 to 107 µm. In general, increasing

the concentration of binder in the coating decreased the thickness of nanocoatings. At the

same NP to binder composition (for example at 1:8 NP to binder weight ratio), thickness

of TiO2 coating with binder C (97 µm) was found to be significantly higher than the

thickness of TiO2 coatings with binders A (74 µm) and B (51 µm). The difference in the

thickness of these nanocoatings may be attributed to the differences in the viscosity of

coating solutions formulated using different binding agents and the relative proportion of

NPs to binder concentration in each type of nanocoating. Li and others (2009) reported

that the thickness of TiO2 membranes developed with PVA binder on stainless steel

decreased with decreasing the molar concentration of TiO2 in the casting solution. They

found that the thickness and micro-pores of the coatings can be controlled by simply

adjusting the concentration of casting solutions instead of applying multiple coats.

Similar results were reported by Cerna and others (2011) for TiO2 coated layers with

varying levels of PEG. Since photocatalytic antimicrobial activity of TiO2 is a surface

dependent phenomenon due to generation of ROS, the thickness of the coating becomes

154

insignificant. However, thickness of the coating may have a significant effect on the

physical stability and other structural properties of the TiO2 coatings.

Surface characteristics of TiO2 coatings

Fig. 5.2 shows scanning electron micrographs of surface of different TiO2

coatings. SEM images give us a detailed look at appearance of the deposited coatings at a

micro level. At the same level of NP to binder concentration, TiO2 coatings with binders

A, B, and C have shown completely different structural characteristics as seen in Fig. 5.2.

TiO2 coating with binder A (TA8) is more compact in nature with aggregated clumps on

the surface (Fig 5.2b). While, the surface of TiO2 coating with binder B (TB8) is compact

with several microscopic pores throughout the coating (Fig 5.2c). Whereas, TiO2 coating

with binder C (TC8) resulted in a compact structure without aggregated clumps with

larger but fewer number of pores on the surface (Fig 5.2e). Inset of the respective SEM

images of TA8, TB8, and TC8 shows the structural arrangement of the TiO2 coatings at

nanoscale. Upon analyzing these SEM micrographs at nanoscale to estimate the surface

coverage of NP vs binder showed a 40, 21, and 39 % coverage of binder and 60, 79, and

61% coverage of TiO2 NPs for coatings TA8, TB8, and TC8, respectively (Fig 5.2).

However, the actual number of TiO2 NPs that are exposed on the surface of the coating

were just a fraction of total percent coverage of TiO2 NPs. For example in Fig 5.3, if we

analyze the magnified image of TiO2 coating with binder C (TC8) at nanoscale; about 39

% of the coating surface was covered with the binder (dark black region), 58 % of the

surface was covered by the unexposed TiO2 NPs which are partly shielded by the binder

particles (blurred grey region), and rest of the 3 % of the surface was covered by the

exposed TiO2 NPs (bright white spots).

155

Further increasing the concentrations of binders B and C in the coating (i.e.

TB16 and TC16) resulted in a more compact surface structure with fewer number of

pores as shown in Figs. 5.2(d), and 5.2(f). This shows that the type and the amount of

binder used in the coating has a significant effect on the morphological and structural

properties of the TiO2 coating. This phenomenon is more obvious when ready-made TiO2

NPs were mixed with different binding agents for coating. Also, it is expected to generate

some irregularities and non-uniformity in the surface of coating while using the solution

deposition technique in the indented stainless steel coupon. However, the results of this

study help to prove the concept of developing durable antimicrobial nanocoatings on food

contact surfaces using appropriate binding agents.

Physical stability of TiO2 coatings

Different test procedures were adopted in order to estimate the physical stability

of the TiO2 coatings for use in food processing environment. Adhesion strength of the

TiO2 coatings was determined by following ASTM D3359-02 standard and the results

were reported in Table 5.3. TiO2 coatings with binder B (TB8 or TB16) showed the

highest adhesion rating of 4B. Here, rating 4B indicates that less than 5 % of the coating

has been removed from the surface as represented in Table 5.2. On the other hand, TiO2

coating with binder C at 1:16 NP to binder weight ratio (TC16) showed the lowest

adhesion strength of 2B which means more than 65% of the coating has been delaminated

from the surface. Further, increasing the concentration of NPs in the coating formulation

to 1:8 NP to binder weight ratio (TC8) significantly enhanced the adhesion strength of the

coating up to 4B. A similar trend was observed for TiO2 coating with binder A (TA8 or

TA4). This indicates that depending upon the type of binder, there exists an optimum

156

concentration of NPs and binder in the coating in order to achieve highest adhesion to the

substrate. For the tested binding agents in this study, a coating suspension at a

concentration of 1:8 NP to binder weight ratio was found to be the optimum for

exhibiting the highest adhesion strength.

The hardness of the coating was determined as per ASTM G171-03 standard

using a scratch resistance test. Preliminary experiments were conducted to determine the

maximum normal force that can be applied on the surface of TiO2 coating and a normal

force of 2 N was found to be the optimum to determine and compare the scratch

resistance of different TiO2 coatings developed in this study. Control samples with only

binder coating failed to withstand the scratch resistance test. Scratch hardness of TiO2

coatings ranged from 0.14 GPa for sample TA8 to 1.08 GPa for sample TB8 (Table 5.3).

TiO2 coatings with binder B showed highest scratch hardness followed by coatings with

binder C and A, respectively. Scratch resistance of the TiO2 coatings developed in this

study using different binders were found to be comparatively much higher than the

chemical vapor deposited TiO2 coatings on stainless steel substrate which was 6.5 GPa at

40 mN (Sobczyk-Guzenda and others 2013) and sol-gel dip coated TiO2 coatings on

polycarbonate sheets which was 0.5±0.04 GPa at 25 µN (Yaghoubi and others 2010).

Wear resistance of the TiO2 nanocoatings after simulated cleaning procedure was

reported in Table 5.3. The weight loss (mg) after 1000 cycles of simulated cleaning

procedure was expressed as wear resistance. The weight loss of the TiO2 nanocoatings

after wear testing ranged from 1.53 (for TC16) to 14 (for TB8) mg. TiO2 coatings with

binder C had the highest wear resistance (less weight loss) followed by TiO2 coatings

with binders A and B, respectively. In general, increasing the binder concentration in the

157

coating increased the wear resistance. However, the difference is not statistically

significant (p<0.05) except for TB8. In addition, subjective analysis of the coatings after

wear testing revealed that all the TiO2 nanocoatings looked physically very stable with

slight scratch marks on the surface. However, after the wear test was followed by drying,

formation of cracks and peeling-off of the coatings from the substrate was noticed for

TB8. This might be attributed to the weak intermolecular bonds between the NPs and the

binder at 1:8 NP to binder weight ratio for TB8.

Effect of test method on bactericidal activity results of TiO2 coatings

Shellac (binder A) is a food-grade natural resin most commonly used in the food

industry for several applications. Also, based on the structural characteristics of the TiO2

coatings with binder A, no significant difference in the total coverage of TiO2 NPs was

observed with decreasing NP concentration in the coating (Fig. 5.2). In addition, these

coatings exhibited good physical stability on stainless steel surface. For this reason, TiO2

coating with binder A was selected as a representative nanocoating to identify a suitable

testing method to determine the bactericidal property. The most widely reported direct

spreading and glass cover-slip techniques were compared with an indented coupon

technique developed in this study. The results of antimicrobial activity of TiO2 coatings

are shown in Table 5.4. Under tested conditions, negative control samples (plain stainless

steel coupons) and positive controls (binder coated stainless steel coupons) under UV-A

light showed a reduction in between 1.5 to 2.5 log CFU/cm2. No significant difference

(p>0.05) in the reduction among control samples was observed for direct spreading, and

indented coupon techniques. This shows that the binder coating itself (based on positive

control results) had no significant antimicrobial property and the observed reduction was

158

only be attributed to the effect of UV-A light. However, a significant difference in the

reduction between the two different control samples was observed in case of glass cover-

slip technique. Also, it should be noted that there was no significant difference (p >0.05)

in reduction between positive control and the TiO2 coatings at different NP

concentrations for the glass cover-slip technique. For the other two methods, TiO2

coatings showed significantly higher microbial reductions when compared with control

samples. This shows that the glass cover-slip technique may not be suitable for the

determination of bactericidal property of TiO2 nanocoatings, especially when TiO2

coatings were created using a binder. This can be explained based on two possible

reasons: i) A cover slip on the inoculated coupon helps to achieve uniform coverage of

bacterial cells on the surface of the nanocoating. However, it also inhibits the presence of

catalyst such as atmospheric oxygen which otherwise plays an important role in the

heterogeneous photocatalysis involving TiO2 to generate ROS, ii) The amount of surface

occupied NPs were limited when nanocoatings were prepared by mixing with a binding

agent as explained in the surface characteristics of nanocoatings in this study (Fig. 5.2).

In such a case, a cover-slip on the inoculated nanocoating promotes only localized

reactions on the coated surface and reduces the efficacy of photocatalytic bactericidal

property of the nanocoating. In addition, leakage of inoculated bacterial culture from the

sides of the coupon is difficult to avoid by using a cover-slip technique. Similar,

concerns has been expressed by Mills et al (2012) and they suggested using an alternative

approach such as a simple well system into which a standard volume of the bacterial

suspension is applied to the sample which lies at the bottom of the well.

159

Increasing the concentration of NPs in the TiO2 coating from 1:16 to 1:8 NP to

binder weight ratio did not significantly increase in the reduction observed using the

direct spreading technique. As per the SEM image analysis results of this study, it is

expected to achieve higher log reduction for coatings with more NPs due to more surface

coverage by TiO2 NPs. However, this did not happened using the direct spreading

technique. This may be due to non-uniform coverage of the inoculum on the entire

surface of TiO2 coating when using direct spreading technique. Whereas, the indented

coupon technique showed a significant increase (p<0.05) in the microbial reduction by

increasing the concentration of NPs in the coating (TA16 vs TA8). Even though there is

was no significant difference in the reduction within the same sample among the three

tested techniques, the indented coupon technique helped to achieve more consistent

results by minimizing variations in the determination of TiO2 antimicrobial property. A

similar technique has been used by Cushnie et al (2010). Mills et al (2012) reported that

the advantage of this type of approach is that it allows the bacterial suspension to be

accurately deployed to a known area of surface under investigation. As per our

observation the major benefits of using the indented coupon technique are: i) to achieve

uniform coverage of inoculated bacterial cells on the entire surface of the coating, ii) to

minimize the sample to sample variation and hence decreases the standard deviation, iii)

to achieve more available surface area, and iv) to allow the presence of oxygen for

efficient photocatalytic disinfection to takes place. Under tested conditions, the results of

current study suggest that the indented coupon technique is a more appropriate method to

determine bactericidal efficacy of photocatalytic TiO2 coatings.

160

Conclusions

This study has identified three promising binding agents to develop physically

stable TiO2 coatings on food contact surfaces. Image analysis of the coated surfaces

revealed that increasing the binder concentration in the coating decreased the exposed

TiO2 NPs on the surface which may reduce the bactericidal property of TiO2 coatings.

TiO2 coatings with polyurethane as a binder showed the highest scratch resistance

followed by coating with polycyclic and shellac, respectively. TiO2 coatings with

polyurethane and polycrylic at 1:8 NP to binder weight ratio showed the highest adhesion

to the substrate. Overall, TiO2 coating with polycrylic showed the highest physical

stability followed by nanocoating with polyurethane and shellac. An indented coupon

technique was found to be the most appropriate to test the bactericidal property of TiO2

coatings. Follow up studies need to be conducted to determine optimum conditions to

exhibit the highest bactericidal property by the developed TiO2 coatings under repeated

use conditions.

Acknowledgments

Funding for this study was provided by Agriculture and Food Research Initiative

grant no 2011-68003-30012 from the USDA National Institute of Food and Agriculture,

Food Safety: Food Processing Technologies to Destroy Food-borne Pathogens Program-

(A4131).

Author Contributions

Authors V. K. Yemmireddy and Yen-Con Hung designed experiments and wrote

the manuscript. V. K. Yemmireddy performed all the experiments and conducted data

161

analysis. Author Glenn D. Farrell provided technical assistance in designing and

fabricating instruments for physical stability assessment.

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Table 5.1. Composition of different TiO2 nanocoatings

1TiO2 nanocoatings with binders A, B, and C.

2Binders A, B, and C are shellac, polyurethane, and polycyclic, respectively.

3Composition of the coating suspension.

Sample code1 Type of binder

2 Composition

3 (weight basis)

TiO2 NPs Binder

TA4 A 1 4

TA8 A 1 8

TB8 B 1 8

TB16 B 1 16

TC8 C 1 8

TC16 C 1 16

165

Table 5.2. ASTM D3359-02 classification of adhesion test results

166

Table 5.3. Physical stability results of TiO2 nanocoatings with different binders

Coating type1 Thickness (µm)

Hardness2(GPa) Adhesion rating

3

Wear resistance4 (mg)

TA4 107 ±17.34a

0.15±0.02d

3B 5.53±0.86b

TA8 74 ±11.67c

0.14±0.11d

3B 3.47±1.94b

TB8 51 ±8.09c

1.08±0.25a

4B 14.0±2.03a

TB16 50 ±7.04c

0.88±0.11ab

4B 5.18±2.87b

TC8 97 ±2.35ab

0.68±0.08bc

4B 1.67±0.83b

TC16 56 ±8.98c

0.55±0.06c

2B 1.53±0.29b

Mean values with the same superscript in the same column are not significantly different

(p >0.05). 1TiO2 coatings with binders A, B, and C at 1:4 to 1:16 NP to binder weight ratio.

2Scratch hardness number at 2 N based on ASTM G171-03 method.

3Adhesion rating (5B: Superior; 0B: Inferior) based on ASTM D3359-02 method-B.

4Weight loss in mg after subjecting to simulated washing procedure.

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Table 5.4. Bactericidal activity of TiO2 nanocoatings using different test

methods

Treatment

Log reduction (CFU/cm2) at 2 mW/cm

2 for 2 h

Direct

spreading

Glass

cover-slip

Indented

coupon

Negative control1

2.42±0.04b

1.47±0.27b

2.19±0.10c

Positive control2

2.45±0.39b

2.15±0.32a

2.31±0.15c

TA16 3.31±0.48a

2.96±0.64a

3.04±0.07b

TA8 3.15±0.47a

2.76±0.60a

3.57±0.48a

Mean values with the same superscript in the same column are not significantly

different (p >0.05). 1Plain stainless steel coupon under UVA.

2Only binder A coated coupon under UVA.

3TiO2 coating with binder A at 1:16 NP to binder weight ratio.

4TiO2 coating with binder A at 1:8 NP to binder weight ratio.

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Fig 5.1. Images of TiO2 nanocoatings with shellac (A), polyurethane (B), and polycrylic

(C) binders at different NP to binder concentrations. Where, TA4 (TiO2 coating with

binder A at 1:4 NP to binder weight ratio), TA8, TB8, TC8 (TiO2 coating with binder A,

B, and C at 1:8 NP to binder weight ratios), TB16, TC16 (TiO2 coating with binder B and

C at 1:16 NP to binder weight ratios).

169

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170

Fig 5.3. SEM image of TiO2 coating with binder C at 1:8 NP to binder weight ratio (TC8)

showing regions of binder, surface exposed TiO2 NPs, and unexposed TiO2 NPs that are

partly covered by the binder.

171

Fig 5.4. In-house fabricated wear resistance tester (a) Cleaning heads fitted to a texture

analyzer to perform reciprocating motion (b) Enlarged image of cleaning heads and

platform to fit coated coupons in place.

172

CHAPTER 6

EFFECT OF BINDER ON THE PHYSICAL STABILITY AND BACTERICIDAL

PROPERTY OF TITANIUM DIOXIDE (TIO2) NANOCOATINGS ON FOOD

CONTACT SURFACES4

4Veerachandra K. Yemmireddy and Yen-con Hung. Food Control (2015) doi:

10.1016/j.foodcont.2015.04.009. Reprinted here with permission of the publisher.

173

Abstract

TiO2 is a promising photocatalyst for use in food processing environment as an

antimicrobial coating. The purpose of this study was to determine the effect of different

binding agents on the physical stability and bactericidal property of TiO2 nanocoatings

created on stainless steel surfaces. A total of six different coating suspensions were

prepared by mixing TiO2 (Aeroxide®

P-25) nanoparticles (NPs) with three different types

of binders (Shellac (A), polyuretahne (B), and polycrylic (C)) at a 1:4 to 1:16 NP to

binder weight ratio. Bactericidal activity of these TiO2 coatings against Escherichia coli

O157:H7 (5-strain) was determined at three different UV-A light intensities (0.25, 0.50

and 0.75 mW/cm2) for 3 h. The type of binder used in the coating had a significant effect

on the log reduction of E.coli O157:H7. TiO2 coatings with binder C showed highest

reduction (> 4 log CFU/cm2) followed by TiO2 coating with binder B and A. Increasing

the binder concentration in the formulation from a 1:4 to 1:16 weight ratio decreased the

log reduction of E.coli O157:H7. Increasing the UV-A light intensity from 0.25 to 0.75

mW/cm2 increased the log reduction of bacteria for all the TiO2 coatings. The physical

stability of the TiO2 coatings was determined using ASTM procedures. TiO2 coatings

with binder B showed highest adhesion strength and scratch hardness when compared to

coatings with other binders. However, on repeated use experiments (1, 3, 5, and 10

times), TiO2 coatings with binder C were found to be physically more stable and able to

retain their original bactericidal property. The results of this study showed promise in

developing durable TiO2 coatings with strong photocatalytic bactericidal property on

food contact surfaces using appropriate binding agents to help ensure safe food

processing environment.

174

Keywords: TiO2; Antimicrobial coating; Physical stability; Binders; E. coli O157:H7.

Highlights:

Most efficient TiO2 coating achieved more than 4 log reduction of E.coli

O157:H7.

Type of binder used in the coating has a significant effect on the log reduction.

Increasing the UVA intensity increased the bactericidal efficacy of TiO2 coatings.

TiO2 coating with polycrylic as binder showed the highest physical stability.

175

1. Introduction

Titanium dioxide (TiO2) is a well-known photocatalyst with excellent

antimicrobial properties under UV-A light. It is widely utilized as a self-cleaning and

self-sterilizing material for surface coatings in many applications (Fujishima, 2000). TiO2

is stable, non-toxic, cheap, and capable of repeated use without substantial loss of

catalytic ability. TiO2 photocatalysts have been added to paints, cements, windows, tiles

or other building products due to its sterilizing and anti-fouling properties (Lan et al.,

2013). Decontamination occurs under ambient conditions utilizing natural oxygen

without forming any photo-induced intermediates (Chong et al, 2010). In addition, TiO2

has been approved by the American Food and Drug Administration (FDA) for use in

human food, drugs, cosmetics, and food contact materials (Maneerat & Hayata, 2006).

Since Matsunaga et al. (1985) reported the application of photocatalysis for the

destruction of Lactobacillus acidophilus, Saccharomyces cerevisiae, and Escherichia coli

using platinum-loaded TiO2, there has been increased interest in the biological

applications of this process. TiO2 photocatalysts have been studied extensively to

inactivate a broad spectrum of microorganisms including viruses, bacteria, fungi, and

algae as well as to kill cancer cells (Kim et al., 2003). Foster et al. (2011) presented a

more comprehensive review on photocatalytic antimicrobial properties of TiO2. TiO2

photocatalysts generate strong oxidizing power when illuminated with UV-A light of

wavelength less than 385 nm. The bactericidal properties of TiO2 are attributed to the

high redox potential of the reactive oxygen species (ROS) such as hydroxyl radical (.OH),

superoxide radical (O2.-), and hydrogen peroxide (H2O2) formed by the photo-excitation.

TiO2-mediated photo-oxidation shows promise for the elimination of microorganisms in

176

areas where the use of chemical cleaning agents or biocides is ineffective or is restricted

by regulations such as pharmaceutical and food industries (Skorb et al., 2008). In

addition, TiO2 becomes superhydrophilic upon irradiation with UV light and this

functionality is reversible and depends on the light exposure (Chen & Mao, 2007). These

properties of TiO2 may help to improve the efficiency of hydrophilic cleaning agents

used in the food industry. Thus, TiO2 photocatalysts offer great potential to develop

antimicrobial coatings on food contact and non-food contact surfaces to avoid cross-

contamination in the food processing environment.

Studies have reported that immobilized TiO2 coatings have the ability to disinfect

Listeria monocytogenes biofilms on stainless steel (Chorianopoulos et al., 2011). Also,

TiO2 coated polypropylene film package can reduce the growth of E. coli on cut lettuce

(Chawengkijwanich & Hayata, 2008), and Pencillium expansum fruit rot on apples and

tomatoes (Manreet & Hayata, 2006). However, most of the earlier studies that reported

antimicrobial activity of TiO2 nanocoatings either used complicated approaches for

coating or did not fully address the issues of durability of the coatings on usage. In our

previous study on nanocoatings, we developed a simple method to create physically

stable TiO2 coatings on stainless steel surfaces using shellac, polyurethane and polycrylic

as binding agents (Yemmireddy et al., 2015). For developing antimicrobial TiO2

nanocoatings on food contact surfaces for the purpose of maintaining a hygienic food

processing environment, the binding agents used must be non-toxic. Shellac is an insect-

produced natural resin most commonly used in food industry for surface

treatment/glazing of confectionary products and citrus fruits to prevent surface damage

during handling and storage (Antic et al., 2010). According to FDA, shellac is only

177

approved for indirect food contact use (21 CFR 175.300). However, it is allowed for food

contact use due to acceptance petition for GRAS status (Baldwin, 2005). Shellac films

show excellent adhesion to a wide variety of surfaces and possess high gloss, hardness

and strength. Alternatively, polyurethane has been extensively studied for several

industrial applications. Notably, waterborne polyurethanes are suitable for paints,

coatings, and adhesive industries due to their inherent advantages of low volatile

compounds, fast drying properties, outstanding flexibility, impact resistance, abrasion

resistance, non-flammability, transparency and easy adherence to a variety of substrates

(Bhargava et al., 2013). As per FDA (21 CFR 177.1680), polyurethane resins are allowed

to use as indirect food additives for use as basic components of single and repeated use

food contact surfaces. Similarly, polycrylics are well known for their wide range of

applications in several paint formulations. As per our earlier study, TiO2 coatings created

using these binders have shown excellent physical stability. However, the photocatalytic

bactericidal property of TiO2 coatings using these binders is not well understood. Hence,

the overall objective of this study was to determine the effect of different binding agents

on physical stability and bactericidal property of TiO2 nanocoatings. Specific objectives

include: To determine:

i) The effect of binder on bactericidal property of TiO2 nanocoatings.

ii) The optimum conditions to create TiO2 nanocoatings with strong bactericidal

property

iii) The durability and bactericidal property of TiO2 nanocoatings on repeated use.

178

2. Materials and methods

2.1 Selection of materials

TiO2 (Aeroxide® P25, Sigma-Aldrich, St. Louis, MO, USA) NPs with an

approximate particle size of 21 nm and specific surface area of 50 m2 g

-1 as per suppliers

specifications were used for developing nanocoatings in this study (Table 1). Three

different binders namely, shellac (A), polyurethane (B) and polycrylic (C) were

purchased from the local supermarket in Griffin, GA (Table 1). Stainless steel (AISI

304L) coupons having an indentation with 46 x 12.5 x 1.25 mm3 dimensions and a total

surface area of 540 mm2 were chosen as a model food contact surface for TiO2

nanocoating. All the coupons were thoroughly cleaned prior to coating first by washing in

acetone followed by ethanol and finally rinsed with deionized water and dried in a hot air

oven at 60̊ C for 30 min.

2.2. Preparation of suspensions for TiO2 coating

Total six different suspensions of TiO2 were prepared by mixing TiO2 NPs with

binder A (1:4 or 1:8 weight ratio), binder B (1:8 or 1:16 weight ratio), and binder C (1:8

or 1:16 weight ratio) in a porcelain mortar for about 15 min. The produced viscous

suspensions were further treated in an ultrasonic water bath (Model # FS60, Fisher

Scientific, Waltham, MA, USA) for about 1 h, in order to avoid formation of TiO2

aggregates. The resultant viscous paste formulations were used for coating on indented

stainless steel coupons.

2.3. Preparation and characterization of TiO2 nanocoatings

TiO2 nanocoatings were created on indented stainless steel coupons by following the

method described in Yemmireddy et al. (2015). Briefly, a sample of 0.25 ± 0.02 g of

179

coating suspension was weighed into the well of an indented stainless steel (SS) coupon

by placing it on a calibrated balance. The deposited coating suspension was evenly spread

across the entire area of the indentation by slowly tilting the coupon sideways or if

needed using a Crayola paint brush by keeping total amount of deposited coating

constant. The coated coupons were air-dried over night at room temperature. The

resultant coatings has a thickness of about 50-100 µm when measured using a handheld

thickness gauge (Elcometer, Model # 345). The morphology and the microscopic

structure of the coating surface was characterized by a variable pressure scanning

electron microscope (VPSEM, Zeiss 1450 EP) with accelerating 25 kV. The SEM images

were further analyzed using image processing software (Paint. NET) to estimate the area

of the coated surface covered by the NPs and the binder.





Becton Dickinson, Sparks, MD, USA) containing 20 % glycerol. Prior to the experiment,

cultures were activated at least twice by growing them overnight in 10 mL of TSB at 37

°C. Later, each bacterial strain was cultured separately in 10 mL of TSB and kept on a

shaking incubator at 230 rpm and 37°C for 16 h. Following the incubation, bacterial cells

were harvested by sedimenting at 4000 x g for 12 min and re-suspended in a sterile

phosphate-buffered saline (PBS, pH 7.2). An equal volume (2 mL) of each strain

suspension was combined to obtain a 10 mL of a five-strain cocktail containing

approximately 106 CFU/mL. Cell concentration was adjusted by measuring the

180

absorbance of bacterial suspension at 600 nm using a UV/Vis spectrophotometer and

confirmed by plating 100 µL portions of the appropriate serial dilution on tryptic soy agar

(TSA) (Difco Laboratories) plates incubated at 37 °C for 24 h.

2.5. Photocatalytic disinfection

Prior to photocatalytic disinfection, the TiO2 coated coupons were pre-sterilized

under germicidal UV light (254 nm) in a biosafety hood for about 1 h. The sterilized

coupons were placed in 90 mm diameter petri-dishes containing moistened filter paper at

the bottom to prevent drying-out of the bacterial culture during the treatment. A 300 µL

aliquot of bacterial culture was pipetted into the indented well of the TiO2 coated coupon

and uniformly spread across the entire surface of the TiO2 coating using a sterile

disposable loop. Later, the inoculated samples were illuminated with a UV-A light

system fitted with four 40 W lamps (American DJ®, Model # UV Panel HP

TM, LL-UV

P40, Los Angeles, CA , USA) from above. The light intensity reaching on top of the

sample was measured using a UV radiometer (UVP®, Upland, CA, USA) with a peak

sensitivity of 365 nm. The light intensity reaching the surface of the sample was adjusted

to 0.25, 0.5 or 0.75 mW/cm2 (±0.05) by changing the distance between the light source

and the sample. Plain SS and only binder coated SS coupons under UV-A light were also

included as negative and positive controls, respectively. The samples were treated for

either 90 or 180 min UV-A light and then immersed in 30 mL of sterile PBS solution

containing 0.1% tween 80 and vortexed for 30 s to re-suspend the bacteria. A viability

count (log CFU/cm2) was performed by appropriate dilution and plating on E.coli

O157:H7 selective Sorbitol-MacConkey agar (SMAC) and incubation at 37 °C for 24 h.

All the experiments were conducted in triplicates.

181

2.6. Measurement of coating physical stability

Hardness of the TiO2 coatings were assessed with the help of a scratch test, based

on ASTM G171-03 method (ASTM, 2009) as described in Yemmireddy et al. (2015) to

make a linear scratch of at least 5 mm length with an applied normal force of 2 N at three

different locations on each sample. The width of each scratch was measured at three

different locations equidistance from each other using a digital microscope pro (20 to

200x magnification, Model # 44308, Celestron LLC,Torrance, CA). Scratch hardness

number (HSp) was calculated as described in the standard and reported in Giga Pascals

(GPa). Further, adhesion strength of the coatings was determined with the help of a tape

test based on ASTM D3359-02 method-B (ASTM, 2002) as described in Yemmireddy et

al. (2015).

2.7. Simulation of repeated use conditions of TiO2 coatings

In order to determine whether the coatings were able to retain their original

bactericidal property and physical stability upon reuse, the coatings were subjected to

multiple use conditions. In this procedure, the coatings were subjected to photocatalytic

disinfection test conditions as described earlier such as pre-sterilization under germicidal

UV light for 1 h followed by photocatalytic disinfection treatment under UV-A light for 3

h and removal of bacterial cells from the coatings using release buffer for 30 sec were

simulated for 1, 3, 5, and 10 times using deionized water in place of actual bacterial

culture. After each treatment cycle the coupons were air-dried before proceeding to the

next cycle. Finally, the dried coupons after 1, 3, 5, and 10 times simulated use were

measured for their bactericidal property and the physical stability as described earlier.

182

2.8. Statistical analysis

Data were analyzed by the analysis of variance (ANOVA) procedure using Statistical

Analysis System (SAS/STAT 9.3, 2011). T-tests were used for pairwise comparisons.

Least significant difference of means tests was done for multiple comparisons, and all

tests were performed with a level of significance 0.05.


3.1. Effect of type and concentration of binder on the bactericidal activity of TiO2

nanocoatings

Fig. 1 shows the effect of type of binder on the log reduction of E.coli O157:H7

produced by TiO2 nanocoatings treated for 3 h at 0.5 mW/cm2 UV-A light intensity.

Control samples with plain stainless steel coupons, and only binder A, B, and C coated

coupons under UV-A light showed a reduction on E.coli O157:H7 population of only

0.17, 0.24, 0.51, and 2.23 log CFU/cm2, respectively. In addition, when these binder

coated coupons were tested in the dark, both binder A and B coatings showed no

significant antibacterial activity; while, binder C coating showed a reduction of less than

1 log CFU/cm2 (data not shown). This shows that under tested conditions, both binder A

and B coatings themselves had no significant bactericidal property. However, binder C

under the tested UVA intensity showed a significant (P ≤0.05) bacterial reduction. This

might be attributed to the possible inherent bactericidal properties of acrylic paint (i.e.

binder C) and its constituents in the presence or absence of UV light. TiO2 coatings with

binders A, B, and C at 1:8 NP to binder weight ratio showed a reduction of 0.96, 3.72,

and 3.92 log CFU/cm2, respectively (Fig 1). Further increasing the concentration of

binders A, B, and C in the TiO2 coating (1:16 NP to binder weight ratio) showed a

183

reduction of only < 0.5 log CFU/cm2 for coating with binder A (TA16, data not shown),

1.73 log CFU/cm2 for binder B (TB16) and 3.35 log CFU/cm

2 for binder C (TC16) (Fig.

1). This is almost a 100, 54 and 15 % decrease in the bactericidal efficacy of TiO2

coatings with binders A, B and C when compared with respective TA8, TB8, and TC8

samples. Alternatively, decreasing the concentration of the binder in the TiO2 coating to

1:4 NP to binder weight ratio (TA4) resulted in an almost 49 % increase in the

bactericidal efficacy (0.95 to 1.45 log CFU/cm2) when compared to TiO2 coating at 1:8

NP to binder weight ratio (TA8). However, TiO2 nanocoatings with binders B and C at a

1:4 NP to binder weight ratio is not a feasible formulation for coating by the solution

deposition technique used in this study. This indicates that the type of binder used in the

TiO2 coating had significant (P ≤0.05) effect on the photocatalytic bactericidal property.

One possible reason for the differences in the antimicrobial activity can be attributed to

the differences in the surface characteristics of the individual TiO2 nanocoatings created

with three different binders.

SEM image analysis of the coatings revealed that the number of TiO2 NPs present

on the surface of each coating varied depending on the type of binder used (Table 2). For

example, in a given area of nanocoating, the amount of TiO2 NPs exposed on the surface

of coating was only 3, 2, and 3 % for TA8, TB8, and TC8, respectively. While, the

corresponding binder coverage was 39, 21, and 39 %, respectively. The remaining

percent coverage of the nanocoating can be attributed to the unexposed TiO2 NPs. The

unexposed TiO2 NPs are believed to be partly shielded by the binder molecules reducing

the ability of UV-A light penetration and bacterial cell contact with the TiO2 NPs. As per

our previous study, the type of binder used in the TiO2 coating has an effect on the

184

structural properties of the resultant coatings (Yemmireddy et al, 2015). SEM analysis of

the coatings revealed that the TiO2 coating with binder-A was more compact in nature

whereas the TiO2 coatings with binders B and C were porous in nature. The porous

structure of the TiO2 coatings with binders B and C might have helped to carryout

efficient oxidation and reduction reactions due to availability of electron donors (H2O)

and the acceptors (O2) from the immediate environment. This condition helps to generate

more ROS for photocatalytic disinfection of bacteria. Thus, the structural characteristics

of TiO2 coating and the number of TiO2 NPs that are directly in-contact with bacterial

cells during photocatalytic disinfection treatment plays an important role in the

generation of ROS responsible for the damage of cell walls and eventual cell death.

Many studies have reported that close contact between the bacteria and the TiO2

increases the extent of oxidative damage (Foster et al., 2011). This explains the reason for

the high bactericidal activity of TiO2 coatings with binder B and C when compared to

TiO2 coating with binder A. Based on these results it is clear that increasing the NP

concentration in the coatings increased the log reduction of bacteria. However, there

exists an optimum level of TiO2 to binder concentration to exhibit greater bactericidal

property depending upon the type of binder used in the coating. TiO2 coatings with

binder C showed the highest bactericidal activity followed by TiO2 coating with binder B

and binder A.

3.2.Effect of light intensity on the bactericidal activity of TiO2 nanocoatings

The effect of UV-A light intensity on the bactericidal activity of different TiO2

nanocoatings was shown in Fig 2. When UV-A intensity range from 0.25 to 0.75

mW/cm2, control samples with plain SS coupon showed a reduction of less than a 1 log

185

CFU/cm2 after 3 h treatment. In a similar experiments by Chawengkijwanich and Hayata

(2008), UV-A light itself showed a 1 log CFU/cm2 reduction of E.coli cells after 3 h

treatment at 1 mW/cm2. Similarly, Kikuchi et al. (1997) reported less than 2 log CFU/cm

2

reduction of E.coli cells after 4 h treatment at 1 mW/cm2. Another study by Krysa et al.

(2011), authors reported that increasing UV-A light intensity from 0.2 to 0.6 mW/cm2,

decreased the survival of E.coli cells from 77 to 38 % after a 3 h treatment. This can be

explained by the fact that UV-A light, with relatively low energy, gradually damages

cells through oxidative stress caused by generation of oxygen radicals within the cells

(Bock et al, 1998). The oxidative stress caused by UV-A light on bacterial cells might be

more pronounced with increasing light intensity and treatment time. This shows that UV-

A light itself has minimal bactericidal activity at low intensity levels used in this study.

Increasing the UV-A light intensity from 0.25 to 0.75 mW/cm2 also increased the

bactericidal activity of all TiO2 coatings (Fig 2). Coating with only binder A has showed

a reduction of 0.12, 0.24 and 1.27 log CFU/cm2 at 0.25, 0.5 and 0.75 mW/cm

2 UVA light

intensities, respectively. This indicates that the binder A coating itself has a negligible

effect on the reduction of bacteria at lower light intensities of below 0.50 mW/cm2 and

followed the reduction trend of the UV-A control. However, further increasing the light

intensity to 0.75 mW/cm2 increased the bactericidal activity of the binder coating

compared to the UVA control. Shellac (i.e. binder A) is a food-grade, insect produced

natural resin and widely used as a glazing agent in the food industry. The binder itself is

non-toxic and used in several other food applications. Antic et al. (2010) studied the

effect of shellac-in-ethanol solutions to reduce the transferability of bacteria from cattle

hide to the beef carcass during slaughter operation by immobilizing the bacterial cells on

186

the hide. They reported that shellac itself did not have significant antimicrobial effects

while shellac-in-ethanol showed some antibacterial effect. Similarly, a possible

synergistic effect between shellac under UV-A light at 0.75 mW/cm2 in the current study

might have resulted in a slightly increased reduction. Similarly, the binder B (i.e.

polyurethane) coating itself under UV-A light had little effect on bactericidal activity (Fig

2). Whereas, binder C (i.e. polycrylic) coating showed significantly (P≤0.05) higher

reduction from 2.5 to 4 log CFU/cm2 after a 180 min UV-A exposure (Fig 2).

Increasing the intensity of UV light from 0.25 to 0.75 mW/cm2 for 180 min,

increased the bactericidal activity of TiO2 coating from 0.63 to 1.69 log CFU/cm2 for

binder A (TA8) and from 2.45 to 3.87 log CFU/cm2 for binder B (TB8). However, no

significant (P>0.05) increase in the log reduction was observed for TiO2 coating using

binder C (TC8) (Fig 2). The minimum detection limit for the current test method is 2 log

CFU/cm2. It should be noted that TiO2 coatings with binder C (TC8) at 0.25 mW/cm

2

already reached the highest possible reductions (4 log CFU/cm2) for an initial bacterial

cell concentration of around 106 CFU/cm

2. This is why no additional reduction was

achieved for binder C (TC8) at a higher UV intensity. In order to determine UV intensity

effect, treatment times for TB8 and TC8 nanocoatings were reduced to 90 min (Fig 2).

This treatment step resulted in almost a 51% (for TB8) and 36 % (for TC8) decrease in

the bactericidal activity of TiO2 coatings with binder B and C when compared with

treatment for 180 min. This demonstrates that the observed reductions are in-fact due to

the pronounced photocatalytic bactericidal effect of TiO2 coatings. Marolt et al. (2011)

reported that the photocatalytic treatment on exposed anatase TiO2 nanoparticles could

result in a reactive species that would destroy the soft organic matter such as binders in

187

the vicinity of NPs, thus exposing even more anatase particles. Increasing the

concentration of NPs and UV-A light intensity might have destroyed and removed a

certain amount of the superficial binder and of the other degradable paint components

from the surface of coating thus increasing the bactericidal property of TiO2 nanocoating.

Chawengkijwanich and Hayata (2008) reported that increasing UV-A light intensity from

0.05 to 1 mW/cm2 increased the antimicrobial efficacy of TiO2 coated polypropylene

films from 0.35 to 3 log CFU/cm2. Similar results were also reported by Krysa et al.

(2011) and Dunlop et al. (2010). This indicates that the type of binder, the relative

proportion of the NP to the binder, and the intensity of UV light all have a significant

effect on the bactericidal property of TiO2 coatings. However, the photocatalytic activity

against pathogens at lower light intensity levels is more relevant to potential real life

applications (Foster et al., 2011). Hence extending the photocatalytic bactericidal

property of TiO2 coating towards lower UV-A light intensities or visible light region is

more beneficial. Based on the results of the current study, an UV-A light intensity of 0.5

mW/cm2 was found to be optimum for exhibiting bactericidal property of TiO2

nanocoatings.

3.3. Bactericidal activity of TiO2 nanocoatings on repeated use

Fig. 3 shows the bactericidal activity of TiO2 nanocoatings with binders A, B, and

C at 1:8 NP to binder weight ratio after the repeated use experiment. Except for the TiO2

coatings with binder B and C, there was no significant (P>0.05) loss of photocatalytic

bactericidal property of the TiO2 coatings with binder A was noticed after the multiple

use experiment. Originally, TiO2 coatings with binders A, B, and C (TA8, TB8, and TC8)

irradiated for 180 min at 0.5 mW/cm2 UVA light intensity exhibited a reduction of 0.96,

188

3.72, and 3.92 log CFU/cm2, respectively. However, after one time simulated use of

coated coupons, no significant difference in the reduction was observed for TiO2 coating

with binder A and the reduction remained around 1 log CFU/cm2 (TA8-1). Whereas,

TiO2 coatings with binders B (TB8-1) and C (TC8-1) had high initial log reduction but

lost almost 73 and 22 % of their original bactericidal property after one time use,

respectively. Further, testing the bactericidal property of TiO2 coatings with binder C for

the 3 (TC8-3), 5 (TC8-5) and 10 (TC8-10) times repeated use experiments did not show

significant further reduction in its bactericidal property. Upon repeated use, the change in

bactericidal efficacy of TiO2 nanocoatings can be attributed to the loss of exposed TiO2

NPs on the surface of coating. This is in part related to the decreased physical stability of

the respective coatings when subjected to the repeated use experimental conditions.

3.4. Physical stability of TiO2 nanocoatings on repeated use

Physical stability results of the TiO2 coatings with binder A (TA8), B (TB8), and

C (TC8) before and after subjecting to the repeated use experiment are shown in Table 3.

The thickness of all the TiO2 coatings decreased after the repeated use experiments. After

the one time use experiment, the thickness of coatings TA8, TB8, and TC8 decreased by

31, 29, and 12 %, respectively when compared with the thickness of original coatings.

Further subjecting the TiO2 coating with binder C (TC8) for 3, 5, and 10 times in the

repeated use experiment resulted in 38, 48, and 54 % decreases in the thickness of the

original coating. Adhesion strength of the TiO2 coatings was assessed based on ASTM

D3359-02 standard method –B. Originally, coatings TA8, TB8, and TC8 showed a mean

adhesion rating of 3B, 4B, and 4B, respectively. As per the ASTM standard, adhesion

strength is rated from 5B to 0B. Where, 5B means the coatings has superior adhesion

189

with 0% loss of coated area, followed by 4B (<5 %), 3B (5-15%), 2B (15-35%), 1B (35-

65%), and 0B (>65%), respectively. It means both the coatings TB8 and TC8 showed

good adhesion strength (4B) before subjecting to repeated use. After 1 time repeated use,

adhesion strength of TB8 decreased to 3B while no significant change in the adhesion

was observed for coatings TA8 and TC8 (Table 3). In addition, TC8 maintained the same

original adhesion strength (4B) even after subjecting for 5 times repeated use. However, a

decrease in the adhesion (from 4B to 3B) was noticed after the 10 times repeated use

experiment for TC8. This can be attributed to the corresponding decrease in the thickness

of the original coating from 97 µm to 45 µm after the 10 times repeated use experiment

as described earlier.

Scratch hardness of the TiO2 coatings with binders A, B, and C before and after

the reuse experiment was reported in Table 3. Originally, TiO2 coating with binder B

(TB8) showed the highest scratch resistance (1.08 GPa) followed by TC8 (0.68 GPa) and

TA8 (0.14 GPa), respectively. After the one time repeated use experiment, scratch

hardness of TB8 and TC8 were reduced to 0.61 GPa and 0.53 GPa, respectively.

Whereas, scratch hardness of TA8 increased to 0.42 after one time use. In a similar

manner, after the 3, 5, and 10 times repeated use experiments, the scratch hardness of

TC8 increased by 32, 32, and 13%, respectively when compared with original coating.

These differences in the scratch hardness among different coatings can be partly

attributed to the nature of the binders used in the coating. Depending on the nature of

binder used in the TiO2 coating the width of the scratch either increased or decreased

after the repeated use experiment. For example, the scratch width of the TiO2 coating

with binder A (TA8) increased from 240 µm to 112 µm after one time repeated use.

190

Since scratch width is inversely proportional to the scratch hardness number (as per the

ASTM standard), the scratch hardness of TA8 increased after the one time use

experiment. Whereas, the width of the scratch for TB8 (70 to 90 µm) and TC8 (90 to 100

µm) increased after one time use which led to a decreased scratch hardness number.

However, as the TiO2 coating with binder C (TC8) subjected for the 3, 5, and 10 times

repeated use experiments, the width of the scratch again decreased to 76, 77, and 82 µm

which resulted in an increase in scratch hardness of the coating.

Bhargava et al. (2013) studied the effect of TiO2 concentration (pigment-to-binder

ratio) and dispersing agent on the peel strength of waterborne-polyurethane based

coatings on aluminum substrates. They found that the adhesion strength of the coating

decreased with increasing pigment-to-binder ratio. This may explain the reason for the

decreased physical stability of TB8 after one time use in the current study. TiO2 coating

with binder B (polyurethane) at a 1:8 NP to binder weight ratio may not be sufficient to

impart high physical stability even though it exhibited good bactericidal property

originally. Kumar et al. (2012) reported that silicone functionalized TiO2 based epoxy

coatings on carbon steel exhibited higher values of scratch hardness, pull-off adhesion

and impact resistance. The synergistic interaction between pigment and polymer matrix

through chemical bonding is believed to be the reason for the high mechanical properties

of TiO2 based epoxy coatings. A similar interaction effect might be one possible reason

for the increased hardness of TA8 and TC8 even after repeated use. Based on these

results, adhesion strength and scratch hardness values of the coating were well correlated

with the retention of original bactericidal property of the TiO2 nanocoatings. Among the

tested nanocoatings, TiO2 coatings with binder C showed high bactericidal property and

191

physical stability after the repeated use experiment. These results indicate that type of

binder and the binder-to-nanoparticle concentration used in the coating has a significant

effect (P≤0.05) on the durability and bactericidal property of TiO2 coatings.

4. Conclusions

As per this study, TiO2 coatings with polycrylic as binding agent showed the highest

bactericidal efficacy followed by TiO2 coatings with polyurethane, and shellac as binding

agents, respectively. Increasing the concentration of binder in the TiO2 coating decreased

the bactericidal efficacy. Increasing the UV-A light intensity from 0.25 to 0.75 mW/cm2

increased the bactericidal activity of the TiO2 coatings. However, an intensity of 0.50

mW/cm2 was found to be optimum to avoid the effect of UV light itself on the bacterial

reduction. TiO2 coating with polyurethane as binding agent showed the highest adhesion

strength and scratch hardness. However, on repeated use experiments, TiO2 coating with

polycrylic was found to be physically more stable and bactericidal when compared with

other TiO2 coatings. The results of this study provide feasibility in development of

durable TiO2 nanocoatings with strong bactericidal properties on food contact surfaces

with appropriate binding agents.

Acknowledgements

Funding for this study was provided by Agriculture and Food Research Initiative

grant no 2011-68003-30012 from the USDA National Institute of Food and Agriculture,

Food Safety: Food Processing Technologies to Destroy Food-borne Pathogens Program-

(A4131). The authors would also like to thank Mr. Glenn Farrell for the technical

assistance.

192

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195

Table 6.1. Details of the binders and the composition of different TiO2 nanocoatings

Sample Description

TiO2 TiO2 Aeroxide® P25, surface area 50 m

2 g

-1 and particle size ~21 nm

Sigma-Aldrich, St. Louis, MO, USA

Binder A Shellac a natural resin

Zinsser Co., Inc. Somerset, NJ, USA

Binder B Water based oil modified polyurethane

Minwax®, Minwax company, Upper saddle river, NJ, USA

Binder C Water based polyacrylic

Minwax®, Minwax company, Upper saddle river, NJ, USA

TA4 Nanocoating with TiO2 and binder A at 1:4 weight ratio

TA8 Nanocoating with TiO2 and binder A at 1:8 weight ratio

TB8 Nanocoating with TiO2 and binder B at 1:8 weight ratio

TB16 Nanocoating with TiO2 and binder B at 1:16 weight ratio

TC8 Nanocoating with TiO2 and binder C at 1:8 weight ratio

TC16 Nanocoating with TiO2 and binder C at 1:16 weight ratio

196

Table 6.2. Estimated surface coverage of the nanocoatings with the binder and the TiO2

nanoparticles

1TA4 is the TiO2 coating with binder A at 1:4 NP to binder weight ratio

TA8, TB8, and TC8 are the TiO2 coatings with binders A, B, and C at 1:8 NP to

binder weight ratio.

TB16, and TC16 are TiO2 coating with binder B and C at 1:16 NP to binder weight ratio.

Sample code1

Percent surface coverage based on SEM image

analysis (Estimate only)

Binder Exposed

TiO2

Unexposed

TiO2

Total

TiO2

TA4 38 8 54 62

TA8 39 3 58 61

TB8 21 2 77 79

TB16 33 5 62 67

TC8 39 3 58 61

TC16 43 2 55 57

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Table 6.3. Physical stability of TiO2 coatings before and after repeated use

experiment

Coating

type1

No of times

used

Thickness (µm) Adhesion rating Hardness (GPa)

Before

After Before

After Before

After

TA8 1 74ab

51bcd

3B 3B 0.14f

0.42e

TB8 1 51bcd

36d

4B 3B 1.08a

0.61dce

TC8 1 97a

85a

4B 4B 0.68dc

0.53de

TC8 3 97a

60bc

4B 4B 0.68dc

0.90ba

TC8 5 97a

50cd

4B 4B 0.68dc

0.90ba

TC8 10 97a

45cd

4B 3B 0.68dc

0.77bc

1TA8, TB8, and TC8 are TiO2 coatings with binders A, B, and C at 1:8 NP to

binder weight ratios.

Mean values with same low case superscript within the same variable are not

significantly different (P>0.05)

198

Fig 6.1. Effect of type and concentration of binder on the log reduction of E.coli

O157:H7 by TiO2 nanocoatings at 0.5 mW/cm2 UV-A light intensity for 3 h.

Where, UV-A is the plain SS coupon under UV-A light. Binder-A, Binder-B, and Binder-

C are the coatings with binders-A, B, and C under UVA light. TA4 is the TiO2 coatings

with binders A at 1:4 NP to binder weight ratio under UV-A light. TA8, TB8, and TC8

are the TiO2 coatings with binders A, B, and C at 1:8 NP to binder weight ratios under

UV-A light. While, TB16, and TC16 are the TiO2 coatings with binders B, and C at 1:16

NP to binder weight ratios under UV-A light. Error bars represent +/- standard deviation

of 3 measurements. Means not labelled with the same letter are significantly different

(P≤0.05)

G GF F

C

E

E D

A

B

A

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

Log r

educt

ion (

CF

U/c

m2)

Treatment type

199

Fig 6.2. Effect of UVA light intensity on the log reduction of E.coli O157:H7 by TiO2

nanocoatings.

Where, UVA is the plain stainless steel under UVA light, Binder-A, Binder-B, and

Binder-C are the coatings with binders-A, B, and C under UVA light. TA8, TB8, and

TC8 are the TiO2 coatings with binders A, B, and C at 1:8 NP to binder weight ratio.

Error bars represent +/- standard deviation of 3 measurements. Means not labelled with

the same letter are significantly different (P≤0.05)

KJL

L

KJI

L

B

GJI

H

BC

A

KL K

JL

KJI

L

CD

GFH

A A

KJI

H

F

GF

A

E

A A

KJI

L

GIH

ED

ED

BC

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

UVA Binder-A Binder-B Binder-C TA8 TB8 TC8

Log r

educt

ion (

CF

U/c

m2)

Treatment type

0.25 mW/cm2 for 180 min 0.5 mW/cm2 for 180 min


200

Fig 3. Bactericidal activity of different TiO2 nanocoatings against E.coli O157:H7 before

and after repeated use experiment at 0.5 mW/cm2 for 3 h.

Where TA8, TB8, and TC8 are TiO2 coatings with binders A, B, and C at 1:8 NP to

binder weight ratio. The number followed by the sample code is the number of times

coating subjected for repeated use experiment. Error bars represent +/- standard deviation

of 3 measurements. Means not labelled with the same letter are significantly different

(P≤0.05)

D D

A

D

A

C

BC B BC

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

TA8 TA8-1 TB8 TB8-1 TC8 TC8-1 TC8-3 TC8-5 TC8-10

Log r

educt

ion (

CF

U/c

m2)

Treatment type

201

CHAPTER-7

CONCLUSIONS

In this research, physically stable TiO2 nanocoatings with strong photocatalytic

bactericidal property were developed on food contact surface of stainless steel through a

systematic approach. Initially, a testing protocol was developed in suspension to select

bactericidal TiO2 NPs. Type and source of TiO2 NPs, bacterial cell harvesting conditions,

volume of reaction mixture, and the intensity of UVA light were found to have

significant effect on the log reduction. As per this study, a 20 mL of suspension with

single wash of bactericidal cells and 2 mW/cm2 UVA light intensity was found to be the

best testing protocol to evaluate the bactericidal efficacy of TiO2 NPs. In addition, it was

also found that photocatalytic oxidation of organic dyes can be used as a quick and easier

way to screen bactericidal TiO2 NPs prior to actual microbiological tests. Later, the effect

of food processing organic matter on the photocatalytic bactericidal efficacy of TiO2 NPs

was studied using produce and meat wash solutions. Factors such as turbidity, total

phenolics, and protein content of the organic matter were found to have significant effect

on the bactericidal efficacy of TiO2. Also, a linear correlation was observed between

chemical oxygen demand (COD) and total phenolics as well as COD and protein

contents. Further, an empirical equation with COD as predictor variable was proposed to

predict the bactericidal efficacy of TiO2 in the presence of food processing organic

matter. These results would help to take effective strategies to improve the bactericidal

property of TiO2 NPs in food processing environment as a coating.

202

A coating method was developed to create TiO2 nanocoatings on stainless steel using

different binding agents. Shellac, polyurethane, and polycrylic were found to be three

most promising binders to develop physically stable TiO2 nanocoatings on stainless steel

when used at 1:4 to 1:16 NP to binder weight ratios. SEM analysis of the coated surfaces

revealed that by increasing the binder concentration in the coating decreased the amount

of surface exposed TiO2 NPs. An optimum concentration of TiO2 NPs to binder is

required to achieve good physical stability and strong bactericidal property. Overall, TiO2

nanocoating with polycrylic showed highest physical stability followed by TiO2 coating

with polyurethane and shellac when subjected to adhesion, scratch and wear resistance

tests.

An indented coupon technique was found to be the most appropriate to test

photocatalytic bactericidal property of TiO2 nanocoatings. Type of binder used in the

coating has significant effect on the bactericidal property of TiO2 nanocoatings.

Increasing the concentration of binder in the TiO2 coating has decreased the bactericidal

property. A layer-by-layer coating method improved to expose more NPs on the surface

has significantly increased the bactericidal property of TiO2 nanocoatings. However,

further studies are needed to optimize this technique to achieve high durability. After 3 h

photocatalytic disinfection treatment, TiO2 coatings with polycrylic as binding agent

showed highest log reduction followed by TiO2 coating with polyurethane, and shellac,

respectively. Intensity of UVA light has significant effect of the bactericidal property of

TiO2 nanocoatings. However, a light intensity of 0.50 mW/cm2 was found to be the

optimum to exhibit high photocatalytic disinfection efficacy and also to avoid the effect

of UVA light itself on bacterial reduction. On repeated use experiments, TiO2 coating

203

with polycrylic at 1:8 NP to binder weight ratio was found to be physically more stable

with high bactericidal property. The results of this research has showed promise to

develop durable TiO2 nanocoatings with strong bactericidal property on food contact

surfaces using appropriate binding agents.

204

APPENDIX-A

STRATAGIES TO IMPROVE PHOTOCATALYTIC BACTERICIDAL

PROPERTY OF TiO2 NANOCOATINGS

205

1. Objective

To determine the effect of coating method on the bactericidal property of TiO2

nanocoatings.

2. Hypothesis

A modified coating method to expose more nanoparticles (NPs) on the surface of coating

may significantly increase the bactericidal efficacy of TiO2 nanocoatings.

3. Rationale

Studies on TiO2 nanocoatings based on Chapters 5 and 6 under different test

conditions revealed that the type of binder used in the coating has significant effect on the

physical stability and photocatalytic bactericidal property. These differences in the

bactericidal property can be attributed to the differences in surface characteristics of TiO2

nanocoatings created using different binding agents. It is also shown that when TiO2 NPs

were mixed with different binding agents for subsequent coating on stainless steel

surface, majority of the coated surface was covered only with the binder and very less

number of TiO2 NPs were actually exposed on the surface. Since the photocatalytic

disinfection mechanism is a surface active phenomenon, presence of more number of

TiO2 NPs on the surface of coating significantly improves its bactericidal property. A

layer-by-layer (LbL) coating approach to expose more number of TiO2 NPs on the

surface is one possibility to achieve high disinfection efficiency. In addition, LbL coating

method may help to reduce the UVA light intensity levels required for the activation of

TiO2 NPs and the total treatment time required to achieve desired bacterial reduction.

206

4. Methodology

Indented stainless steel coupons as described in Chapter 5 were used for the

coating. In this study, two different approaches were followed for creating TiO2 coating

(Fig. A1). In the first approach, 0.25 ± 0.01 g of TiO2 coating suspension at 1:8 NP to

binder weight ratio which is prepared as described in Chapter 5 was deposited in to the

well of SS indentation by placing the coupon on a calibrated balance. The deposited

suspension was evenly spread across the entire area of the indentation by slowly tilting

the coupon sideways or if needed using Crayola paint brush by maintaining the constant

weight of deposited coating. Later, the coated coupons were air-dried over night at room

temperature. This approach of coating was refereed as direct coating method. In the

second approach, a layer of binder coating equivalent to the weight of binder used in

coating formulation of direct coating method was first dispensed inside the well of

indentation. Immediately, the coupon was placed underneath a sieve (U.S. mesh # 60)

which was fixed with a template of indented coupon on the top and TiO2 NPs (equivalent

weight in coating formulation of direct method) was pushed through the sieve. In this

way a uniform layer of TiO2 NPs were spread on top of the binder layer. After that, the

coupon was removed out and the NPs spread on top of binder layer were slightly pressed

to adhere and strongly attach to the binder coating. Later, the coatings were air-dried over

night at room temperature and tapped off loosely bound NPs on coating before weighing

them on a calibrated balance mentioned before. This approach of coating was referred as

layer-by-layer (LbL) coating method (Fig. A1). E.coli O157:H7 (5-strain) was prepared

at around 107 CFU/mL cell concentration and used in the photocatalytic disinfection

treatment by following methodology reported in Chapter 6.

207

5. Results

Fig. A2 compares the bactericidal efficacy of TiO2 nanocoatings with binders A,

B, and C prepared by the direct coating (TA8, TB8 and TC8) vs the layer-by-layer

coating (AT8, BT8 and CT8) methods. As reported in the Chapter 6, increasing the

intensity of UVA light from 0.25 to 0.75 mW/cm2 significantly increased bactericidal

activity of TiO2 nanocoatings with binder A (TA8) and B (TB8). However, no significant

effect of light intensity has been observed for TiO2 nanocoating with binder C (TC8).

This is attributed to the inherent bactericidal property of binder-C under UV-A light as

discussed earlier in Chapter 6. This shows that the TiO2 nanocoatings created by direct

coating method with binder-C (TC8) have highest bactericidal activity followed by

binder-B (TB8) and binder-A (TA8) (Fig. A2).

By using Layer-by-Layer (LbL) coating approach, TiO2 coating on binder-A

(AT8) showed up to 389, 227, and 137% increase in the log reduction at UVA light

intensities of 0.25, 0.5, and 0.75 mW/cm2, respectively when compared with the direct

coating method (TA8) (Fig. A2). This can be explained by the fact that the TiO2 NPs are

masked in presence of binder while they are freely available for contact with bacteria in

the absence of binder. Faure et al (2011) reported that the bactericidal efficacy of

coatings involving mixture of TiO2, zeoliths, and inorganic binders on a commercial

support material is much less than the TiO2 coatings on quartz support without any binder

even at 87.5% less concentration of TiO2 NPs. No significant difference in the log

reduction of TiO2 nanocoatings on binder-C (CT8) was observed with respect to coating

method after 3 h treatment (Fig. A2). However, method of coating has significant effect

on the log reduction when the treatment time was reduced to only 90 min (CT8>TC8).

208

Whereas, LbL coating method did not improved the bactericidal activity of TiO2 coatings

on binder-B (BT8) when compared with direct coating method (TB8). In contrast, the

bactericidal activity of BT8 decreased by 35 % (at 0.5 mW/cm2) and 5 % (at 0.75

mW/cm2) when compared with TB8. This result can be explained based on our

observation while developing TiO2 nanocoating with binder-B using LbL approach. We

have noticed that the TiO2 NPs sprinkled on the surface of binder-B layer eventually

submerged in the binder layer resulting in fewer surface exposed TiO2 NPs (Fig. A3).

This further reduced the bactericidal property of original TiO2 nanocoatings (TB8)

created by direct coating method even at comparatively higher UVA light intensities.

6. Conclusions

Direct coating of TiO2 by mixing the NPs together with a binding agent as a

suspension may help to achieve high physical stability and durability. However, this

approach of coating limits the photocatalytic bactericidal efficacy of TiO2 nanocoatings

due to possible shielding of binder on the surface of TiO2 NPs. This condition limits the

extent of ROS generated by TiO2 NPs during photo-treatment and takes longer times to

achieve desired disinfection level. On the other hand, increasing the available surface

area of NPs on the surface through Layer-by-Layer coating method significantly

increased the photocatalytic bactericidal property of TiO2 nanocoatings using different

binders. However, this property depends on the nature of the binder used in the coating.

Moreover, binding agents are required to form strong bond with the NPs to achieve

physically stable and durable coatings for use in commercial applications. Further studies

need to be conducted in order to optimize the process of Layer-by-Layer coating to

209

achieve high bactericidal property of TiO2 nanocoatings without affecting the coating

physical stability.

References:

Faure, M., Gerardin, F., André, J.-C., Pons, M.-N., & Zahraa, O. (2011). Study of

photocatalytic damages induced on E. coli by different photocatalytic supports

(various types and TiO2 configurations). Journal of Photochemistry and

Photobiology A: Chemistry, 222(2–3), 323-329.

210

Fig A1. TiO2 nanocoating on stainless steel surface (SS) using (a) Direct coating, (b)

Layer-by-Layer coatings methods

211

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

TA8 AT8 TB8 BT8 TC8 CT8

Log r

educt

ion (

CF

U/c

m2)

Treatment type



Fig A2. Effect of coating method on bactericidal activity of TiO2 coatings

Where TA8, TB8, and TC8 are TiO2 coatings with binders A, B, C at 1:8 NP to binder weight

ratios, respectively as deposited by direct coating method; AT8, BT8, and CT8 TiO2 coatings

with binders A, B, C at 1:8 NP to binder weight ratios, respectively as deposited by layer-by-

layer coating method.

212

Fig A3. Comparison of TiO2 nanocoatings with binders A (TA8/AT8), B

(TB8/BT8), and C (TC8/CT8) at 1:8 NP to binder weight ratio created by (i)

Direct coating method (TA8, TB8, and TC8), and (ii) Layer-by-Layer coating

method (AT8, BT8, and CT8).

213

APPENDIX-B

STUDIES ON BACTERICIDAL ACTIVITY OF VISIBLE LIGHT ACTIVATED

IRON OXIDE (Fe2O3) NANOPARTICLES AND NANOCOATINGS

214

1. Objective

To understand the bactericidal properties of iron oxide (Fe2O3) nanoparticles (NPs) in

suspension and as a nanocoating.

2. Hypothesis

Fe2O3 NPs and nanocoatings with potent bactericidal property under visible light

activation can be created using various chemical and physical synthesis and deposition

techniques.

3. Rationale

Fe2O3 has attracted a lot of attention for photocatalytic applications due to its short

band gap energy (Eg = 2.2 eV) and the ability to absorb a large part of the visible light

spectrum (λ= 564 nm). In addition, Fe2O3 NPs are chemically stable, non-toxic, cheap,

and readily available. Very limited information is available in the literature regarding

photocatalytic bactericidal properties of Fe2O3 NPs. Recently, Basnet et al (2013)

reported that the physical vapor deposited (PVD) Fe2O3 nanocoatings has showed

excellent bactericidal property under visible light illumination. They also reported that

the oblique angle deposited (OAD) coatings are more bactericidal compared to thin film

(TF) coatings. However, the effect of method of NP synthesis and deposition techniques

on bactericidal property of Fe2O3 NPs in suspension as well as in coating form is still not

clear. Understanding the effect of fabrication method on bactericidal property of visible

light activated Fe2O3 NPs would be a great use in several food safety applications.

215

4. Methodology

4.1. Fe2O3 NPs and nanocoatings

Several samples of chemically synthesized, ball milled, silicon dioxide (SiO2) and

tungsten oxide (WO3) doped Fe2O3 NPs of known characteristics and photocatalytic

activity were provided by Prof. Yiping Zhao’s lab at the Nanoscale Science and

Engineering Center, The University of Georgia, Athens, GA. In addition, sputter

deposited thin film (TF), OAD nanorod (NR), SiO2 or WO3 doped Fe2O3 coatings were

also provided by Prof. Zhao’s lab. A commercial sample Fe2O3 NPs (Alfa Aesar®, Ward

Hill, MA) was also included for comparison.

4.2. Bacterial strains & inoculum preparation

Bacterial strains either E.coli (ATCC 1428) or E.coli O157:H7 (5-stain) at around

107-8

CFU/mL were used in these studies. Bacterial inoculum for photocatalytic

disinfection treatments was prepared by following the procedure described in Basnet et al

(2013).

4.3. Photocatalytic disinfection in suspension:

Photocatalytic bactericidal activity of Fe2O3 NPs in suspension was determined by

following method described in Yemmireddy and Hung (2015). However, UVA light

source was replaced with a visible light (UTILITECH) in the current study. The

experiments were conducted at different concentration of NPs (1 to 10 mg/mL), volume

of reaction mixture (5 to 30 mL), and light intensities (25 to 100 mW/cm2). One positive

(only aqueous suspension of NPs in dark) and a negative (only visible light without NPs)

control samples were also included in each treatment. The log reductions over a 3 h

216

treatment time were determined by plating appropriate dilutions on tryptic soy agar

(TSA) or sorbital macconkey agar (SMAC) followed by incubation at 37°C for 24 h.

4.4. Photocatalytic disinfection as a coating

Fe2O3 coated glass coupons were first pre-sterilized under 30W germicidal UV

light (254 nm) in a biological safety cabinet (Class II Type A/B3, NuAire, Inc.,

Plymouth, MN) for about 1 h. The sterilized coupons were placed in a 90 mm diameter

petri-dishes containing moistened filter paper at the bottom to prevent drying-out of the

bacterial culture during the treatment. Later, a 100 µL aliquot of bacterial culture was

uniformly spread across the entire surface of Fe2O3 coating using a sterile disposable

loop. These samples were treated under visible light illumination at 50 to 100 mW/cm2

for up to 2- 4 h. The light intensity reaching the surface of each coupon was measured

with a optical power meter (ThorLabs PM100D/S310C). Appropriate positive and

negative controls were also included. During the treatment the filter paper is frequently

moistened to avoid drying-out of inoculum. After the specified treatment time, the

coupons were taken out and immersed in 10 mL of sterile PBS solution containing 0.1%

tween 80 and vortexed for 30 s to re-suspend the bacteria. A viability count (log

CFU/cm2) was performed by appropriate dilution and plating on either TSA or SMAC

and incubation at 37 °C for 24 h.

5. Results

The treatment conditions and the results of various experiments involving Fe2O3

NPs and their doped structures in suspension as well as in coating were summarized in

Table B1. The results indicate that under tested conditions, Fe2O3 NPs in suspension were

not shown significant bacterial reduction. In the first stage, several studies have been

217

conducted using commercial and chemically synthesized Fe2O3 NPs in suspension at

different concentration of NPs, volume of reaction mixture, visible light intensity, initial

bacterial concentration, and photocatalytic treatment time in order to improve the

bactericidal efficacy and to optimize test conditions for maximum bactericidal property.

However, even after subjecting to variable test conditions the tested Fe2O3 NPs were

unable to exhibit significant reduction. At this stage we suspected that the method of

synthesis of NPs may have an effect on their photocatalytic activity. In the second stage

of experiments, Fe2O3 NPs synthesized by ball milling (physical synthesis method) were

again tested for photocatalytic bactericidal property in suspension under different test

conditions. The results did not show an improvement in bactericidal property when

compared with chemically synthesized Fe2O3 NPs. At this stage we suspected that the

charge separation efficiency of Fe2O3 NPs might be severely hindered in the suspension.

As a reason the recombination rate of photo-generated valence band holes and conduction

band electrons were so high that not enough reactive oxygen species (ROS) were

generated from Fe2O3 NPs for bacterial inactivation. Later, in order to increase the charge

pair separation ball milled Fe2O3 NPs mixed with SiO2 and WO3 NPs. However, no

significant difference in the bactericidal activity was observed. Agglomerated NPs in the

suspension was believed to be one possible reason for the limited photocatalytic

bactericidal property of Fe2O3 NPs and their doped powders. Studies reported by Tuchina

et al (2014), and Zhang et al (2011) further support this hypothesis. In the next stage,

inactivation studies were focused on using immobilized Fe2O3 NPs on glass substrate as a

coating. The results indicate that only Fe2O3 NR coatings are showing good bactericidal

activity followed by Fe2O3 thin films further supporting results of Basnet et al (2013).

218

However, doped structures of WO3 core and Fe2O3 shell NRs and WO3-Fe2O3 side coated

NRs were unable to exhibit bactericidal property. Photocatalytic activities by dye

degradation of different nanostructures as provided by Dr. Yiping Zhao’s lab are in the

order of TiO2 NRs/NPs under UVA light >> Fe2O3 NRs under vis-light > Fe2O3 NPs

under vis-light > TiO2 NPs under vis-light. The same trend can be attributed to the high

bactericidal activity of Fe2O3 NRs when compared to Fe2O3 NPs.

6. Conclusions

Fe2O3 is a promising photocatalyst for several food safety applications due to its non-

toxicity and ability to activate under visible light. However, developing Fe2O3

nanostructures on different surfaces using simple approaches for practical application is a

challenge. We made an effort to understand the bactericidal property of Fe2O3 NPs in

suspension and create nanocoating with strong bactericidal activity. We investigated the

effect of fabrication method of Fe2O3 NPs and nanocoatings on bactericidal activity under

different test conditions. Our preliminary results shows that Fe2O3 NPs in suspension are

photo-catalytically not very active to exhibit significant bacterial reduction in contrast to

Fe2O3 nanorod coatings. The possible reason for the differences in photocatalytic

bactericidal activity of Fe2O3 under visible light activation is still not fully understood.

More systematic studies need to be conducted at mechanistic point of view to better

understand the Fe2O3 bactericidal properties under different scenarios.

219

References

Basnet, P., Larsen, G. K., Jadeja, R. P., Hung, Y.-C., & Zhao, Y. (2013). alpha-Fe2O3

Nanocolumns and Nanorods Fabricated by Electron Beam Evaporation for

Visible Light Photocatalytic and Antimicrobial Applications. Acs Applied

Materials & Interfaces, 5(6), 2085-2095.

Tuchina, E. S., Kozina, K. V., Shelest, N. A., Kochubey, V. I., & Tuchin, V. V. (2014).

Iron oxide nanoparticles in different modifications for antimicrobial phototherapy.

Proc. Of SPIE, Vol (8955), 1-12.

Zhang, W., Rittmann, B., & Chen, Y. (2011). Size Effects on Adsorption of Hematite

Nanoparticles on E. coli cells. Environmental Science & Technology, 45(6), 2172-

2178.

220

B1. Summary of photocatalytic bactericidal activity of various types of Fe2O3 in

suspension and as a nanocoating

Type of Fe2O3 NPs Type of

study

Type of bacterial

culture

Test conditions

(Volume of

suspension, light

intensity)

Log reduction/

treatment time

Chemically

synthesized

Suspension E.coli (ATCC 1428) 20 mL

7 mW/cm2

<0.5 log/ 2 h

Commercial &

Chemical

Suspension E.coli (ATCC 1428) 30 mL

65 or 100 mW/cm2

<0.25 log/ 4 h

Ball milled Suspension E.coli (ATCC 1428) 30 mL

100 mW/cm2

<0.25 log/ 3 h

Ball milled Fe2O3

mixed with SiO2

Suspension E.coli (ATCC 1428) 5, 10, 20 mL

100 mW/cm2

<0.25 log/ 2-6 h

Ball milled Fe2O3 -

WO3

Suspension E.coli (ATCC 1428) 10, 20 mL

100 mW/cm2

0.71 log / 3 h

Ball milled WO3-

Fe2O3

Suspension E.coli (ATCC 1428) 10, 20 mL

100 mW/cm2

0.46 log/ 3 h

Commercial

Suspension E.coli O157: H7 20 mL

50 mW/cm2

<0.5 log/ 3 h

Ball milled

Fe2O3- SiO2

Fe2O3 -WO3

Suspension E.coli O157: H7 20 mL

50 mW/cm2

<0.5 log/ 3 h

Fe2O3- WO3 thin film Coating E.coli O157: H7 100 µL

10 mW/cm2

1.15 log / 3 h

Fe2O3 OAD NRs Coating E.coli O157: H7 100 µL

10 mW/cm2

>3.75 log /3 h

WO3 (core) - Fe2O3

(shell) NRs

Coating E.coli O157: H7 100 µL

50 mW/cm2

<0.25 log /2 h

WO3 - Fe2O3 side

coated NRs

Coating E.coli O157: H7 100 µL

50 mW/cm2

<0.25 log /2 h

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