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Antibacterial and Antiviral Study of Dialdehyde Polysaccharides

Permanent Link: http://ufdc.ufl.edu/UFE0023936/00001

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Title: Antibacterial and Antiviral Study of Dialdehyde Polysaccharides
Physical Description: 1 online resource (136 p.)
Language: english
Creator: Song, Le
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: antibacterial, antivirial, dialdehyde, polysaccharides
Materials Science and Engineering -- Dissertations, Academic -- UF
Genre: Materials Science and Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Concerns for microbial contamination and infection to the general population, especially the spread of drug-resistant microorganisms, have greatly increased. Polymeric biocides have been found to be a feasible strategy to inactivate drug-resistant bacteria. However, current polymeric biocide systems involve multi-step chemical reactions and they are not cost-effective. Desirable antimicrobial systems need to be designed to be environmentally friendly, broad-spectrum effective against microorganisms, flexible for various delivery methods and economically affordable. We demonstrated that dialdehyde polysaccharides (including dialdehyde starch and dialdehdye cellulose) were broad-spectrum polymeric biocides against gram-positive/negative bacteria, bacteriophages and human virus. These polymers can be easily converted from starch and cellulose through one-step periodate oxidation. Destructions of microorganism by dialdehyde polysaccharides have been achieved in aqueous suspension or by solid surface contact. The dialdehdye functions of dialdehdye polysaccharides were found to be the dominant action against microorganism. The reactivity of the dialdehyde functionality was found to be pH-dependent as well as related to the dispersion of dialdehyde polysaccharides. Degradation of dialdehyde starch during cooking was confirmed. Degradation of dialdehyde starch was more liable in alkaline condition. Carboxylic acid and conjugated aldehyde functionalities were the two main degradation products, confirmed from the spectroscopic studies. The pH effect on the polysaccharide structure and the corresponding antimicrobial activity was very complicated. No decisive conclusions could be obtained from this study. Liner inactivation kinetics was found for dialdehyde starch aqueous suspension against bacteria. This linear inactivation kinetics was derived from the pseudo-first chemical reaction between the dialdehyde starch and the bacteria. The established inactivation kinetics was successfully predicated the response of bacteria to dialdehyde starch with time. Inactivation of bacteria by dialdehyde starch was speculated to be the crosslinking-interaction between the dialdehyde starch and the bacterial surface. Amino groups of bacterial surfaces were blocked by dialdehyde starch. This crosslinking action was also suggested from the preliminary study of the bacterial dehydrogenase activity. However, membrane damage was found in the dialdehdye starch treated bacteria from the fluorescent study.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Le Song.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Baney, Ronald H.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0023936:00001

Permanent Link: http://ufdc.ufl.edu/UFE0023936/00001

Material Information

Title: Antibacterial and Antiviral Study of Dialdehyde Polysaccharides
Physical Description: 1 online resource (136 p.)
Language: english
Creator: Song, Le
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: antibacterial, antivirial, dialdehyde, polysaccharides
Materials Science and Engineering -- Dissertations, Academic -- UF
Genre: Materials Science and Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Concerns for microbial contamination and infection to the general population, especially the spread of drug-resistant microorganisms, have greatly increased. Polymeric biocides have been found to be a feasible strategy to inactivate drug-resistant bacteria. However, current polymeric biocide systems involve multi-step chemical reactions and they are not cost-effective. Desirable antimicrobial systems need to be designed to be environmentally friendly, broad-spectrum effective against microorganisms, flexible for various delivery methods and economically affordable. We demonstrated that dialdehyde polysaccharides (including dialdehyde starch and dialdehdye cellulose) were broad-spectrum polymeric biocides against gram-positive/negative bacteria, bacteriophages and human virus. These polymers can be easily converted from starch and cellulose through one-step periodate oxidation. Destructions of microorganism by dialdehyde polysaccharides have been achieved in aqueous suspension or by solid surface contact. The dialdehdye functions of dialdehdye polysaccharides were found to be the dominant action against microorganism. The reactivity of the dialdehyde functionality was found to be pH-dependent as well as related to the dispersion of dialdehyde polysaccharides. Degradation of dialdehyde starch during cooking was confirmed. Degradation of dialdehyde starch was more liable in alkaline condition. Carboxylic acid and conjugated aldehyde functionalities were the two main degradation products, confirmed from the spectroscopic studies. The pH effect on the polysaccharide structure and the corresponding antimicrobial activity was very complicated. No decisive conclusions could be obtained from this study. Liner inactivation kinetics was found for dialdehyde starch aqueous suspension against bacteria. This linear inactivation kinetics was derived from the pseudo-first chemical reaction between the dialdehyde starch and the bacteria. The established inactivation kinetics was successfully predicated the response of bacteria to dialdehyde starch with time. Inactivation of bacteria by dialdehyde starch was speculated to be the crosslinking-interaction between the dialdehyde starch and the bacterial surface. Amino groups of bacterial surfaces were blocked by dialdehyde starch. This crosslinking action was also suggested from the preliminary study of the bacterial dehydrogenase activity. However, membrane damage was found in the dialdehdye starch treated bacteria from the fluorescent study.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Le Song.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Baney, Ronald H.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0023936:00001


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1 1 ANTIBACTERIAL AND ANTIVIRAL STUDY OF DIALDEHYDE POLYSACCHARIDES By LE SONG A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2 2008 Le Song

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3 3 To my wife, Fei Dong; my daughter, Katherine C. Song in Houston, TX; and my family members in China

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4 4 ACKNOWLEDGMENTS I gratefully acknowledge my a dvisor, Dr.Baney, for the opportunity to work in his group. His guidance, knowledge and critical thinking have inspired me to dedicate myself to research work. Financial support from Dr. Baneys research funds was vital for my study, especially for my PhD research in the last year and a -half. I especially appreciate Dr. Farrah in the Department of Microbiology, who advised me on the antiviral experiments and allowed me to use his lab and facilitates. I am also grateful to Dr. Koopmen for allowing me to use his lab for antimicrobial experiments, and Dr. C.Y.Wu for conducting the virus aerosols test; both are in the Department of Environmental Engineering. I also thank Dr. Vjay in Particle Engineering Research Center (PERC) for help and many valuable discussi ons. Thanks also go to my committee members (Dr. Batich, Dr. Beatty, Dr. Wagener and Dr. Whitney) for their appreciated comments and help. I express my gratitude to Jame Rocca at the Brain Institute for NMR, Liao Chih -Wei in the Department of Materials Sci ence and Engineering for Xgal, Wu Pei Hsun in the Department of Chemical Engineering for fluorescent microscopy, Steve McClellan in Interdisciplinary Center for Biotechnology Research (ICBR) for flowcytomery and Dr.Shi YongCheng at Kansas State University for GPC measurement. I would like to thank all my labmates and friends for their discussions and help. Finally, I express my deepest appreciation to my wife, my one -year old daughter, and my whole family for their unconditional love and support.

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5 5 TABL E OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................................... 4 LIST OF TABLES ................................................................................................................................ 8 LIST OF FIGURES .............................................................................................................................. 9 ABSTRACT ........................................................................................................................................ 12 CH A P T E R 1 INTRODUCTION ....................................................................................................................... 14 2 LITERATURE REVIEW ........................................................................................................... 18 2.1 Chemistry of Dialdehyde Polysaccharide ........................................................................... 18 2.2 Toxicity of Dialdehyde Polysaccharide ............................................................................... 22 2.3 Biomedical Application of Dialdehyde Polysaccharide ..................................................... 22 2.4 Antimicrobial Studies of Glutaraldehyde ............................................................................ 24 2.5 Design Antimicrobial Solid Surface .................................................................................... 27 2.5.1 Anchor a Small Molecular Biocide to a Substrate ................................................... 28 2.5.2 Polymeric Biocide ...................................................................................................... 29 2.5.3 Comments ................................................................................................................... 30 2.6 Microbial Inactivation Kinetics ............................................................................................ 31 3 ANTIMICROBIAL ACTIVITY OF DIALDEHYDE SUSPENSION ................................... 36 3.1 Introduction ........................................................................................................................... 36 3.2 Materials and Experiments ................................................................................................... 37 3.2.1 Materials ..................................................................................................................... 37 3.2.2 Preparation of Starch Suspension .............................................................................. 37 3.2.3 Preparation of Microbial Stock Solution .................................................................. 37 3.2.4 Degree of Oxidation of As -received Dialdehyde Starch ......................................... 38 3.2.5 X ray Diffraction ........................................................................................................ 38 3.2.6 Sonication ................................................................................................................... 39 3.2.7 Solubility ..................................................................................................................... 39 3.2.8 Fourier Transform Infrared Spectroscopy (FTIR) .................................................... 39 3.2.9 Ultraviolet -Visible Spectroscopy (UV -VIS) ............................................................ 39 3.2.10 Particle Size Measurement ...................................................................................... 39 3.2.11 Viscosity Measurement ............................................................................................ 40 3.2.12 Nuclear Magnetic Resonance Spectroscopy (NMR) ............................................. 40 3 .2.13 Antimicrobial Test .................................................................................................... 40 3.2.14 Bacterial Inactivation Study .................................................................................... 41 3.3 Results and Discussion ......................................................................................................... 42 3.3.1 Characterization of the As Received Dialdehyde Starch ......................................... 42

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6 6 3.3.2 Cooking Effects on the Physicochemical Transition of the Dialdehyde Starch ..... 42 3.3.3 The pH Effect on Physicochemical Change of As -Prepared DAS Aqueous Suspension ........................................................................................................................ 47 3.3.4 Antimicrobial Study of DAS Suspensions ................................................................ 49 3.3.5 Bacterial Inactivation Kinetics .................................................................................. 58 3.4 Conclusion ............................................................................................................................. 61 4 DESTRUCTION OF MICROORGANISMS BY SURFACE CONTACT ............................ 78 4.1 Introduction ........................................................................................................................... 78 4.2 Materials and Methods ......................................................................................................... 79 4.2.1 Materials ..................................................................................................................... 79 4.2.2 Anchor of Trialkoxysilyl Compounds onto Cotton Textile ..................................... 80 4.2.3 Periodate Oxidation of Cellulose Filter Paper (Dialdehyde Cellulose) .................. 80 4.2.4 Deposition of Starch Film .......................................................................................... 80 4.2.5 Determination of Oxidation Extent of Dialdehyde Cellulose (DAC) ..................... 81 4.2.6 Characterization of Cellulose Filter Paper ................................................................ 81 4.2.7 Antibacterial and Antiviral Assessment by Surface Contact ................................... 81 4.2.8 Evaluation of DAC Filter Paper for Virus Aerosols ................................................ 83 4.3 Results and Discussion ......................................................................................................... 84 4.3.1 Antibacterial Activity of Cotton Textile ................................................................... 84 4.3.2 Characterization of Dialdehyde Cellulose (DAC) Filter Paper ............................... 86 4.3.3 Destruction of Microorganisms by Dialdehyde Polysaccharide Surface Contact .............................................................................................................................. 89 4.3.4 Preliminary Results of DAC Filter Paper for MS2 Virus Aerosols ........................ 92 4.4 Conclusions ........................................................................................................................... 94 5 STUDY THE ANTIBACTERIAL MECHANISMS OF DIALDEHYDE STARCH .......... 107 5.1 Introduction ......................................................................................................................... 107 5.2. Materials and Experiments ................................................................................................ 108 5.2.1 Antibacterial Experiments ....................................................................................... 108 5.2.2 FTIR Spectra ............................................................................................................. 108 5.2.3 Electrophoretic Mobility .......................................................................................... 109 5.2.4 Dehydrogenase Act ivity ........................................................................................... 109 5.2.5 Fluorescent dye Studies ........................................................................................... 109 5.2.5.1 Fluorescence microscopy .............................................................................. 110 5.2.5.2 Fluorescence spectroscopy ............................................................................ 110 5.2.5.3 Flow cytome try .............................................................................................. 110 5.3 Results and Discussion ....................................................................................................... 110 5.3.1 Bacterial Surface Properties .................................................................................... 110 5.3.2 Inhibition of Enzyme Activity of Bacteria ............................................................. 112 5.3.3 Fluorescent Dye Study ............................................................................................. 113 5.4 Conclusions ......................................................................................................................... 116

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7 7 6 CONCLUSIONS AND FUTURE WORK .............................................................................. 123 6.1 Conclusions ......................................................................................................................... 123 6.2 Future Work ........................................................................................................................ 124 LIST OF REFERENCES ................................................................................................................. 126 BIOGRAPHICAL SKETCH ........................................................................................................... 136

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8 8 LIST OF TABLES Table page 1 1 Chemical structures of biocides in antisepti cs and disinfectants ........................................ 17 2 1 Mechanism of antimicrobial action of glutaraldehyde ........................................................ 33 2 2 Antimicrobial textile treatments ............................................................................................ 33 3 1 Selected microorganisms in current study ............................................................................ 63 3 2 Some physical properties of as received dialdehyde starch ................................................ 63 3 3 Viscosity, pH and solubility of DAS aqueous suspensions cooked at 95 C in different time .......................................................................................................................... 63 3 4 Summary of volume mean particle size ................................................................................ 63 3 5 Effect of the pH on the solubility of the DAS aqueous suspensions, the absorption intensity ratio of FTIR and UV ............................................................................................. 64 3 6 The MLCs of 1 hr sonicated DAS aqueous suspensions and the corresponding pH values ..................................................................................................................................... 64 3 7 Kinetic parameters of DAS aqueous suspension against bacteria ....................................... 64 4 1 Antibacterial activity of free AEM 5700 compound ........................................................... 95 4 2 Antimicrobial activity of DAS films ..................................................................................... 95 4 3 Prelimina ry results of DAC filter paper for MS2 virus aerosol .......................................... 95 5.1 Intensity ratio of C=O stretching vibration over N H deformation .................................. 117 5 2 Flow cytometer analysis of the treated-bacterial systems ................................................. 117

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9 9 LIST OF FIGURES Figure page 2 1 Chemical structures of simple starch and cellulos e ............................................................. 34 2 2 Periodate oxidation of AGU to generate dialdehyde function ............................................ 34 2 3 Some elementary structures of dialdehyde starch ................................................................ 34 2 4 Bacterial structure of gram -positive and gram -negative bacteria ....................................... 35 2 5 Schematic illustration of AEM 5700 onto surfaces to for m surface bound antimicrobial activity ............................................................................................................. 35 2 6 Synthesis of 3 triethoxysilylpropyl 5,5 dimethylhydantoin ................................................ 35 3 1 Yellow color deve lopment of DAS aqueous suspension cooked at 95 C for different time .......................................................................................................................................... 65 3 2 X ray diffraction patterns of as received DAS granule DAS aqueous suspension. ........... 65 3 3 X ray diffraction patterns of native (NS) and dialdehyde potato starch ............................. 65 3 4 Particle size measurement by Coulter LS13320 and by Brookhaven Zetaplus. ............... 66 3 5 Fourier transform infrared spectra of the DAS samples ...................................................... 67 3 6 Degradation mechanisms of DAS in alkaline media ........................................................... 68 3 7 Ultraviloet -visible spectra of the DAS samples ................................................................... 68 3 8 Yellow color development of as prepared DAS aqueous suspension in different pH values ...................................................................................................................................... 69 3 9 Fourier transform infrared spectra of DAS solids from supernatants (a) and sedimentations (b) in the carbonyl region. ........................................................................... 69 3 10 Ultraviolet-visible spectra of DAS aqueous suspensions at different pH values ............... 69 3 11 The 1H NMR of DAS supernatant at different pH values ................................................... 70 3 12 The 13C NMR of DAS supernatants (bottom pH 3, up pH 8.7) .......................................... 71 3 13 Oxidation of starch by different oxidizing agents ................................................................ 71 3 14 Antimicrobial activity of DAS granular suspension, as -prepared DAS aqueous suspension and as -prepared oxidized starch aqueous suspension ....................................... 72

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10 10 3 15 Effect of sonication time on the antibacterial activity of DAS aqueous suspension against E.coli and S.aureus .................................................................................................... 72 3 16 The pH dependent behavior of PBS and 1 -hr sonicated 2.7% DAS aqueous suspension against gram -negative a nd gram -positive bacteria ........................................... 73 3 17 Antiviral test results of PBS and 2.7% DAS aqueous suspension, pH and mixing time dependent test ......................................................................................................................... 74 3 18 One hour sonciated2.7% DAS aqueous suspension, 4.5% oxidized starch (OS) aqueous suspension and PBS, all at pH 4.8 against bacteria ............................................... 75 3 19 Ultarviloet -visible spectra of as -prepar ed 3% DAS with different dilutions (a) and the calibration curve (b) ......................................................................................................... 75 3 20 The DAS aqueous suspensions against bacteria at different time ....................................... 76 3 21 Relationship between 1/MLC vs time at room temperature ................................................ 76 3 22 Comparison of inactivation of DAS aqueous suspension against S.aurues and E.coli with developed model ............................................................................................................ 77 4 1 Chemical structures of trialkoxysilyl compounds ................................................................ 96 4 2 Schematic illustration of anchoring trialkoxysilyl compounds onto cotton textile ............ 96 4 3 Antimicrobial experimental set up for textile ....................................................................... 96 4 4 Experimental setup for viable removal efficiency and physica l removal efficiency of the test filters (from Dr. C.Y.Wu). ........................................................................................ 97 4 5 Antibacterial activities of aldehyde compounds against E.coli at different time ............... 98 4 6 Antimicrobial activity of cotton textile treated by AEM 5700 with various concentrations against E.coli in deionized water and in artificial seawater ....................... 98 4 7 Antibacte rial activities of 1% trialkoxysilyl aldehdyes treated cotton textiles at different time .......................................................................................................................... 99 4 8 Calibration curve for the determination of sodium periodate ............................................. 99 4 9 Effect of reaction time on the oxidation extent of cellulose filter ....................................... 99 4 10 X ray diffraction patterns of cellulose filter paper at different oxidation time (hr) ......... 100 4 11 Relationship between oxidation extent and crystallinity of DAC ..................................... 100 4 12 Fourier transform infrared spectra of DAC filter paper at different regions .................... 101

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11 11 4 13 Ultraviloet -visible spectra of cellulose filter paper ............................................................ 102 4 14 Antibacterial activity of DAS film against bacterial suspension ...................................... 102 4 15 Antivirial activity of DAS film against bacteriophages ..................................................... 103 4 16 Antibacterial activity of DAC filter paper .......................................................................... 103 4 17 Post -treated DAC filter paper against S.aureus .................................................................. 104 4 18 Ultraviolet-visible spectra of le achates of DAC ................................................................. 104 4 19 Antivirial activity of DAC filter paper against MS2 .......................................................... 105 4 20 Antiviral activity of cellulose filter pape r ........................................................................... 106 5 1 Schematic illustration of the reaction between DAS and bacterium ................................ 118 5 2 Fourier transform infrared spectra of bacteri a and pure DAS ........................................... 118 5 3 The pH -mobility behavior of bacteria ................................................................................. 119 5 4 Effect of DAS on viability and dehydrogenase activity of bac teria .................................. 119 5 5 Fluorescent microscopy pictures of S.aureus and E.coli ................................................... 121 5 6 Fluorescence spectroscopy of bacterial samples (A E .coli B S.aureus ), pure 0.3% DAS and 0.1% AEM 5700 (C) ............................................................................................ 122

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12 12 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ANTIBACTERIAL AND ANTIVIRAL STUDY OF DIALDEHYDE POLYSACCHARIDES By Le Song December 2008 Chair: Ronald H.Baney Major: Materials Science and Engineering Concerns for microbial contamination and infection to the general population, especially the spread of drug resistant microorganisms, have greatly increased. Polymeric biocides have been found to be a feasible strategy to inactivate drug resistant bacteria. However, current polymeric biocide systems involve multi -step chemical reactions and they are not cost -effective. Desirable antimicrobial systems need to be designed to be environmentally friendly, broad spectrum effective against microorganisms, flexible for various delivery methods and economically affordable. We demonstrated t hat dialdehyde polysaccharides (including dialdehyde starch and dialdehdye cellulose) were broad -spectrum polymeric biocides against gram -positive/negative bacteria, bacteriophages and human virus. These polymers can be easily converted from starch and cel lulose through one -step periodate oxidation. Destructions of microorganism by dialdehyde polysaccharides have been achieved in aqueous suspension or by solid surface contact. The dialdehdye functions of dialdehdye polysaccharides were found to be the domin ant action against microorganism. The reactivity of the dialdehyde functionality was found to be pH dependent as well as related to the dispersion of dialdehyde polysaccharides. Degradation of

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13 13 dialdehyde starch during cooking was confirmed. Degradation of dialdehyde starch was more liable in alkaline condition. Carboxylic acid and conjugated aldehyde functionalities were the two main degradation products, confirmed from the spectroscopic studies. The pH effect on the polysaccharide structure and the corresp onding antimicrobial activity was very complicated. No decisive conclusions could be obtained from this study. Liner inactivation kinetics was found for dialdehyde starch aqueous suspension against bacteria. This linear inactivation kinetics was derived fr om the pseudo-first chemical reaction between the dialdehyde starch and the bacteria. The established inactivation kinetics was successfully predicated the response of bacteria to dialdehyde starch with time. Inactivation of bacteria by dialdehyde starch w as speculated to be the crosslinkinginteraction between the dialdehyde starch and the bacterial surface. Amino groups of bacterial surfaces were blocked by dialdehyde starch. This crosslinking action was also suggested from the preliminary study of the ba cterial dehydrogenase activity. However, membrane damage was found in the dialdehdye starch treated bacteria from the fluorescent study.

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14 14 CHAPTER 1 INTRODUCTION Antiseptics and disinfectants are used extensively in daily life. Various types of biocides u sed in antiseptics and disinfectants are shown in Table 1 1 (Gao and Cranston 2008; Kim et al. 2006; McDonnell and Ru ssell 1999) Despite the variety of biocides and their different antimicrobial mechanisms, the widespread use of biocides has prompted microbes to develop antimicrobial resistance. Resistance can be a natural property of an organism (i.e., the intrinsic r esistance) or can be achieved by mutation, acquisition of new genetic information, expression of previously silent genes and other phenotypic alterations (Chapman 2003; McDonnell and Russell 1999) Antimicrobial resistance has become a major public health concern. For example, Staphylococcus aureus has de veloped resistance to almost all currently available antibiotics including methicillin (Levy and Marshall 2004) A cost comparison of treating methicillin resistant S.aureus (MRSA) versus methicillin -susceptible S.aureus (MSSA) was investigated in New York City (Rubin et al. 1999) This study found that a threefold increase in mortality and 22% economic cost increase associated with MRSA. For all hospitalized individuals with MRSA in New York City, the cost was estimated to be millions of dollars. Approximately 90,000 people die in the United State from nosocomial infections by pathogens such as MRSA each year (Lewis and Klibanov 2005) .The economic cost of the major resistant pathogens has been estimated to b e several billion dollars a year in the United States by the US Office of technology Assessment advisory committee. If the community infections were considered, the cost would be even greater (Levy and Marshall 2004) With the increasing concern toward the potential of the microbial contamination and the infection risks to the general population, the development of new biocides is necessary and

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15 15 critical. The developed new biocides need to either overcome the microbial resistance mechanisms, or attack new targets of microbes. For example, bacterial resistance to small molecular quaternary ammonium compounds (QAC) is one of the best -studied resistance mechanisms. Multi -drug resistant pumps have been developed in the QAC resistant bacteria (Chapman 2003) These efflux pumps are membrane -bound, proton -motive force -dependent cation export proteins. These pumps are able to actively expel small molecular QACs, which result in resistance. In order to counter this QAC resistance, the incorporation of QAC moiet y to a polymer chain has been developed (Lin et al. 2003; Lin et al. 2002b) The authors believed that the drugr esistant pumps would fail to efflux a polymeric biocide because of the immobilized polycations. This developed polymeric biocide with QAC functional moiety has been found to be active against QAC resistant S.aureus The QAC type biocides have been reported to be inactive against non -envelope viruses. They are commonly used as antibacterial agents (McDonnell and Russell 1999) The antibacterial and antiviral activity of small molecular dialdehyde, i.e., glutaraldehyde has been well studied and reviewed (Gorman et al. 1980; McDonnell and Russell 1999) We believed that introduction of dialdehyde functional moiety to a polymer chain; a novel polymeric biocide with broad -spectrum antib acterial and antiviral activity could be developed. Polyglutaraldehyde has been synthesized from the glutaraldehdye monomer. It was characterized for protein immobilization and cell separation in the late 70s (Margel and Rembaum 1980; Margel et al. 1979; Rembaum et al. 1978) However, the antimicrobial study of polyglutaraldehyde has not been reported yet. Another well known chemistry of introducing dialdehyde functional moiety to polymer chain is the periodate oxidation of polysaccharide. Periodate oxidation of starch and cellulose is

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16 16 a c ommon method to perapre dialdehdye polysaccharide (Jackson and Hudson 1937) Like the polyglutaraldehdey case, the study of the antimicrobial a ctivity of dialdehyde starch (DAS) and dialdehdye cellulose (DAC) is very limited to date. A method to prepare antibacterial surface was described by an early US patent (Siragusa 1977) Dialdehyde polysaccharide granules were first suspended in an agar solution. Once the agar solution solidified, the surface was cross streaked with bacterial suspension. Inhibition of gram -positive/gram negativ e bacterial growth was observed for the surface containing DAC. Inhibition of gram -positive bacteria, but not gram negative bacteria was observed for the surface with DAS. Aldehyde polymers can be prepared from the polymerization of glutaraldehyde or peri odate oxidation of polysaccharides. The focus of this research is on the dialdehyde polysaccharides. Primary concern is the toxicity of glutaraldehyde (Beauchamp et al. 1992) Secondly, the chemistry of the periodate o xidation has been well established. The reaction can be performed in a mild condition and the degree of the oxidation can be readily controlled. Lastly, the antimicrobial activity of dialdehyde polysaccharides can be delivered in various methods such as aq ueous suspension and solid surface. The purpose of our work was to examine the antibacterial and antiviral activity of dialdehyde polysaccharides. Two delivery methods such as the aqueous suspension and the solid surface contact were selected to evaluate their antibacterial and antiviral activity. Additional objectives were to characterize the physicochemical characteristics of the dialdehyde polysaccharides associated with their antibacterial and antiviral activity. The antibacterial mechanisms and the ba cterial inactivation kinetics of dialdehyde starch aqueous suspensions were also investigated.

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17 17 Table 1 1 Chemical structures of biocides in antiseptics and disinfectants Biocide Example Chemical structure Alcohols Ethanol CH 3 CH 2 OH Isopropanol (CH 3 ) 2 CHOH Silanols Triethylsilanol (CH 3 CH 2 ) 3 SiOH Aldehdyes Glutaraldehdye O=CH(CH 2 ) 3 HC=O Formaldehyde O=CH 2 Phenols Phenol Triclosan Quaternary ammonium compounds Benzalkonium chloride AEM 5700 Peroxygens Hyd rogen peroxide H 2 O 2 Peracetic acid CH 3 COOOH Heavy metals Silver compounds Ag Halogen releasing agents Chlorine compounds HOCl 5,5 dimethylhydantoin (N halamine) Biguanides Polyhexamethylene biguanide (PHMB) Chito san

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1 8 18 CHAPTER 2 LITERATURE REVIEW 2.1 Chemistry of Dialdehyde Polysaccharide Polysaccharides such as starch and cellulose are carbohydrate polymers consisting of monosaccharide units. All of the common polysaccharides contain glucose as the monosaccharide unit. Starch and cellulose are the two largest biomass produced on earth (Figure 2 1). Starch and cellulose are environmentally friendly and cost competitive. The major structural feature of starch and cellulose is the 1,4 linkage of their anhydrog lucose units (AGU). The main differences in their physicochemical properties are based on the glycosyl linkage in starch and the -glycosyl linkage in cellulose. Periodic acid and periodates are specific for the oxidative cleavage of 1,2 -glycol stru ctures in carbohydrates such as starch and cellulose to produce aldehydes (Jackson and Hudson 1937) (Figure 2 2). Starch is oxidized readily a t room temperature by aqueous periodic acid solution. Cellulose can also be oxidized in a similar condition but at a slower rate than starch (Ja ckson and Hudson 1937) A commercial process to manufacture dialdehyde starch has already been developed using electrolytically regenerated periodic acid (Radley 1976) In this process, the oxidizing agent periodic acid is reduced to iodic acid during periodate oxidation. This iodic acid can be reconverted to periodic acid by electrolysis. The regenerated periodic aci d is re used to convert another batch of dialdehyde starch. Not all of the carbonyl functions of the dialdehyde polysaccharides are free. In the case of dialdehyde starch, they may form hemiacetal linkages with primary alcohol groups (Figure 2 3, a, b), or hemialdal linkages with water (Figure 2 3, c), or one or both carbonyl groups may be hydrated (Figure 2 3, d) (Guthrie 1961; Haaksman et al. 2006; Radley 1976) For the dialdehyde

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19 19 cellulose, many studies suggest that the majority of its aldehyde groups are in the hemi aldal form and the remainder as hemiacetal or hydrated aldehyde (Guthrie 1961) However, the carbonyl function of dialdehyde polysaccharides, in whatever form it may exist, is quite reactive. These hemiacetal and hemialdal linkages are fairly weak and are readily broken to liberate the c arbonyl groups to react with other functional groups (Mehltretter 1964; R adley 1976) Since they are carrying two aldehyde groups on each glucose unit, dialdehyde polysaccharides can react with other molecules through crosslinking reaction with amino ( NH2), hydroxyl ( -OH) and sulfurhydryl ( SH) functional groups (Jane 1995) The reactivity of the dialdehyde polysaccharides is pH -dependent. Dialdehyde starch has been reported not to react with urea at room temperature in neutral or slightly acid solution, but to react under alkaline condition (Sloan et al. 1956) At elevated temperature, the reaction between DAS and urea occurred at neutral or acidic pH. The in teresting observation in this study was that only one molecule of urea reacted with each dialdehyde unit. One aldehyde group per dialdehyde unit remained in the products. The authors believed that the dialdehyde starch reacted in a hemiacetal form. In anot her study, the reaction of dialdehdye starch with thiourea gave a product with two thiourea per dialdehdye unit (Guthrie 1961) This kind of controversy triggered more studies to investigate the relative reactivity of the two aldehyde groups presented in the dialdehyde starch. In the study of the oxidation of dialdehyde starch with peracetic acid and sodium bromide at pH 5, only partial oxidation was achieved. Mono aldehyde -carboxy starch with approximately 50% carbonyl groups and 50% carboxylic groups was obtained. When it was oxidized b y sodium chlorite and hydrogen peroxide at the same condition, dialdehyde starch was completely oxidized to dicarboxy starch (Haaksman et al. 2006) These results suggest that in some cases both aldehyde

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20 20 groups are reactive, in other cases only one of the aldehyde groups is reactive or both aldehyde gr oups are partially reacted. The authors found that the reaction of dialdehyde starch with sodium chlorite/hydrogen peroxide occurred mainly at the C 3 aldehyde group at the beginning of the reaction. In contrast, both aldehyde groups were reactive with per acetic acid/sodium bromide, whereby C 2 aldehyde was preferentially reacted in the initial stage. The difference in the dialdehyde starch oxidized by different oxidation agents is related to the oxidation mechanism. Sodium chlorite can oxidize aldehyde groups occurring as hydrated and nonhydrated forms. The oxidation of dialdehyde starch by bromine proceeds through the hydrated form and is catalyzed by base (Haaksman et al. 2006) Haaksman believed that the aldehyde group on C 2 would be expected to be more hydrated than the aldehyde group on C 3 becau se of the electron -withdrawing oxygen at the adjacent C 1 atom. The difference of reactivity of the two aldehyde groups in dialdehyde cellulose has been also reported in the literature. Oxidation of dialdehyde cellulose with chlorous acid showed that one a ldehyde group was oxidized faster than the other (Guthrie 1961) In the study of oxidation of dialdehyde cellulose with bromine water, dialdehyde cellulose was suggested to react preferentially in the hemiacetal form with C 3 present as a free aldehyde group (Guthrie 1961) Kim studied the chromatographic separation of aromatic primary amines based on their interactions with aldehyde groups of dialdehyde cellulose gel in a stationary phase (Kim and Kuga 2000) They found the interaction was related to the acid dissociation constant (pKa) of amine and the pH of elunet In the high pKa case (pKa >6) at pH range 4 5.5, amines showed no interaction with aldehyde groups. The authors believed that amines at these large pKas were highly protonated in the test pH range and Schiff base reaction did not occurred. When amines wi th medium pKas (pKa 4 5.3) and low pKas (pKa<3.4) at the test pH range 45.5, the

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21 21 interaction between amines and aldehyde groups were significant. However, the pH -dependence of the interaction was different. For medium pKas amines, the interaction was high er with increasing pH. Whereas, for low pKas amines, the interaction was lower with increasing pH. The authors proposed that in the medium pKa amines, the proportion of protonated amines increased with decreasing pH in the test pH range as the pKas of amin es were in the test pH range. For low pKas amines in the test pH range, amines were believed to be present in the free form. The authors gave a possible explanation that the enhanced interaction was caused by the protonation of resultant imines. The compl ex structure of dialdehyde polysaccharide, i.e., a great number of possible structures, the equilibriums among different structures, and the differences in reaction mechanisms and reaction species seem to be responsible for the reactivity of dialdehyde pol ysaccharide in different conditions. Dialdehyde polysaccharides such as dialdehdye starch and dialdehyde cellulose are alkaline liable. Cannizzaro reaction between two aldehyde functions and alkoxycarbonyl elimination at C 5 have been found during alkali ne degradation (Fry et al. 1942; Whistler et al. 1959) The pH effect on the degradation of dialdehdye starch in water heated at 90 C in a pH range from 3 to 7 have been studied (Plunkett 1968; Veelaert et al. 1997a) The degradation of dialdehyde starch was very limit ed at pH range 3 4 for up to five hours heating. At pH 7, extensive chemical degradation occurred by a decrease of the aldehyde content, accompanied by acid release and a decrease of the average molecular weight. At pH 5, degradation still occurred but to a much lower extent. The physical properties of dialdehyde polysaccharides have been reviewed (Guthrie 1961) With increasing degree of oxidation, crystallinity of the dialdehyde polysaccharides

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22 22 decreases. They become completely amorphous in the high degree of oxidation. For example, w hen degree of oxidation of dialdehdye starch is above 80%, it becomes completely amorphous. 2.2 Toxicity of Dialdehyde Polysaccharide Dialdehyde starch was found to display no toxicity when fed to Guinea pigs (Gaddy et al. 1975) It was applied as a therapeutic agent and a feed additive to control ammonia toxicity in ruminants (Gaddy et al. 1974) The acute oral toxicity of 100% oxidized dialdehyde starch can not been detected when given to young adult rats at 1g/1kg dose (Wilson 1959) The oral acute toxicity of 10% dialdehyde starch (100% oxidized) aqueous suspension was reported to be LD50 (Radley 1976) Dialdehdye starch also showed low toxicity to rats by dermal and respiratory routes (Tang et al. 2003) The biologic al toxicity of dialdehyde starch nanoparticles up to 0.05% weight percentage loading were found to be the same as natural starch toward mammary cells in vitro biological toxicity analysis (Yu et al. 2007) 2.3 Biomedical Application of Dialdehyde Polysaccharide Application of dialdehyde polysaccharides depends on their rich reactivity of aldehyde groups. Due to its crosslinking ability, dialdehdye starch has been employed in the paper, leather and textile industry (Fiedorowicz and Para 2006; Jane 1995; Kanth et al. 2006; Mehltretter 1964; Radley 1976) The potential application of dialdehyde starch as a component of biodegradable plastics was also invest igated (Jane et al. 1994; Langmaier et al. 2008; Spence et al. 1995; Ustunol and Mert 2004) Dialdehyde cellulose can serve as intermediates for cellulose derivatives. Dialdehyde cellulose was reported as the column packing and chromatogra phy material (Kim and Kuga 2000; Pommerening et al. 1992) The focus of the review of application of dialdehyde polysaccharides is their biomedical application.

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23 Having the reactive aldehyde groups, dialdehyde starch has been grafted onto different substrates such as poly vinyl alcohol and polystyrene (Ikada et al. 1979; Onyiriuka 1996) After the surface was activated by the dialdehyde starch, it demonstrated effective binding ability to biological active molecules such as proteins and cells with no loss in activity. This approach can be used for the surface m odification for the protein absorption and cell culture. Surface modification by dialdehyde starch has been utilized as a urea adsorption agent for the medical treatment of chronic kidney disease (Onishi and Nagai 1986) Because of its low toxicity, dialdehyde starch has been studied as a drug delivery carrier (Onishi and Nagai 1986; Yu et al. 2007) Dialdehdye starch can form a Schiff base or conjugation with drugs such as doxorubicin or isoniazid. After the drug loading, the drug can have controlled released because of the biodegradability of dialdehyde starch or gradual dissolution of the conjugate. The in vitro study showed by this approach an effective and sustained drug release delivery system was achieved. Dialdehdye cellulose has also been explored as a drug release carrier and an adsorbent of heavy metal ions and protein (Kim et al. 2004) Dialdehyde cellulose has been investigated as a hemostatic agent in an osseous environment (Laurence et al. 2005) This material was found to have cytotoxicity, biocompatibility and resorption properties similar to control (comme rcialized Surgicel gauze) but its hemostatic power was higher. Dialdehyde cellulose was also studied as a wound dressing material (Edwards et al. 2001) Neutrophil elastase is present in a relatively high level in chronic pressure ulcers (Edwards et al. 2001) Elastase may impede wound healing by degrading extracellular matrix proteins, peptide growth factors and cell surface receptors. It was found that dialdehyde cellulose significantly removed elastase from the chronic wound and inhibited the elastase activity by the selectively absorption of neutrophil elastase in the wound fluid.

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24 Unlike the increasing study of dialdehyde polysaccharide in biomedical application, antimicrobial studies are very limited. Studies have been rep orted on the antimicrobial activity using dialdehyde polysaccharide (Hou et al. 2008; Tang et al. 2003) .However, the antimicrobial activity was provided by chiotsan. Dialdehyde stach was employed as a crosslinking agent to improve the mechanical and water -swelling properties of chiosan film, or chiotsan was grafted onto the dialdehyde cellulose fibers. Other antimicrobial activity studies have been focused on the derivatives of dialdeh yde starch (Barabasz et al. 1986; Para et al. 2004) Only a patent in 1977 described a method of inhibiting microbial activity using insoluble dialdehyde polysaccharide (Siragusa 1977) Polysaccharide powders such as dialdehdye starch or dialdehyde cellulose were added to molten nutrient agar at approximately 45 C. The agar medium was spread onto sterile Petri dish followed by the solidification. The solidified agar surface was cross -streaked with 10 l aliquot of a microbial suspension. The agar plates were incubated to determine the growth of the test microbe. The data in this patent indicated that the dialdehyde cellulose inhibited the growth of gram -negative/positive bacteria, whereas, dialdehyde s tarch only inhibited the growth of gram -positive bacteria. 2.4 Antimicrobial Studies of Glutaraldehyde Dialdehyde polysaccharides have the similar dialdehyde functions as glutaraldehyde. It is helpful to understand the potential biocide application of dia ldehyde polysaccharides by surveying of the antimicrobial activity and mechanism of glutaraldehdye. Glutaraldehyde has a broad -spectrum activity against bacteria, spores, fungi and viruses. Several excellent reviews of the antimicrobial action of glutarald ehyde can be found in literature (Gorman et al. 1980; McDonnell and Russell 1999) Antimicrobial mechanism of glutaraldehyde is summarized in

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25 Table 2 1 (McDonnell and Russell 1999). In general, antimicrobial mechanism of glutaraldehyde involves a strong association with the reactive sites of microorganism. Bacteria are defined into two groups based on the staining procedure developed by Christian Gram. Crystal violet can stain gra m -positive bacteria to purple, but not on gram negative bacteria. The bacterial structure is illustrated in Figure 2 4 (Kim 2006) Gram -positive bacteria have a thick dense cell wall, mainly peptidoglycan (4090%). Gram -negative bacteria have a thin cell wall (5 10% peptidoglycan) with an extra outer membrane (lipopolysaccharide, porin and phospholipids). The peptidoglyca n retains the cell shape and prevents osmotic lysis. It does not have the function as permeability barrier. In contrast, the outer membrane of gram negative bacterial cell is a permeability barrier. The plasma membrane in both types of bacteria is also a p ermeability barrier, mainly composed of phospolipid and proteins (Kim 2006) Regarding the gram positive bacteria, co nsiderable corsslinking reaction between glutaraldehyde and the abundant peptidoglycan in the cell occurs. This crosslinking reaction results in a strengthening and sealing effect on the wall. Glutaraldehyde can also react with cell wall protein to enhance the sealing effect. Transport processes such as nutrient is inhibited to affect enzyme activity. Although the peptidoglycan layer is more loose and thin in the gram negative bacteria, it still offers numerous reactive sites to react with glutaraldehyde. I n addition to that, the outer membrane (lipoprotein components) can also react with glutaraldehyde. Certain cell wall associated or periplasmic located enzymes can be inactivated by glutaraldehyde (Gorman et al. 1980) In the study of antiviral action of glutaraldehyde against bacteriophages, glutaraldehyde was fo und to bind to virus DNA as well as to react with proteins (McDonnell and Russell 1999) For aldehydes without the crosslinking ability to form the protein DNA crosslinks, these

PAGE 26

26 aldehydes have no effect on DNA synthesis (McDonnell and Russell 1999) .Low concentration (<0.1%) alkaline glutaraldehyde are effective against purified poli ovirus, whereas poliovirus RNA is highly resistant to glutaraldehyde up to 1%. The complete poliovirus particle is much more sensitive than poliovirus RNA (McDonnell and Russell 1999) Based on this information, the antiviral action has been inferred to the interaction between glutaraldehyde an d capsid proteins of poliovirus. Glutaraldehyde is more active at alkaline pHs than at acidic pHs. The enhanced biocidal activity is proposed to be the pH effect on the glutaraldehyde structure, or the outer layers of the microbial cell, or the both (Gorman et al. 1980) As the pH is changed from acidic to alkaline, more reactive sites such as free amino groups will be formed at the cell surface leading to a more rapid bactericidal effect (McDonnell and Russell 1999) This new reactive sites was supported by the greater uptake of glutaraldehyde by bacteria in alkaline condition than in acid condition (Gorman and Scott 1977) The pH effect on the structure of glutaraldehyde is more complicated. In the acid condition, gl utaraldehyde monomer is in equilibrium with the various hydrates. Aldol -type unsaturated polymers have been confirmed in alkaline solution (Gorman et al. 1980) Adol type polymers have been shown to react with proteins readily in alkaline solution, but not to react with proteins under acid state (Gorman et al. 1980) A similar observation was also found for the reaction between g lutaraldehyde and malt extract broth at 37 C (Gorman and Scott 1977) Acid glutaraldehyde was shown to have negligib le reactivity over a 6 hour period compared to the considerable reaction in the alkaline solution. Even though alkalination can enhance the antimicrobial activity of glutaraldehyde, extensive loss of reactive aldehyde groups by

PAGE 27

27 polymerization in alkaline s olution, is responsible for the rapid loss of biocidal activity of alkaline solution on storage (Gorman et al. 1980) The toxicity of glutaraldehyde has already been reviewed (Beauchamp et al. 1992; Takigawa and Endo 2006) Many adverse health effects of glutaraldehyde exposure on humans have been reported. The Oral LD50 in rats for a 25% glutaraldehyde solution was reported to be 60mg/ kg (Gorman et al. 1980) 2.5 Design Antimicrobial Solid Surface Transmission of infectious pathogens by surface contact is a growing concern to medical providers and the public health. Antimicrobial surface design is helpful to protect the spread of pathogens in a variety of applications such as in health care facilit ies, textiles and buildings. For example, in textile industry, the production of antimicrobial textiles was 100,000 tones worldwide in 2000. Furthermore, the production of antimicrobial textile (increased more than 15% a year) was one of the fastest growing sectors of the textile market in Western Europe between 2001 and 2005 (Gao and Cranston 2008) Some commercially used biocides and those under development for the treatment of various fibers are summarized in Table 2 2 (Gao and Cranston 2008) The common design is to select a biocide illustrated in Table 1 1. The antimicrobial textile treatment is obtained whether by direct biocide incorporati on such as silver, polyhexamethylene biguanide and triclosan or by surface bound of biocide to the textile fiber such as AEM 5700, N -halamine and peroxyacid. Conventional approaches to prevent the growth of microorganisms on the substrates are achieved by controlled release of bioactive molecules from the substrate (Haufe et al. 2005; Mahltig e t al. 2004; Mahltig et al. 2005) They are usually made in two ways: 1) Incorporation of a biocide to a substrate, the migration or diffusion of biocide out of the substrate provides the antimicrobial activity. 2) A biocidal compound is grafted to a subst rate by a bond sensitive to

PAGE 28

28 hydrolysis (Hazzizalaskar et al. 1993) However, the biocidal activity by this approach depends on the liberation of the biocide into the environment A progressive loss of activity with time and a hazard to the environment is inevitable. Because of this drawback of controlled release, another approach to design a non -leaching bioactive surface is our interest. The strategic question to design a non -leaching antimicrobial surface is how to introduce the antimicrobial functional moiety to the substrate by covalent bond. Two common approaches have been reported to design a nonleaching antimicrobial surface. One is a small molecular biocide can be a nchored to a substrate by covalent bond. The other is chemical modification of the material of substrate with antimicrobial functional moiety. 2.5.1 Anchor a Small Molecular Biocide to a Substrate The frequently used small biocides are already summarized in Table1 1. The functional antimicrobial groups are quaternary ammonium salts (QAC), N -halamines, aldehydes and others. The most studied systems are QAC and N -halamine. They have the common structure as: X -R Y. X is the anchor group, usually is trialkoxys ilyl group, which can form oxane bond to the substrate containing hydroxyl group. Y is the antimicrobial group, which can be QAC, N halamine and other antimicrobial functional moiety. R is the main structure, usually C4 to C18 alkyl chain. The well studied QAC type is AEM 5700 (Isquith et al. 1972; Isquith and McCollum 1978; Walters et al. 1973) (Figure 2 5). AEM 5700 can be bonded to various substrates including glass, ceramic, cotton, wool and linen. The treated substrates exhibit broad -spectrum antimicrobial activity including gram positive and gram -negative bacteria, alage, fungi and yeast (Isquith et al. 1972) It has already been a commercial product applied in textile and other areas for two decades. Hydantoin (N -halamine) can also be bound to a substrate by introduction of trialkoxysilyl group in the hydantoin chemical structure demonstrated in Figure 2 6 (Barnes et al. 2006; Liang

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29 et al. 2005) Trialkoxysilyl type N -halamine compound can be bound to different substrates following the similar scheme as AEM 5700. The treated substrates also demonstrated broadspec trum antimicrobial activity. Furthermore, because of the reversible reaction chemistry of N -halamine (Akdag et al. 2006; Qian and Sun 2005; Sun and Worley 2005) the antimicrobial activity can be regenerated. New hydantoin derivatives have also been developed (Qian and Sun 2003; Sun et al. 2001a) such as monomethylol 5,5, dimethylhydantoin and dimethylol 5,5 dimethylhydabtoin. These N halamine derivatives can be directly grafted onto cellulose textile during chemical finishing process. 2.5.2 Polymeric Biocide Instead of anchor ing the small molecular antimicrobial agent onto a substrate, chemical modifications of the materials of substrates, mainly polymers, have been received great attention to prepare polymeric biocides. Biocidal polymers can be incorporated into other polymer s, or extrude themselves. They can be employed for surface contact disinfectants in many applications, such as sterile bandages and clothing in the biomedical industry. The advances of biocidal polymers have been summarized in several reviews (Gao and Cranston 2008; Kenawy e t al. 2007; Klibanov 2007; Lewis and Klibanov 2005; Worley and Sun 1996) Preparation of biocidal polymers in general can be classified into two categories. One is the polymerization or copolymerization of monomer bearing antimicrobial functional groups the other is introduction of antimicrobial functional groups to synthetic or natural polymers by chemical modification. Two antimicrobial functional groups, quaternary ammonium salt (QAC) and N halamine, are still the most studied. Polymerizations of mo nomers containing QAC moieties (Dizman et al. 2004; Imazato et al. 1999; Imazato et al. 1995; Punyani and Singh 2006) have been studied a lot. A relatively new

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30 class of bicodial polymers with cyclic N -halamines has been developed at University of Auburn (Sun et al. 1995; Sun and Sun 2002; Sun et al. 2001b; Sun and Sun 2001) The monomers containing cyclic N -halamine s were synthesized. These monomers were copolymerized with other monomers such as acrylonitrile, methyl methacrylate and vinyl acetate. Polyglutaraldehyde was also prepared from the polymerization of glutaraldehdye monomer (Margel and Rembaum 1980) However, no antimicrobial study of this aldehyde type polymer has been reported in literature. Quaternization of synthetic and natural polymers is a common method to chemically modify polymers to render their antimicrobial activity. Qu aternization of chitson can be obtained whether direct N alkyl quaternization of amine functional groups in chitosan structure or reaction of quaternary ammonium salt containing reactive functional group with amine (Kim et al. 1997; Nam et al. 1999) Quaternization of various polymer s have been reported extensively in literature including polyurethane, polysiloxane, polyvinyl N -hexylpyridinium, polyethylenimine and other polymers (Hazzizalaskar et a l. 1993; Lin et al. 2002a; 2003; Lin et al. 2002b; Nurdin et al. 1993a; Sauvet et al. 2000) N halamine or hydantoin chemical modification of polymers has also gained increasing interest in research for various polymers such as polystyrene, polyamide, pol yurethane, polyesters and others (Chen et al. 2004a; b; Grunzinger et al. 2007; Grunzinger and Wynne 2006; Lin et al. 2001; Lin et al. 2002c; Williams et al. 2005) Sometimes, these two chemical modification were combined to prepare biocidal polymer (Liang et al. 2006) Only an earlier US patent described the antimicrobial activity of dialdeh yde polysaccharide (Siragusa 1977) 2.5.3 Comments As in the aforementioned discussion, the dominant study of the design of a non -leaching a ntimicrobial surface is focusing on the QAC and N -halamine types. The QAC -type

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31 antimicrobial polymer is basically a polycationic material. The salinity of media could affect the antimicrobial activity of QAC type non leaching antimicrobial surface. Antimicrobial action of small molecular QAC can be blocked by ion -pairing and precipitation of quaternary cations with bulky anions (Kopecky 1996) The advantage of N -halamine is the regeneration of its antimicrobial activity by chlorine bleach. However, this regeneration also causes problem. After the recharge, a substantial amount of adsorbe d chlorine remains on the surface. A reduction step (i.e. with sodium sulfite) need to be used to remove the unbound residual chlorine from the surface (Gao and Cranston 2008) Furthermore, the preparation of polymeric biocide usually involves multip le chemical reactions as well as costly polymers and other reactants. A simple and convenient method needs to be developed to prepare polymeric biocide. 2.6 Microbial Inactivation Kinetics Microbial inactivation kinetics, whether through chemical, thermal, radioactive or other processes, are quite complex. Three variables including the concentration/dose of the inactivation agent (C), intensity of mciroorganism response and the exposure time (t) must be considered in a kinetics study. We only present the re view of microbial inactivation kinetics by chemical agent here. Various mathematical models have been proposed for microbial inactivation dependent on the nature of the lethal chemical agent (Peleg 2002; Peleg et al. 1997; Peleg and Penchina 2000) The situation becomes more complicated if the agents concentration decreases as a result of evaporation, chemical reaction or both during exposure. The diminishing effective concentration may affect the survival pattern of the microbe (Peleg 2002; Peleg et al. 1997; Peleg and Penchina 2000) When the lethal chemical agent concentration remains practically unchanged during the continuous exposure, mic robial mortality has been considered a first order kinetics (Peleg 2002; Peleg and Penchina 2000; Winks 1984; Winks and Waterford 1986) First -order inactivation

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32 kinetics has been observed in various inactivation systems (Peleg and Penchina 2000) Inactivation of Bacillus subtilis spores by glutaraldehyde, formaldehyde, hydrogen peroxide, peracetic acid, sodium hypochlorite and phenol has been reported to follow first order (Retta and Sagripa nti 2008) It was acknowledged that the empirical model, i.e., Habers rule is satisfactory to estimate the time response of an organism to an agent, when the quantity of the agent available for reaction is in abundance (Bunce and Remillard 2003; Winks 1984; Winks and Waterford 1986) Habers Rule: Ctm = K (3 2) C is the concentration of dose, t is the time of exposure, K is a constant. When m=1, Habers rule can describe a first order kinetics. When m 1, the effect of exposure time on the response is significant. When m 1, the effect of dose concentrat ion on the response is dominant. In fact, Habers rule is only an empirical description without any theoretical derivation.

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33 Table 2 1 Mechanism of antimicrobial action of glutaraldehyde Microorganism Mechanism Bacteria Strong association with outer lay ers of cell, crosslinking of amino groups in protein, inhibition of transport processes into cell Viruses Actual mechanisms unknown, but involve protein DNA crosslinking and capsid changes Bacterial spores Probably as a consequence of strong interactio n with outer cell layers Fungi Fungal cell wall appears to be a primary target site, with postulated interaction with chitin Table 2 2 Antimicrobial textile treatments Biocide Commercial product Comments Silver Yes Slow release, durable but Ag can be depleted AEM 5700 (QAC type) Yes Covalent bond, very durable, possible QAC bacterial resistance PHMB Yes Large amount required, potential bacterial resistance Triclosan Yes Large amount needed, bacterial resistance, breaks down into toxic dioxin, banned in some European countries Chitosan No Low durability, poor handle on textile N halamine No Need regeneration, odor from residual chlorine Peroxyacids No Needs regeneration, poor durability

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34 Figure 2 1 Chemical structures of simple star ch and cellulose Figure 2 2 Periodate oxidation of AGU to generate dialdehyde function Figure 2 3 Some elementary structures of dialdehyde starch glycosyl 1,4 linkage Simple Starch glycosyl 1,4 linkage Cellulose

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35 Figure 2 4 Bacterial structures of gram -positive and gram -negative bacteria Figure 2 5 Schematic illustration of AEM 5700 onto surfaces to form surface bound antimicrobial activity Figure 2 6 Synthesis of 3 triethoxysilylpropyl 5,5 dimethylhydantoin (CH (CH2 2) )3 3NC NC18 18H H37 37Cl Cl (CH (CH3 3) )2 2+ + O O (O (O Si Si O) O)x x Cotton textile Cotton textile Biocidal Biocidal action action Escherichia coli Escherichia coli (CH (CH2 2) )3 3NC NC18 18H H37 37Cl Cl (CH (CH3 3) )2 2+ + O O (O (O Si Si O) O)x x Cotton textile Cotton textileSurface recovery Surface recovery (CH (CH2 2) )3 3NC NC18 18H H37 37Cl Cl (CH (CH3 3) )2 2+ + O O (O (O Si Si O) O)x x Cotton textile Cotton textile (CH (CH2 2) )3 3NC NC18 18H H37 37Cl Cl (CH (CH3 3) )2 2+ + O O (O (O Si Si O) O)x x (CH (CH2 2) )3 3NC NC18 18H H37 37Cl Cl (CH (CH3 3) )2 2+ + O O (O (O Si Si O) O)x x (CH (CH2 2) )3 3NC NC18 18H H37 37Cl Cl (CH (CH3 3) )2 2+ + (CH (CH2 2) )3 3NC NC18 18H H37 37Cl Cl (CH (CH3 3) )2 2+ + (CH (CH2 2) )3 3NC NC18 18H H37 37Cl Cl (CH (CH3 3) )2 2+ + (CH (CH2 2) )3 3NC NC18 18H H37 37Cl Cl (CH (CH3 3) )2 2+ + O O (O (O Si Si O) O)x x O O (O (O Si Si O) O)x x (O (O Si Si O) O)x x Cotton textile Cotton textile Biocidal Biocidal action action Escherichia coli Escherichia coli (CH (CH2 2) )3 3NC NC18 18H H37 37Cl Cl (CH (CH3 3) )2 2+ + O O (O (O Si Si O) O)x x Cotton textile Cotton textile (CH (CH2 2) )3 3NC NC18 18H H37 37Cl Cl (CH (CH3 3) )2 2+ + O O (O (O Si Si O) O)x x (CH (CH2 2) )3 3NC NC18 18H H37 37Cl Cl (CH (CH3 3) )2 2+ + O O (O (O Si Si O) O)x x (CH (CH2 2) )3 3NC NC18 18H H37 37Cl Cl (CH (CH3 3) )2 2+ + (CH (CH2 2) )3 3NC NC18 18H H37 37Cl Cl (CH (CH3 3) )2 2+ + (CH (CH2 2) )3 3NC NC18 18H H37 37Cl Cl (CH (CH3 3) )2 2+ + (CH (CH2 2) )3 3NC NC18 18H H37 37Cl Cl (CH (CH3 3) )2 2+ + O O (O (O Si Si O) O)x x O O (O (O Si Si O) O)x x (O (O Si Si O) O)x x Cotton textile Cotton textileSurface recovery Surface recovery

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36 CHAPTER 3 ANTIMICROBIAL ACTIVITY OF DIALDEH YDE SUSPENSION 3.1 Introduction Dialdehyde starch retains the original parent starch granular form. Proper cooking can disperse dialdehyde starch granules in water with minimal depolymerization. The optimal condition to cook a high degree oxidation of dial dehyde starch with minimal degradation has been reported (Plunkett 1968; Veelaert et al. 1997a; Veelaert et al. 1997b) When dialdehyde starch was cooked at 90 C up to 5 hours at pH values be tween 3 4, the degradation of dialdehyde starch was limited. Gelation of dialdehdye starch was observed during the cook (Veelaert et al. 1997b) A gelation mechanism was proposed. Upon heating in the water, the DAS granules first swelled, followed by the disruption of the granules to release the DAS polymeric molecules. Finally, the gel form ation occurred by the physical entanglements and chemical crosslinks. The effective use of dialdehyde polysaccharides depend on the interaction between dialdehyde functions and the targets. It is proposed that the gel structure can be disrupted by dispersi on technology such as sonication. In this chapter, studies are aimed at the processing method such as the cooking and sonication effect on the antimicrobial activity of dialdehyde starch. The dominant antimicrobial action of dialdehyde starch was identifi ed. Bacterial inactivation kinetics was studied and a model to describe the inactivation kinetics was established. The physicochemical changes of dialdehyde starch by processing methods as well as by pH were characterized. The pH effect on the antimicrobia l activity of dialdehyde starch was also studied.

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37 3.2 Materials and Experiments 3.2.1 Materials Dialdehdye starch was purchased from Sigma (P9265). High-degree oxidized corn starch D17F was kindly supplied by Grain Processing Corporation, Iowa, USA. Both s tarches are in granular forms and were used without further purification. 3.2.2 Preparation of Starch Suspension The dialdehyde starch granular suspension was prepared by simply mixing the as received DAS with deionized water at room temperature (DAS granular suspension). The cooking condition was selected based on the literatures that degradation or depolymerization occurred to a very low extent (Plunkett 1968; Veelaert et al. 1997a) The dialdehyde starch aqueous suspension was prepared by stirring the dialdehyde starch in deionzied water at 9095 C for two hours i n an oil bath with reflux. Typically, three grams of dialdehyde starch was mixed with 97 gram deionzied water. There was negligible total weight change before and after the cook. The suspension was then cooled down to room temperature to obtain a 3% dialde hyde stach aqueous suspension (DAS aqueous suspension). 5 wt% oxidized starch was also prepared in the same way (OS aqueous suspension). 3.2.3 Preparation of Microbial Stock Solution The studied microorganisms are list in Table 3 1. All microorganisms were obtained from the Department of Microbiology at the University of Florida. The bacterial suspensions were prepared according to the procedure by Kim (Kim et al. 2006) The bacteria were inoculated in the Columbia broth overnight at 37 C with constant agitation whi le under the aerobic condition. The bacterial cells were collected by centrifugation at 1000g RCF (relative centrifugal force) for 10min at 4 C and washed three times with sterilized

PAGE 38

38 deionized water. The obtained bacterial pellet was resuspended in sterili zed deionized water after final washing to obtain 2 6109 CFU/ml (colony forming unit) stock concentrations. The preparation of bacteriophages (bacterial viruses) is more complicated than the preparation of bacteria. First, a host bacterial culture in earl y log phase of growth was inoculated with a sample containing bacteriophages in Tryptic soy broth (TSB broth). The culture was incubated for four to ten hours followed by centrifugation to remove bacterial debris until there was no observation of any bacte rial pellets. The supernatant fraction was filtered through a 0.2 m pore size filter to remove any remaining bacteria. The supernatant was used as a virus stock and diluted by 1% TSB broth with approximately 109 PFU/ml (Plaque Forming Unit) concentrations. The human virus, i.e., Poliovirus 1 stock, was provided by the D epartment of Microbiology at the University of Florida with approximately 107 PFU/ml concentrations. 3.2.4 Degree of Oxidation of As -received Dialdehyde Starch The oxidation extent of the as received DAS was analyzed by the elemental analysis using the method described by Kim (Kim and Kuga 2001) Typically, hydroxylamine hydrochloride (0.02 mol) was dissolved in 100ml of pH 4.4 acetate buffer (0.1M) and then 100mg dialdehyde starch granules were added. The mixtue was stirred at room temperature for 48 hours. In th is prolonged time, all the aldehyde functiona groups were converted to oxime by a Schiff base reaction. The product was washed with deionized ware, filtered and dried at room temperature in vacuum oven for two days. The carbon and nitrogen content of the o ximes obtained were determined in the CHN lab in the Department of Chemistry at the University of Florida. 3.2.5 X -ray Diffraction X ray diffraction patterns were recorded with a Philips APD3720 X ray diffractometer with Cu K radiation at 40KV and 20mA in the 2 range of 5 70 in MAIC.

PAGE 39

39 3.2.6 Sonication Sonication was carried out using a Misonix S3000 sonicator with output power 120w. The temperature during sonication was kept constant at room temperature. The dialdehyde starc h suspensions were sonicated one hour, if not otherwise specified. 3.2.7 Solubility The solubility of dialdehyde starch in deionized water was performed by the combination of centrifugation and freeze -drying. The as -prepared dialdehyde starch aqueous suspe nsions were adjusted in different pH values. These DAS aqueous suspensions were centrifuged at 10,000g RCF at 4 C for 30min to obtain a sedimentation fraction and a supernatant. The sedimentations and supernatants were freeze -dried for 24 hrs. Solid weight in each fraction was measured. Solubility was expressed as the weight percentage of the solid in the DAS supernatant over total solid weight of DAS. 3.2.8 Fourier Transform Infrared Spectroscopy (FTIR) FTIR spectra of dialdehyde starch solids (1% in KBr) were recorded by a Thermo electron magna 760 FTIR with a DTGS detector in a diffuse reflection mode using 128 scans at resolution of 4cm1 in PERC. 3.2.9 Ultraviolet Visible Spectroscopy (UV VIS) UV-VIS spectra of the DAS suspensions and DAS solids were obtained by a Perkin -Elmer Lambda 800 UV -VIS spectrometer in the transmission and reflectance modes respectively in PERC. Quartz cuvettes were used for the suspension measurement. 3.2.10 Particle Size Measurement Light scattering instruments in PERC including Coulter LS13320 and Brookhaven Zetaplus were employed to measure the particle size of DAS suspensions using refractive index

PAGE 40

40 1.56 calculated from the refractive index increment data of 100% oxidized DAS reported by Levine (Levine et al. 1959) 3.2 .11 Viscosity Measurement Three percentage (wt) dialdehyde aqueous suspensions cooked at 90 95 C in different time were prepared. The suspensions were cooled down to the room temperature and the viscosities of the suspensions were measured by a Brookfield CAP2000 viscometer in 500RPM speed in PERC. The viscosity data was reported as the mean of triplicate tests. 3.2.12 Nuclear Magnetic Resonance Spectroscopy (NMR) DAS supernatants were analyzed using 1H and 13C liquid NMR. The 500MHZ NMR spectrometer in t he McKnight Brain Institute at the University of Florida was used. DAS supernatants were prepared in deionized water as aforementioned. The DAS supernatant was mixed with D2O in a NMR tube. For the 1H NMR, the chemical shift of H2O was suppressed. The purp ose of the NMR study was to identify aldehyde and C=C chemical shifts of DAS supernatant. 3.2.13 Antimicrobial Test The antimicrobial tests were carried out by adding 0.1ml microbial aliquot into 9.9g test medium. Control runs were also conducted under t he same conditions except the DAS was not included. The suspensions were stirred for one hour and four hours at room temperature. Samples were taken out after one hour and four hours for the plate count. Bacterial samples were serial diluted by a phosphate buffer saline (PBS) and then plated on Tryptic soy agar. After 24 48 hrs incubation at 37 C, the colonies that grew on the agar were counted in order to estimate the number of viable bacteria. Samples containing bacterial virus were serial diluted by 1% T SB broth. The numbers of phage were determined by using the appropriate host bacterium and a soft agar overlay after 12 24 hrs incubation at 37 C. Poliouvirus 1 was grown on Buffalo Green

PAGE 41

41 Monkey (BGM) kidney cells. Viruses were serial diluted in Minimal Es sential Medium (MEM) with 2% fetal calf serum (FCS) and plated on BGM cells using an agar overlay procedure and the number of poliovirus was determined after 2448 hrs incubation at 37 C (Lukasik et al. 2000) For virus experiments, the number of virus PFU in the zero time was same for the control and as -prepared DAS aqueous suspension. We also found the residual DAS in zero dilution had no effect on the viability of host bacteria for up to four -hour exposure. The time effect on the microbial viability was negligible for the controlled samples during the antimicrobial test up to four hours. Minimum lethal concentration ( MLC) and log reduction were employed to evaluate the antimicrobial activity of DAS against test organisms. MLC was defined as the lowest concentration of DAS aqueous suspension to completely inactivate the test organism in one -hour exposure. In this case, a 7 -log reduction was achieved to determine the MLC for bacteria. The number of microorganism for the controlled in one -hour exposure was chosen as baseline to calculate the log reduction. The log reduction was determined as: Log reduction = Log N,c1Log N,tt (3 1) Where N,c1 was the numbers of microbe in the controlled sample at time 1 hour, N,tt was the numbers of survival microbe in the tested sample at time t (t= 1 or 4 hour). The MLC or the log reduction was determined by at least triplicate tests. 3.2.14 Bacterial Inactivation Study E.coli and S.aureus were selected for the bacterial inactivation kinetics study. The experiments were performed in an incubator. The temperature of the incubator can be adjusted from 4 C to 37 C and be kept constant at the set temperature. The MLCs of dialdehyde aqueous suspension to achieve 7 log reduction at different exposure time in four -chosen temperatures were determined. In the studied temperatures, the experimental time had no effect on the

PAGE 42

42 bacterial viability fo r the controlled samples. The dialdehyde starch aqueous suspensions for the kinetics study were non -sonicated. All the MLCs of bacterial experiments were determined at least triplicate repeats. 3.3 Results and Discussion 3.3.1 Characterization of the As -Re ceived Dialdehyde Starch The physical properties of the as received dialdehyde starch are shown in Table 3 2. The as received dialdehyde starch was a high degree oxidation and amorphous material. 3.3.2 Cooking Effects on the Physicochemical Transition of t he Dialdehyde Starch DAS aqueous suspension prepared in our study became homogeneous and transparent with a yellow color upon heating at 95 C for 2 -hour. After several hours storing at room temperature, gel formation was observed in the yellow color liqui d phase. However, in the DAS granular suspension (zero time), the DAS granules were observed to settle to from a clear colorless water phase. The solubility of DAS before and after 2 -hr cooking is presented in Table 3 3. Most of the 2 hr cooked DAS stayed in water phase after high -speed centrifugation. The faint yellow color of DAS aqueous suspension was developed with cooking time (Figure 3 1). The viscosity of the DAS suspension was reported to decrease because of the swelling and fragmentation of the DA S granules upon heating (Levine et al. 1959) The viscosity of dialdehyde starch cooked in different time is also presented in Table 3 3. The viscosity of dialdehyde starch suspension decreased with increasing cooking time, but the difference was not v ery significant. Because the diluted suspensions (only 3%) were examined in this study, the measured viscosities were close to the viscosity of deionized water. Further studies were focused on the dialdehdye granular suspension (zero time cooked) and the dialdehdye aqueous suspension (2 hour cooked). X ray pattern of the as -received dialdehyde

PAGE 43

43 granular and the film deposited from the dialdehyde aqueous suspension are displayed (Figure 32). The studied dialdehyde starch was purchased; the information of t he source starch to prepare the dialdehyde starch was not available from the manufacturer. The manufacturer did not want to sell the source starch as well as disclose any information regarding to it. A literature study was used as reference (Figure 3 3) (Fiedorowicz and Para 2006) Native potato starch exhibited a strong diffraction pattern centered around 13 15 2 A complete lack of a diffraction pattern was observed for DAS15 and DAS25 samples, which is a typical characteristic of amorphous samples. The diffraction pattern obtained for the as received DAS granule was similar to the DAS15 and DAS25 samples in the reference. The total loss of crystallinity of the as -received DAS granule was anticipated, because of the high degree of oxidation. The degree of oxidation for the as received DAS granule was 90% provided by the manufacturer as well as by the elemental measurement shown in Table 3 2. The prepared DAS aqueous suspension was deposited on a glass slide at room temperature, f ollowed by vacuum dry also at room temperature. X -ray pattern of this sample is illustrated (Figure 3 2). The diffraction pattern of this sample was lower and wider than that of the as received DAS granule. This behavior indicated a continuing collapse of the starch granule organization during cooking The cooking effect on the DAS particle size was investigated by the light scattering instruments. A Coulter LS13320, an instrument measuring particle size greater than 40nm, was used to analyze the particle s ize of the DAS granular suspension and the as -prepared DAS aqueous suspension. Based on the solubility data in Table 3 3, most of the DAS stayed in the aqueous phase after high -speed centrifugation. This supernatant was analyzed by Brookhaven

PAGE 44

44 Zetaplus usin g multimodal size distribution. Zetaplus can detect particle size below 40 nm. The particle size measurements are shown in Figure 3 4 and Table 3 4. No significant particle size change between the as -prepared DAS aqueous suspension and the DAS granular sus pension was detected by the Coulter LS13320. In the gelation mechanism proposed by Veelaert (Veelaert et al. 1997b) aggregation and swelling of DAS granules first occurred during cooking in deionized water. With continuous heating, most of the granules disappeared. A low viscosity gel suspension was obtained when it was cooled and stored at room temperature. With this gel formation, the particle size of the as -prepared DAS aqueous suspension was similar to the as received DAS granular suspension. In our study, the dominant portion of the DAS stayed in the water. A dramatically decrease of th e DAS particle size was observed for the supernatant. Furthermore, the freeze-drying solids of the supernatant can be easily dispersed in deionized water at room temperature to obtain a homogeneous suspension. Whereas, the freeze -drying solids of the sedim entation cannot form a homogeneous aqueous suspension. It is clear that the solubility and particle size of dialdehyde starch were significantly affected by the selected cooking condition. The development of a faint yellow color during cooking indicated that chemical transition might also occur. FTIR and UV -vis were used to analyze possible chemical change. The FTIR spectra are shown in Figure 3 5. The interpretation of the IR of the as -received DAS granule was as follows (Margel and Rembaum 1980; Yu et al. 2007) : around 3400 cm1, stretching of the OH; double peaks at ca. 2950/2900cm1, asymmetric and symmetric of the CH2; 1735cm1, carbonyl grou p stretching; 1640cm1, stretching of the OH of water; 1323 and 779 cm1, vibration of the C C and C H bonds of C CHO and CHO respectively. For the DAS solids after cooking, a new

PAGE 45

45 absorption band appeared at 1693cm1. The absorbance band of carbonyl stret ching at 1735cm1 split into two peaks, with a strong absorbance at 1716cm1. We have assigned the new peak at 1693cm1 to the stretching of the carbonyl group of conjugated aldehyde ( -C=C C=O). In the study of synthesis and characterization of polyglutara ldehyde (Margel and Rembaum 1980) FTIR spectra of poylglutaraldehyde exhibited two bands at 1720 and 1689cm1. Those authors assigned the 1720cm1 absorbance peak to the nonconjugated aldehyde and the 1680cm1 to the conjugated aldehyde. Those authors further stated it was difficult to distinguish the C=C bond and the OH of water. Both absorbances are at the same wavelength of 1640cm1. Even after being dried at high vacuum, polyglutaraldehyde still contained variable amounts of water. We proposed that the 1735 and 1693 cm1 in our DAS samples after cooking were the stretching of the C=O of the non-conjugated aldehyde and conjugated aldehyde respectively. The absorbance band at 1716cm1 in DAS sedimentation was probably the stret ching of the carboxyl group of carboxylic group as discussed in a later section. Degradation mechanisms of dialdehyde starch in alkaline media have been proposed in literature. Cannizzaro reaction and -elimination are frequently employed to understand the degradation of DAS as shown in Figure 3 6 (Fry et al. 1942; Veelaert et al. 1997a; Whistler et al. 1959) These mechanisms predicate a conjugated aldehyde formation, with accompanying generation of an acid. During the cooking, we observed development of a faint yellow color and decrease of pH (see Table 3 3). The proposed mechanisms have been explained for the degradation of DAS in alkaline conditi on. In acid condition, degradation of DAS could also happen. Veelaert studied the available aldehyde content of 100% oxidized DAS after cooking

PAGE 46

46 (Veelaert et al. 1997a) Compared to the starting DAS, they found approxima tely 10% reduction of available aldehyde when it was cooked at 90 C for 5 hour at pH 3. Some aldehydes were believed to convert into carboxylic acid by the Cannizzaro reaction. The formation of the conjugated aldehydye could explain the color change to ye llow of DAS aqueous suspension during the cooking. The formation of the conjugated aldehyde functionality was further confirmed by the UV -Vis spectra (Figure 3 7). Transmission and reflectance modes were employed for the samples of suspensions and solids respectively. For the suspensions, no absorbance peak could be observed for the DAS granular suspension. This observation was probably caused by the sedimentation of the DAS granules. However, a strong absorbance peak at 238 nm wavelength was observed in t he 0.03% DAS aqueous suspension supernatant (as -prepared DAS aqueous supernatant diluted 100 times). A strong peak at 246 nm wavelength and a weak peak at 300 nm wavelength were observed in the reflectance mode of the freeze-drying 3% DAS aqueous suspensio n supernatant (the amount of DAS solids from freezedrying sedimentation was not enough to run the reflectance mode) and the as received DAS granule solid samples respectively. It is a known phenomenon that commercial glutaradehyde solution exhibits two a bsorption maxims commonly at 235nm and 280nm. After carefully purification, the absorption at 235nm can be eliminated. Reported studies indicate that the monoglutaraldehyde (non -conjugated aldehdye) and polymeric glutaraldehyde (conjugated aldehdye) are re sponsible for the absorbance at 280 and 235nm respectively (Anderson 1967; Gillett and Gull 1972; Ranly 1984) The 238 and 246 nm absorbency of the 3% DAS aqueous supernatant in the transmission and reflectance modes support our FTIR results that a conjugated aldehyde function with an ethylenic linkage (C=C -C=O) was formed during cooking. The absorbance of the non-

PAGE 47

47 conjugated aldehyde in the range of 270290nm was not detected, even though it was confirmed by the FTIR. This result can be explained by the high extinction coefficient ( =18.6 L g1 cm1) of the conjugated aldehyde compared to the non-conjugated aldehyde ( =4.202 L g1 cm1) (Margel and Rembaum 1980) A weak absorption at 300nm was observed for the as received DAS granule in the reflectance mode. This absorption could be the nonconjugated aldehyde function. The conjugated aldehyde absorption was not observed in the DAS granule case, as it should have been much stronger than the non -conjugated aldehyde absorption based on the values of extinction coefficient. FTIR and UV-vis spectra clearly demonstrated that conjugated aldehyde was formed during the cooking, probably caused by the elimination reaction as illustrated in Figure 3 6. 3.3.3 The pH Effect on Physicochemical Change of As -Prepared DAS Aqueous Suspension The yellow color further developed when the pH of the as -prepared DAS aqueous suspension was adjusted from acidic to al kaline condition (Figure 3 8). This color development indicated more degradation would occur in the alkaline condition. The pH effect on the physicochemical change of the DAS aqueous suspension was investigated. The solubility of the DAS aqueous suspension at different pH values was examined as shown in Table 3 5. The change of solubility of the DAS aqueous suspension at different pH values was negligible. A distinct chemical change of the DAS at different pH values was observed by FTIR and UV -vis (Figure 3 9, 3 10). Intensity ratio of non -conjugated aldehyde (1735cm1) and conjugated aldehyde(1693cm1) at different pH values was calculated for the DAS supernatants. Intensity ratio of conjugated aldehyde (238nm) absorbance for same concentration of DAS aque ous suspension at different

PAGE 48

48 pH values was also determined, using absorbance of the as -prepared DAS aqueous as reference. As indicated in the spectra, conjugated aldehyde significantly increased in the alkaline condition. The 1716cm1 absorbance in the FTIR spectra of sedimentation was assigned to the stretching of C=O group of carboxylic acid. When the pH of DAS aqueous suspension was adjusted to the alkaline condition, this band disappeared. A new band was observed at 1570cm1. This band could be the symme tric stretching of the C=O of the carboxylate group (sodium salt) (Margel and Rembaum 1980) It is anticipated that the carboxylic acid from degradation can form the salt in the alkaline condition. This band was not observed in the FTIR spectra of supernatants. One possible reason was that the aldehyde content in the supernatant was much higher than that in the sedimentation. This band was overlapped by the absorbance of aldehyde. 1H and 13C NMR spectra of DAS supernatants at diff erent pH values are presented (Figure 3 11, 3 12). For the 1H NMR, the same acquisition time (7 min.) was used. For the 13C NMR, due to the weak signals, much longer acquisition time (around 20h) was used. Chemical shifts of protons from C2 and C3 aldehyde s of DAS have been reported at 9.36 and 9.56 ppm (Zhang et al. 2007) A conjugated aldehyde compound was studied (Iwagawa et al. 1994) Chemical shifts of protons from aldehyde and C=C were assigned at 9.30 and 6.38 ppm respectively. In the 1H NMR spectra, we as signed the resonances between 9 -10 ppm were protons from aldehdyes and the resonances between 6 7 ppm were protons from C=C. Sufficient information to identify each resonance was not obtained, but one can speculate the shoulder around 9.30 ppm is the proton resonance from the conjugated aldehyde. A very weak resonance at 190 ppm was recorded in the 13C NMR spectra of the DAS supernatant at pH 3. This resonance was intensified for DAS supernatant at pH 8.7, meanwhile some new resonances between 130 150 ppm were observed. In the solid state 13C NMR study of

PAGE 49

49 dialdehyde cellulose, the expected carbonyl chemical shifts were not detected (Kim et al. 2000; Princi et al. 2004) In the 13C liquid NMR spectra of conjugated aldehyde compound (Iwagawa et al. 1994) aldehyde was assigned at 192.70 ppm and C=C bonds were assigned at 130150 ppm. In our current study, the chemical shift at 190 ppm was believed to be an aldehyde. The resonances between 130 150 ppm were assigned to C=C. The NMR stud ies confirmed the formation of the C=C bond for the DAS supernatant. The formation of C=C was enhanced in the alkaline condition. It was not possible to distinguish the normal aldehyde and conjugated aldehyde from the NMR spectra. Because of the complex st ructure of DAS and a variety of degradation mechanisms, a complete composition of the DAS aqueous suspension cannot be determined in this study. However, the formation of carboxylic acid and conjugated aldehyde has been confirmed. Furthermore, the pH also affected the formation of conjugated aldehyde. The conjugated aldehyde function (C=C -C=O) significantly increased in the alkaline condition confirmed by the various spectroscopic studies as -elimination has been reported to be favorable in the alkaline condition. 3.3.4 Antimicrobial Study of DAS Suspensions Generation of carboxylic acid function in the DAS structure was observed after the cooking. In addition to DAS, we used another starch, i.e. oxidized starch in our antimicrobial study. The purpose to select this starch was to investigate the effect of starch polymer structure and the carboxylic acid function on the antimicrobial activity. The difference of DAS and oxidized starch was exp lained in Figure 3 13 (Zhang et al. 2007) Unlike the selective oxidation of C2 C3 to form dialdehyde functions by periodate oxidation; the oxidation by hypochlorite is non-selective. A ll reducing hydroxyl groups can be

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50 oxidized to carboxylic group to obtain the so -called oxidized starch. Oxidized starch is prepared in the alkaline condition. A high degree oxidized starch D17F was kindly supplied by Grain Processing Corp.. Density, oxidation extent and pH of as received oxidized starch are 1.5g/cm3, 90% and 7.5 respectively from the manufacturer. Antimicrobial activity of a 2.7wt% DAS granular suspension, a 2.7wt% DAS as prepared aqueous suspension and a 4.5wt% oxidized starch (OS) as pr epared aqueous suspension were examined first. The antimicrobial results are shown in Figure 3 14. The antimicrobial activity of the DAS granular suspensions was very limited. DAS granular suspension showed no activity against gram -negative bacterium E.col i in one hour and four hour exposure tests. A very weak inactivation of gram positive bacterium S.aureus was observed in four hour exposure test. In one -hour test, DAS granular suspensions showed no activity against the test viruses. When the test time was increased to four hour, ca 1.5 log reduction was observed for MS2 virus, but no antiviral activities were observed for other two viruses. The reference oxidized starch showed no activity against E.coli and S.aureus in one hour and four -hour experiments. However, the DAS aqueous suspensions showed a significant antibacterial activity against E.coli and S.aureus In one -hour test, the DAS aqueous suspension completely inactivated S.aureus and ca. two log reduction against E.coli. When the exposure time inc reased to four hour, DAS aqueous suspension also completely inactivated E.coli Similar antiviral activity of the DAS aqueous suspensions against viruses was observed. A significant antiviral activity against these three viruses in one -hour test was found. Furthermore, the antiviral activity was significantly enhanced in four hour test.

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51 Before any conclusion is drawn of the antimicrobial activity among these three systems, it is necessary to point out that the pHs were different. The pHs of these three syst ems were: pH 3.8 for DAS granular suspension, pH 3.0 for DAS aqueous suspension and pH 5.2 for OS aqueous suspension. The pH effect on the antimicrobial activity needs to be considered. In the present study, it was found that the pH change of the as prepar ed starch aqueous suspension after sonication was negligible. The sonication effect on the antimicrobial activity of the as -prepared DAS aqueous suspension was investigated (Figure 3 15). Without sonication, the DAS aqueous suspensions already demonstrated antibacterial activity, approximately 2 log reduction for the 2.7% DAS aqueous suspension against E.coli and 4 log reduction for the 1% DAS aqueous suspension against S.aureus Antibacterial activity of the DAS aqueous suspension was significantly enhance d by sonication. A 1% DAS aqueous suspension achieved 7 log reduction against S.aureus after 15min. sonication, while, 2.7% DAS aqueous suspension also completely inactivated E.coli after 30min. sonication. Effect of sonication on the antibacterial activit y was further supported in the MLC values of the DAS aqueous suspension against three gram -positive bacteria and three gram -negative bacteria as shown in Table 3 6. After one -hour sonication, the MLCs of the DAS aqueous suspension against E.coli and S. aureus both were 0.8%. Further demonstrated in Table 36, it can be seen that the DAS aqueous suspension exhibited a significant broad -spectrum antibacterial activity. However, after one hour sonication of the 2.7% DAS aqueous suspension, no enhancement of the antiviral activity was found compared to that of non -sonicated DAS aqueous suspension in our study. The difference of the sonication effect on the antibacterial and antiviral activity was probably caused by the particle size of microorganism. Particle size of E.coli or S.arueus is about 2 m, whereas

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52 viruses are much smaller. For example, MS2 is a single -strand RNA, unenveloped, icosahedra shaped with 27.5nm size. The dispersion of the DAS aqueous suspension by sonication might enhance the interaction b etween the DAS particles and big size bacteria, whereas had negligible effect on the interaction between the DAS particles and much smaller size viruses. The gel formation of the as -prepared DAS aqueous suspension has already been presented before. This ge l structure was observed to break down to form homogeneous suspension during sonication. Sonication could enhance the dispersion of DAS aqueous suspension to significantly improve its antibacterial activity. The pH change of the as -prepared DAS aqueous sus pension by sonication was negligible; while the antibacterial activity was significantly enhanced. This observation indicated that the antimicrobial activity of DAS aqueous suspension might be from the reactivity of dialdehyde function, instead of the pH. A complete study of the pH effect on the antimicrobial activity of the DAS aqueous suspension was conducted. For antibacterial study, one hour sonicated 2.7% DAS aqueous suspension and 4.5% oxidized starch aqueous suspension were employed. For antiviral st udy, the as prepared 2.7% DAS aqueous suspension without sonication was used. The pH effect on the antimicrobial activity was studied by adjusting the pH values of phosphate buffer saline (PBS buffer as control) and 2.7% DAS aqueous suspension using Hcl/Na OH as shown in the Figure 3 16 and 317. In the selected pH range, DAS aqueous suspension exhibited significant antibacterial activity against the test gram -positive bacteria, especially in the acidic and alkaline conditions. It was noticed that in the mil d pH range, the antibacterial activity was minimal. Controlled PBS buffer only showed antibacterial activity against gram -positive bacteria in the acidic condition with the log reductions significantly smaller than the DAS aqueous suspensions in the same c ondition.

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53 Antibacterial activity of the DAS against gram -negative bacteria followed the similar tendency. However, DAS showed less effectiveness against Salmonella typhimurium and Pseudomonas aeruginosa. Controlled PBS buffer also exhibited antibacterial activity against gram -negative bacteria in the acidic condition. As indicated in Figure 3 14, the pH of the as -prepared DAS aqueous suspension was ca.3. It showed a 7 log reduction against S.aureus higher than that (approximately 5) of PBS at pH 3. Howev er, the as prepared DAS aqueous suspension only showed around a 2 log reduction against E.coli lower than the log reduction (around 4) of PBS at pH 3. Probably the salt at pH3 of PBS buffer also contributed some activity against E.coli at pH 3. In genera l, the DAS aqueous suspension was more effective against gram -positive bacteria than the gram -negative bacteria in a broad pH range. For the antiviral study, weak inactivation of the MS2 and PRD1 was observed in the PBS at pH 3.0 and 3.4. No inactivation w as found for MS2 and PRD1 at other pH values. Furthermore, in the selected pH range, inactivation against Polio of the PBS buffer could not be detected. DAS aqueous suspensions showed a much stronger antiviral activity against chosen viruses in the studied pH range, especially in the acidic and alkaline conditions. The antiviral activity was significantly enhanced with increasing test time. These results strongly suggested that the dominant inactivation mechanism could be attributed to the activity of alde hyde groups instead of the acidity of the DAS aqueous suspension. A better understanding of the inactivation mechanism was to select a pH which PBS buffer did not exhibit any antibacterial activity In the mild pH range, for example, at pH=4.8, the antimic robial activity was minimal in the selected pH values for the DAS aqueous suspensions at one -hour exposure. Meanwhile, no

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54 antibacterial activity of PBS buffer at this pH value in one hour was observed. In the antiviral study indicated in Figure 3 17, incre asing exposure time to four hour, an enhancement of antiviral activity was observed. In order to fully understand the DAS inactivation mechanism, DAS aqueous suspension, oxidized aqueous suspension (both were sonicated for one hour) and PBS buffer in pH 4 .8 were studied for a longer time exposure against bacteria. When the exposure time became four -hour, DAS aqueous suspension completely inactivated E.Coli Pseudomonas aeruginosa and S.aureus and achieved at least 5 log reductions for Salmonella typhimuri um Enterococcus faecalis and Bacillus cereus Meanwhile, no antimicrobial activity of PBS and oxidized starch was found at pH 4.8 regardless of the exposure time. The difference of antibacterial activity of the DAS aqueous suspension, oxidized starch su spension and PBS buffer at pH 4.8 strongly support that the dominant bacterial inactivation is from the dialdehyde functions. In summary of the antimicrobial activity of DAS suspensions, the antimicrobial activity is related to the reactivity of DAS. The reactivity of DAS can be affected by the dispersion technology or be pH -dependent. The difference of antimicrobial activity between the DAS granular suspension and the as prepared DAS aqueous suspension was proposed to be the physicochemical change during the cooking. Cooking significantly increased the DAS solubility in the water, while at the same time, decreased the DAS particle size. Sonication can also enhance the dispersion of the as -prepared DAS aqueous suspension. Based on the antimicrobial activit y of the reference oxidized starch, the carboxyl functions generated during the cooking might not play role in the antimicrobial activity of DAS aqueous suspension.

PAGE 55

55 Commercial glutaraldehdye usually contains polymeric glutaraldehyde (conjugated aldehyde) (Gillett and Gull 1972) Several studies of the glutara ldehyde on the enzyme activity indicated that the pure glutaraldehyde was the least effectiveness compared with the non purified glutaraldehyde containing polymeric glutaraldehyde (Anderson 1967; Ranly 1984) However, it is not clear whether the formation of the conjugated aldehyde could improve the antimicrobial activity of DAS aqueous suspension in this study. It was found that the antimic robial activity of DAS was pH dependent. In general, it was more effective in the acidic and basic conditions than in the mild conditions. This observation was similar to the pH effect on the antibacterial activity of glutaraldehyde (Gorman et al. 1980; McDonnell and Russell 1999) The solubility of the DAS aqueous suspension at different pH values was almost the same. This result excluded t he effect of solubility on the pH dependent behavior of the DAS antimicrobial activity. A distinct chemical change of the DAS aqueous suspension at different pH values was observed. The conjugated aldehyde function (C=C C=O) was significantly increased in the basic condition as indicated by spectroscopic studies. Formation of conjugated aldehyde by aldol condensation was observed in the study of the chemistry of glutaraldehyde (Gorman et al. 1980) Aldol -type polymers in glutaraldehyde alkaline solution have been reported to react readily with protein (Gorman et al. 1980) Again, it is not clear whether the formation of the conjugated aldehyde could improve the antimicrobial activity of DAS in this study. Though, the antibacterial activity of DAS exhibited significant higher antibacterial activity in alkaline condition compared to the mild pH range. Crosslinking reaction between the dialdehyde functions of glutaraldehyde and the functional groups in the bacterial structure such as amino function of proteins has been

PAGE 56

56 determined to be a bacterial inactivation mechanism of glutaraldehyde (Gorman et al. 1980; McDonnell and Russell 1999) Considering the similar dialdehyde functions of DAS and glutaraldehyde, it is believed that the bacterial inactivation mechanism of DAS may follow the similar type of crosslinking reaction as glutaraldehyde. Sloan has studied the reaction between the DAS aqueous suspension and urea (Sloan et al. 1956) It was found at room temper ature, that there was no reaction at neutral or slightly acidic pH. Kim proposed a chromatographic method for the separation of amines based on their interactions with dialdehyde cellulose gel (Kim and Kuga 2000) The interaction of amine and dialdehyd e functions was dependent on the acid dissociation constant (pKa) of amines as well as the pH of eluent. The pH -dependence of the interaction between the dialdehyde function and the amine function may be a plausible explanation for the pH -dependent antibac terial activity of DAS. Another possible reason for this pH -dependent behavior is that the carbonyl groups in DAS are not all free functionalities. Hemiacetal or hemialdal linkages are also formed (Haaksman et al. 2006) These linkages are fairly easy to break to liberate free carbonyl groups, especial ly in the acid condition (Anderson and Fife 1971) Similar results have been reported for the glutaraldehyde in the acidic condition. In the acid condition, glutaraldehyde monomer is in equilibrium with the various hydrates (Gorman et al. 1980) This equilibrium can be affected by temperature a nd pH. Cell wall surface structure of bacteria can also be affected by the pH. Bacteria have heterogeneous surface with various surface functional groups. In general, Gram positive bacteria have a greater binding capacity due to their thicker layer of pep tidoglycan, which contains the major binding sites. The variation in structural cell wall architecture and chemistry between

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57 gram -positive and gram -negative bacteria probably is the reason that the DAS aqueous suspension was more effective against gram pos itive bacteria than gram negative bacteria in a broad pH range. Bacterial surfaces consist of a complex, heterogeneous mixture of potential binding sites. The types of sites available include carboxylic, phosphoric, phosphodiester, amino and hydroxyl group s (Martinez et al. 2002) The available sites are affected by the pH. A s the pH is changed from acidic to alkaline, more reactive sites such as free amino groups will be formed at the cell surface leading to a more rapid bactericidal effect by glutaraldehyde (McDonnell and Russell 1999) Because of the complex structure of dialdehyde starch, heterogeneous bacteri al surface and different inactivation mechanism, the decisive conclusion for the pH -dependence of the dialdehyde starch aqueous suspension against microorganism cannot be determined in the current study. Bacterial recovery experiments were also performed. The purpose of this study was to understand the action of DAS aqueous suspension against bacteria to be permanent or not. Two sets of experiments have been conducted. The first set was to evaluate the recovery of the completely killed bacteria. After comp letely inactivation of E.coli and S.aureus by the as prepared DAS aqueous suspension, the bacterial suspensions were plated onto TSA agar and incubated at 37 C to confirm the complete inactivation. Meanwhile, the bacterial suspensions were also incubated i n TSB broth at 37 C to check the re -growth of bacteria. After 1 2 days incubation, the TSB broths containing treated bacteria were still clear, indication of no growth of bacteria. The TSB broths containing controlled bacteria became turbidity. TSA plate c ounting confirmed no growth of treated bacteria, whereas recovery of controlled bacteria was found.

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58 Another set of experiments was to study the non-completely killed bacteria. Approximate 2 log reduction of E.coli and S.aureus was achieved by the as -prepar ed DAS aqueous suspension. The bacterial suspension was plated onto different agar plates, i.e., TSA and Brain Heart Infusion (BHI) agar. BHI agar has more nutrient than TSA agar. In general, damaged bacteria (not completely killed bacteria) may not grow o n TSA agar, but can grow on BHI agar. If there is significant difference in the numbers of colonies onto these two agars, a recovery of bacteria happens. In our experiments, the numbers of colonies counted from these two agar plates were almost the same fo r the controlled and treated bacteria respectively. These experiments demonstrated that the inactivation of bacteria by the DAS aqueous suspension was irreversible. 3.3.5 Bacterial Inactivation Kinetics Habers rule has been frequently employed to predic ate dose/time effect on the toxicity of an agent against an organism. Successful application of Habers rule needs to satisfy several requirements. The action of the agent against an organism is a continuous and irreversible process. The quantity of the ag ent available for reaction is in abundance (Bunce and Remillard 2003; Winks 1984) Once an agent/organism system fit these conditions, empirical model, i.e. Habers rule is satisfactory to estimate the response of an organism to an agent with time. Habers rule is expressed as: Habers Rule: Ctm = K (3 2) C is the concentration of agent, t is the time of exposure, K is a constant. When m=1, Habers rule can describe a first order kinetics. When m is g reater than 1, the effect of exposure time on the response is significant. When m is smaller than 1, the effect of dose concentration on the response is dominant.

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59 In the antibacterial study of glutaraldehdye, the uptake of glutaraldehdye by bacteria was l imited compared to the bulk glutaraldehdye concentration (Gorman and Scott 1977) Glutaraldehyde concentration can be determined from the 235nm absorbance of the glutaraldehyde solution (Munton and Russell 1970) The relationship between the DAS concentration and its UV absorbance at 238nm was first examined. A linear relationship was established as shown in Figure 3 19. Later, the change of 238nm UV absorbance was m onitored for the DAS E.coli and DAS S.aureus systems at the starting and end points. DAS concentration change during exposure time was negligible as demonstrated in Figure 3 20. This result indicated that DAS was in abundance during reaction. The irreversi ble inactivation between bacteria and DAS was already found during the bacterial recovery experiments. The results described above indicated Habers rule could be employed to describe the bacterial inactivation kinetics, probably a linear kinetics. We chos e a simple and convenient method to study the inactivation kinetics of the DAS aqueous suspension against bacteria. The minimum lethal concentration (MLC) of the as prepared DAS aqueous suspension was determined for five different exposure times in a given temperature. The relationship between the MLC and exposure time was obtained to establish the inactivation kinetics. Furthermore, four temperatures were selected to investigate the temperature effect on the inactivation kinetic. A typical illustration is present in Figure 3 20.The kinetics data are summarized in the Table 3 7. A linear relationship was found between 1/MLC and exposure time for the DAS against E.coli and S.aureus at room temperature (Figure3 20). This linear relationship was found in ot her temperatures as indicated in Table 3 7. The relationship between the MLC (weight percentage)

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60 and exposure time (hour) could be expressed as 1/MLC=k*t for each temperature with a correlation coefficient R2=0.99 in this study. A first order inactivation kinetics was established here for the two studied bacteria. The rate constant k was temperature and bacterial strain dependent. Increasing temperature accelerated the inactivation of the DAS aqueous suspension against bacteria. The DAS aqueous suspension was more effective against gram positive bacteria S.aureus than the gram negative bacteria E.coli The obtained activation energy was 17.4 and 16.6 kcal/mol respectively for E.coli and S.aureus These values were almost the same for the studied bacteria an d were very close to the activation energy of the glutaraldehdye against Bacillus subtilis spore (20kcal/mol) reported in the literature (Sagripanti and Bonifacino 1996) The first order kinetics was found for the bacterial inactivation kinetics of the DAS aqueous suspe nsion as seen in Table 3 7. A simple chemical reaction model is proposed to describe this first order kinetics. It is speculated that the bacterial inactivation of DAS was due to the crosslinking reaction between the DAS and bacterium. DAS + Bacterium DAS Bacterium (3 3) By applying chemical reaction kinetics, the change of bacterial concentration during inactivation can be expressed as: d[Bacterium]/dt=k[DAS][Bacterium] (3 4) Bracket symbol represents the concentrat ion of the reactant, k is the reaction constant, and t is the reaction time. The concentration of DAS was kept constant during the inactivation as indicated in Figure 3 20. This chemical reaction is a pseudo-first order reaction. After integration, we can obtain: Log[Bacterium]t/[Bacterium]0=k[DAS]*t (3 5)

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61 Subscript symbols t and 0 represent the bacterial concentration at the time t and at the initial. This can be rewritten as: Log reduction=k[DAS]*t (3 6) For complete inactivation of the bacteria with initial bacterial concentration at 107CFU/ml, Log reduction equals to 7. Replacing the concentration of DAS by the MLC (minimum lethal concentration), one gets: 1/MLC=t*k/7 (3 7) This equation predicates the li near relationship between 1/MLC vs. time t, which is the same form of the bacterial inactivation kinetic equation illustrated in Table 3 7. The Arrhenius equation can be applied to study the temperature dependence of the rate constant, i.e. k=Ae( Ea/RT) (3 8) The activation energy Ea of the DAS aqueous suspension against bacteria was determined (Table 3 7). The obtained mathematical model was verified by the predication of the response of bacterial inactivation at other conditions. This model (Equ 3 6) predicates that the log reduction is the same for different starting bacterial concentration during the same exposure time for the same concentration of the DAS aqueous suspension at the same temperature. The calcu lated log reductions from this model predicated the measured log reductions of S.aureus and E.coli at room temperature well (Figure 3 22). This observation strongly supports the validity of the developed model. 3.4 Conclusion The DAS aqueous suspension wa s demonstrated to be a broad -spectrum biocide against gram -positive/negative bacteria, bacteriophages and a human virus. Antimicrobial activity of the DAS aqueous suspension was related to its dialdehyde reactivity. The reactivity of DAS was found to be af fected by the dispersion technology or be pH -dependent. Degradation of DAS

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62 during cooking was observed with the generation of carboxylic acid and the formation of conjugated aldehyde functions. Solubility and particle size were significantly affected by co oking. Degradation of DAS was found be more liable in the alkaline condition. Antimicrobial activity of DAS was also identified to be pH -dependent. However, no decisive conclusion could be obtained to explain the pH -dependent antimicrobial activity in this study. Bacterial inactivation kinetics by DAS was found to be first -order kinetics. This behavior was explained by pseudo-first order reaction kinetics.

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63 Table 3 1 Selected microorganisms in current study Gram negative bacteria Escherichia coli C3000 ATC C 15597 ( E.coli EC) Salmonella typhimurium ATCC 19585 ( S.typh, ST) Pseudomonas aeruginosa (PA) Gram positive bacteria Staphylococcus aureus ( S.aureus SA) Enterococcus faecalis (EF), Bacillus cereus (BC) Bacteriophage MS2 (ATCC 15597 B1) Host: E.coli (ATCC 15597) PRD1 Host: S. typh (ATCC 19585) Human virus Poliovirus 1 Note: Microorganism without ATCC number was laboratory strain Table 3 2 Some physical properties of as received dialdehyde starch Density 1.5g/cm 3 a Oxidation content 90% a,b Ref ractive index 1.56 c Crystallinity Amorphous b see Figure 3 2 a: Manufacturers data b: measured, c: calculated Table 3 3 Viscosity, pH and solubility of DAS aqueous suspensions cooked at 95 C in different time Time (hr) 0 0.5 1 2 Viscosity (Poise) 0.26 0.08 0.04 0.03 Solubility (wt%) 0 Not measure Not measure 98 pH 3.8 Not measure Not measure 3.0 Table 3 4 Summary of volume mean particle size DAS GS DAS AS DAS AS Supernatant Particle size 135 m 108 m 9 nm

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64 Table 3 5 Effect of the pH on the solubility of the DAS aqueous suspensions, the absorption intensity ratio of FTIR and UV pH 3.0 4.8 7.0 8.7 Solubility (%) 98 98 98 98 FTIR Intensity ratio 1.67 1.31 1.14 0.72 UV Intensity ratio 1 1.01 1.24 3.88 Table 3 6 The MLCs of 1-hr sonicated DAS aqueous suspensions to achieve the 7 log reduction in one -hour exposure and the corresponding pH values for the DAS aqueous suspensions EC ST PA SA EF BC MLC(wt%) 0.8 2.1 1.0 0.8 1.0 0.2 pH 3.2 3 .0 3.1 3.2 3.1 3.5 Note: MLC values were average of at least tripicates within 0.05 0.1%. Table 3 7 Kinetic parameters of DAS aqueous suspension against bacteria Temperature ( C) Kinetic equation E.coli S.aureus 4 1/MLC=0.015*t, R 2 =0.99 1/MLC=0.052* t, R 2 =0.99 14 1/MLC=0.025*t, R 2 =0.99 1/MLC=0.23*t, R 2 =0.99 23 1/MLC=0.14*t, R 2 =0.99 1/MLC=0.36*t, R 2 =0.99 34 1/MLC=0.21*t, R 2 =0.99 1/MLC=1.39*t, R 2 =0.99 Activation energy (kcal/mol) 17.4 16.6

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65 Figure 3 1 Yellow color development of DAS aqueous suspension cooked at 95 C for different time, A: zero time, B: 0.5 hour, C: 1 hour, D: 2 hour. 0 100 200 300 400 500 600 700 800 5 10 15 20 25 30 35 40 2 Intesnity As-received DAS granule DAS aqueous deposited film Figure 3 2 XRD patterns of as -received DAS granule and deposited film of 2hr 95 C cooked DAS aqueous suspension. F igure 3 3 XRD patterns of native (NS) and dialdehyde potato starch of 1.5% (DAS1 5), 5% (DAS5), 15% (DAS15) and 25% (DAS25) degree of oxidation A B C D

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66 0 10 20 30 40 50 60 70 80 90 100 3.65.58.412.819.429.444.667.8103156237 Particle size (nm)Volume fraction Figure 3-4 Particle size measurement by Cou lter LS13320 (a), A: as-prepared DAS aqueous suspension (DAS-AS), B: DAS granul ar suspension (DAS-GS), by Brookhaven Zetaplus (b) for supernatant of as-prepa red DAS aqueous suspension after high speed centrifugation (DAS-A S-Supernatant). A B a b

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67 500 1000 1500 2000 2500 3000 3500 4000 Wavenumber (cm-1) Absorbance 1480 1580 1680 1780 Wavenumber (cm-1) Absorbance Figure 3 5 FTIR s pectra of as received DAS granule, DAS solids from the supernatant and sedimentation of as -prepared DAS aqueous suspension after centrifugation and freeze -drying, (a) full spectra, (b) enlarged spectra in the carbonyl region Sedimentation Supernatant As received Supernatant Sedimentation As received 1735 1693 1640 1716 a b

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68 Figure 3 6 Degradation mecha nisms of DAS in alkaline media 220 240 260 280 300 320 340 360 Wavelength (nm) Absorbance A B C D 238 246 300 Figure 3 7 UV-vis spectra of the DAS samples, Reflectance mode: as -received DAS granular (A), freeze -drying of 3% as -prepared DAS aqueous supernatant (B), Transmission mode:3% as -prepared DAS aqueous supernatant ,diluted 1 00 times by deionized water (C), 0.3% DAS granular suspension (D)

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69 Figure 3 8 Yellow color development of as prepared DAS aqueous suspension in different pH values, pH=3, 4.8, 7, 8.7 from left to right 1480.00 1580.00 1680.00 1780.00 Wavenumber (cm-1) Absorbance pH 3 pH 4.8 pH 7 pH 8.7 1735 1693 1480.00 1580.00 1680.00 1780.00 Wavenumber (cm-1) Absorbance pH 3 pH 4.8 pH 7 pH 8.7 1735 1716 1693 1570 Figure 3 9 FTIR spectra of DAS solids from supernatants (a) and sedimentations (b) in the carbonyl region. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 210 230 250 270 290 310 330 350 Wavenumber (nm) Absorbance pH 8.7 pH 7 pH 4.8 pH 3 238 Figure 3 10 UV -vis spectra of DAS aqueous suspensions at different pH values a b

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70 DAS supern atants at pH 3, 4.8, 7, 8.7 (bottom to up), expand scale Figure 3 11 1H NMR of DAS supernatant at different pH values DAS supernatant at pH3, full scale

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71 Figure 3 12 13C NMR of DAS supernatants (bottom pH 3, up pH 8.7) Figure 3 13 Oxidation of star ch by different oxidizing agents PeriodateHypochloriteStarch Dialdehyde starch Oxidized starch PeriodateHypochloriteStarch Dialdehyde starch Oxidized starch

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72 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 E.coli S.aureus MS2 PRD1 POLIO Log reduction DAS-GS 1hr DAS-GS 4hr DAS-AS 1hr DAS-AS 4hr OS-AS 1hr OS-AS 4hr Figure 3 14 Antimicrobial activity of DAS granular suspension, as prepared DAS aqueous suspension and as -prepared oxidized starch aqueous suspension in one hour and four hour exposures against various microorganisms 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 0.00 15.00 30.00 45.00 60.00 Sonication Time (min) Log Reduction 2.7% DAS-EC 1% DAS-SA Figure 3 15 Effect of sonication time on the antibacterial activity of DAS aqueous suspension against E.coli and S.aureus in one hour exposure

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73 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 pH Log reduction EC ST PA 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 pH Log reduction EC ST PA 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 pH Log reduction SA EF BC 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 pH Log reduction SA EF BC Figure 3 16 pH dependent of PBS and 1 -hr sonicated 2.7% DAS aqueous suspension against gram -negative and gram positive bacteria in one hour exposure PBS DAS PBS DAS

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74 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 pH Log reduction PBS,1hr DAS,1hr PBS,4hr DAS,4hr 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 pH Log reduction PBS,1hr DAS,1hr PBS,4hr DAS,4hr 0.00 1.00 2.00 3.00 4.00 5.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 pH Log reduction PBS,1hr DAS,1hr PBS,4hr DAS,4hr Figure 3 17 Antiviral test results of PBS and 2.7% DAS aqueous suspension, pH and mixing time dependent test PRD1 MS2 Polio

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75 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 EC ST PA SA EF BC Log Reduction PBS4.8, 1hr PBS4.8, 4hr DAS4.8, 1hr DAS4.8, 4hr OS4.8, 1hr OS4.8, 4hr Figure 3 18 One -hour sonciated2.7% DAS aqueous suspension, 4.5% oxidized st arch (OS) aqueous suspension and PBS, all at pH 4.8 against bacteria in one hour and four hour exposure experiments. (Oxidized starch was only test against EC and SA) 0 0.5 1 1.5 2 2.5 210 260 310 360 wavenumber (nm) Absorbance y = 779.39x R2 = 1 0 0.5 1 1.5 2 2.5 0.00% 0.05% 0.10% 0.15% 0.20% 0.25% 0.30% 0.35% Weight percentage Absorbance (a) (b) Figure 3 19 UV -vis of as -prepared 3% DAS with different dilutions (a) and the calibration curve (b) 0.3% 0.15% 0.03% 0.003% 238

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76 0 0.1 0.2 0.3 0.4 0.5 0.6 210 260 310 360 Wavenumber (nm) Absorbance SA 0hr SA 8hr EC 0hr EC 10hr Figure 3 20 DAS aqueous suspensions against bacteria at different time (0.35% DAS against S.aureus and 0.7% DAS agains t E.coli ), absorbances were for 10 times dilution y = 0.3648x R2 = 0.99 y = 0.1407x R2 = 0.99 0 0.5 1 1.5 2 2.5 3 3.5 0 2 4 6 8 10 12 Time (hr) 1/MLC E.coli S.aureus Figure 3 21 Relationship between 1/MLC vs time at room temperature, the MLC values were average at least triplicates within 0.05 0.1%

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77 0 1 2 3 4 5 4 5 6 7 Log starting S.aureus concentration Log reduction 2hr 3hr calculated 2hr calculated 3hr 0 1 2 3 4 5 4 5 6 7 Log starting E.coli concentration Log reduction 1hr 3hr calculated 1hr calculated 3hr Figure 3 22 Comparison of inactivation of 0.6% DAS aqueous suspe nsion against S.aurues and 0.9% DAS aqueous suspension against E.coli with different starting concentration at different time with developed model

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7 8 CHAPTER 4 DESTRUCTION OF MICROORGANISMS BY SURFACE CONTACT 4.1 Introduction Dialdehyde starch showed a broad -spectrum antimicrobial activity in aqueous suspension discussed in Chapter 3. As a polymeric biocide, we believe it can also destruct microorganism by surface contact. In Chapter 2, we discussed two methods to design a non-leaching antimicrobial surf ace. One strategy is to anchor a small molecular biocide onto a substrate; the other is using polymeric biocide. Almost 30 years ago, Isquith demonstrated contact kill when surfaces was functionalized with alkylammonium (AEM 5700) (Isquith et al. 1972; Isquith and McCollum 1978) Absence of the zone of inhibition was observed in Isquith work, leaching of biocide might be still in question during long term application. Nurdin studied the ageing of biocidal polyurethane in water (Nurdin et al. 1993b) Biocidal polyurethane was prepared with pendant quaternary ammonium salts (QAC) in the polymer structure. They found a two-stage antibacterial activity of the prepared biocidal polymer after aging in water. Immersion o f the biocidal polymer in water caused a decrease of activity with time regardless of the various pendant QACs. Relatively fast decrease of activity in the first stage (short time immersion) was believed to be the diffusion of some synthesis QAC residues. Even though, for some QACs chemical modifications, diffusion zone were not observed in the first stage. The slower decrease of activity in the second stage was proposed to be the equilibrium between the QACs and amine. The antibacterial activity in the sec ond stage was believed to be a polymer -bacteria contact kill. Antimicrobial action of small molecular QAC was reported to be blocked by ion-paring and precipitation of quaternary cations with bulky anions in an aqueous solution (Kopecky 1996)

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79 A significant amount of research has been conducted on the non leaching antimicrobial surface. Most of the research focused on QAC and hydantoin systems. An early US patent described a method to prepare antimicrobial surface using dialdehyde polysaccharide granules (Siragusa 1977) However, the antimicrobial activity of dialdehyde polysaccharide in other forms, such as polymer film and filter paper has not yet been reported. The interest of our current study is to investigate an aldeh yde type surface modifier to destruct microorganisms by contact. Contact between an antimicrobial surface and the microorganism is critical to achieve antimicrobial activity. ASTM E2149 01 shake flask method and AATCC 100 static contact method are the two common methods to determine the antimicrobial activity of a surface. This chapter focuses on the destruction of microorganisms by surface contact. Two different strategies were employed. One was to anchor trialkoxysilyl compounds containing biocidal func tional groups. QAS (AEM 5700) and aldehyde type compounds were chosen to understand the longterm antimicrobial performance. The other was to design antimicrobial surface by dialdehyde polysaccharide modification. Deposit of dialdehyde starch film on a sub strate and direct periodate oxidation of cellulose filter paper were employed. 4.2 Materials and Methods 4.2.1 Materials Trialkoxysilyl compounds were triethoxysilylbutyraldehyde and triethoxysilylundecanal obtained from Gelest, and AEM 5700 received from Aegis. Chemical structures of these compounds are present in Figure 4 1. Sodium periodate (NaIO4) and Whatman 50 plain cellulose filter paper (particle retention >2.7 m, 5.5cm diameter) were purchased from Fisher. Dialdehyde starch and oxidized starch used here were the same products as described in Chapter 3. All chemicals were used without further purification.

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80 4.2.2 Anchor of Trialkoxysilyl Compounds onto Cotton Textile Anchoring of trialkoxysilyl compounds is schematically illustrated in Figure 42. Th e first step involved the hydrolysis of these compounds, followed by the condensation onto a substrate. Typically, trialkoxysilyl compounds were hydrolyzed in deionized water at room temperature for 24 hours. Various concentrations of these compounds in aq ueous solution were prepared. Cotton textiles were immersed in the prepared aqueous solution for 30 minutes at room temperature. They were taken out to condense in an oven at 70 C for two hours. The textiles were washed and rinsed several times to remove u nbound residues. 4.2.3 Periodate Oxidation of Cellulose Filter Paper (Dialdehyde Cellulose) Twelve pieces of cellulose paper (total weight ca. 2.72g) was immersed into 100ml deionized water containing 0.2M sodium periodate. The pH of the periodate solution was 3 4. The reaction was kept dark at 37 C in a shaker for certain time. The speed of the shaker was 200RPM. After the reaction, the filter papers were first washed five times with deionized water. These filter papers were immersed into 100ml deionized water in a shaker at room temperature overnight. They were again washed five times with deionized water. Two milliliters of a 0.5% (w/v) sodium metabisulfite aqueous solution was spread onto a filter paper, no color change was observed. This indicated no p eriodate residues. After completely rinse of residual periodate, the treated filter papers were dried in a hood at room temperature for 24 h. 4.2.4 Deposition of Starch Film A 3% DAS and a 5% oxidized starch aqueous suspensions were prepared as described i n Chapter 3. Two milliliters of the suspension were spread onto a 100*15mm polystyrene Petri dish. The suspension was dried in the hood at room temperature for 24 h to form a starch film on the polystyrene surface.

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81 4.2.5 Determination of Oxidation Extent of Dialdehyde Cellulose (DAC) Two methods were employed to determine the oxidation extent of DAC. The first was elemental analysis as described in Chapter 3. The second was to monitor the consumption of sodium periodate during the reaction. The amount of pe riodate consumed was determined by measuring the absorbance of periodate at 221nm of the supernatant liquid (Maekawa and Koshijima 1984) Untreated cellulose filter paper wa s used as a blank control for the elemental analysis. The periodate solution itself was another blank control for the second method. 4.2.6 Characterization of Cellulose Filter Paper The cellulose fitter papers (untreated and treated) were analyzed by XRD a nd UV -vis (reflectance mode) as described in Chapter 3. FTIR spectra were recorded for the treated cellulose filter paper on a reflectance stage. Untreated cellulose filter paper was chosen as the background. 4.2.7 Antibacterial and Antiviral Assessment by Surface Contact ASTM E214901 shake flask method was used to evaluate the antibacterial activity of treated textile as illustrated in Figure 4 3. A modified AATCC 100 static contact method was employed to examine the antimicrobial activity of the diald ehyde polysaccharide surface. Determination of the number of bacteria in the test and control samples was obtained from the recovery of bacteria from the surface in the original AATCC 100 method. After certain times of incubation, bacteria were rinsed off form the samples by shaking in a know amounts of neutralizing solution. The number of bacteria presented in the liquid was determined by plate count. This method was modified by direct observation of microorganism onto a surface without a recovery step. T riphenyltetrazolum chloride (TTC) or Xgal (5 bromo 4 chloro 3 indolyl -D galactoside)/IPTG (isopropyl D -thiogalactoside) was employed to examine the bacteria or

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82 MS2 virus growth on the surface respectively. The TTC was prepared in a 5% deionized water s olution; Xgal/IPTG was prepared in dimethylformamide (DMF) solution (1.25g IPTG/1g Xgal in 25ml DMF) and was kindly supplied from Dr. Gowers group in the Department of Materials Science and Engineering. The TTC or Xgal/IPTG was added in a nutrient agar wi th volume ratio at 1/1000. At this concentration, TTC or Xgal/IPTG had no effect on the viability of bacteria or virus. TTC can react with live bacteria to form red colonies. When a host bacterium is attacked by a live bacteriophage, a hole is left. The nu mbers of holes are counted as the number of bacteriophage. If a substrate is opaque such as cellulose filter paper, it is very difficult to distinguish the hole on the substrate. When Xgal/IPTG is added into nutrient agar, the hole with a green background in the MS2/ E.coli system can help in counting the survival numbers of MS2 bacteriophage. A typical procedure for the antimicrobial assessment by direct surface contact of dialdehyde polysaccharide was: An aliquot of 0.1ml of the microorganism suspension w as pipetted onto the test surface. After one hour, TTC or Xgal was added into 40ml nutrient agar medium hold at 50 C. Nutrient agar was poured onto the surface. After solidification of the agar, the plates were incubated at 37 C for 24h. They were examined for the microbial growth, and the numbers of colonies or plaques were counted. Two bacterial strains ( E.coli and S.aureus ) and two bacteriophages (MS2 and PRD1) were used in the study. The employed pathogen suspension contained about 107CFU/ml for bacteri a or 104PFU/ml for viruses. Polystyrene Petri dish itself and oxidized starch surface were used as the control in the antimicrobial test of DAS surface. Untreated cellulose filter paper was the control for treated -cellulose filter paper. All experiments we re conducted at least in triplicates.

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83 4.2.8 Evaluation of DAC Filter Paper for Virus Aerosols This study was a preliminary attempt to evaluate the performance of DAS filter paper against virus aerosols (MS2). The experiments were conducted by JinHwa Lee in Dr. Chang -Yu Wus lab in the Department of Environmental Engineering Science, University of Florida. The experimental setup is illustrated in Figure 4 4. As demonstrated in Figure 4 4, the experimental set up had two components. One was a control without filter, the other was experiment with filter. Pressure drop, physical removal efficiency (PRE), viable removal efficiency (VRE) and infectivity of virus on the filter were obtained. PRE was determined as: PRE (%) = 100 1 E pN N (4 1) where NE is the number of particles entering the filter and Np is the number of particles penetrating the filter. VRE was determined by counting plaques of virus collected from control and experimental impingers. VRE (%) = 100 1 test ctrC C (4 2) where Cctr is the number of virus collected from the control impinger and Ctest is the number of virus collected from the experimental impinger. Infectivity of virus on the filter paper was determined by counting plaques of virus recovered from untreated and treated filter paper. The extracted fraction is defined as the ratio of the infectivity count in the extract solution to the total viruses collected on the filter. Experiments were conducted at room temperature and t wo relative humidities (RH): medium RH (55 5%, MRH) and high RH (90 5%, HRH). Experiments were conducted in triplicates in each environmental condition.

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84 4.3 Results and Discussion 4.3.1 Antibacterial Activity of Cotton Textile E.coli was the only bacterial strain tested in this study with ca.106 CFU/ml concentration during experiment. MLC data was determined in one -hour experiment to completely kill E.coli i.e., 6 log reduction. Before the trialkoxysilyl compounds were anchored to the cotton textile, the a ntibacterial activity of the free compound was examined. MLCs of AEM 5700 compounds against E.coli in deionized water were very low as indicated in Table 4 1. Trace amount of leaching AEM 5700 compound during antimicrobial test may have significant effect on the antibacterial activity of treated textile. Antibacterial action of AEM 5700 was significantly reduced in artificial seawater. Precipitation of QAC compound was observed. Antimicrobial action of small molecular QAC was reported to be blocked by ionparing and precipitation of quaternary cations with bulky anions in the aqueous solution (Kopecky 1996) Based on this study, the salinity of media significantly affected the antimicrobial activity of AEM 5700 compound. Asreceived triethoxysilylbutylaldehyde or triethoxysilylundecanal at 5% concentration was tested against approximatel y 106CFU/ml E.coli suspension. Antibacterial activities of these two compounds were very limited. No inactivation against E.coli was observed in one -hour exposure for both compounds. Antimicrobial activity of triethoxysilylbutylaldehyde was stronger than t riethoxysilylundecanal. Increasing exposure time can enhance the antibacterial activity of triethoxysilylbutylaldehdye. Only two log reduction was achieved in four -hour exposure. Based on the high concentration and long exposure time of these two compounds against E.coli, they were considered not to be effective biocides. Valeraldehyde (O=CH(CH2)3CH3) has a similar structure as triethoxysilylbutylaldehyde except the difference in the end group.

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85 However, valeraldehyde demonstrated strong antibacterial activity in one hour -exposure against E.coli with MLC at 0.6 wt%. Difference in solubility of these two compounds in water may cause the significant difference in their antibacterial activities. Triethoxysilylbutylaldehdye is not soluble in water, whereas valer aldehyde is miscible in water. The MLC of AEM 5700 was found to be very low against E.coli We believed that for the textile treatment by anchoring AEM5700, the leaching of trace amount of AEM 5700 might have significant effect on the antimicrobial activ ity of the treated textile. Antimicrobial activities of unbound trialkoxysilyl aldehdye compounds were not found in a one -hour exposure against E.col i. Once they were anchored onto textile, it was hoped that the bound aldehyde might have some antibacterial activity, as there was no solubility issue. Further studies focused on the antimicrobial activity of textile treated by anchoring these trialkoxylsilyl compounds. In general, the antibacterial of AEM 5700treated cotton textile displayed two stage behaviors as seen in Figure 4 6. In the first stage, the antibacterial activity was the combined effect from the leachate and surface -bound AEM 5700. After a certain number of cycles, the leachate lost its antimicrobial activity. The remaining antibacterial activ ity was caused by the surface-bound AEM 5700. The remaining antibacterial activity was very close for cotton textile treated by different concentrations of the AEM 5700. In each condition, approximately twolog reduction remained in the level off stage. Th e MLC of AEM 5700 against E.coli was very high in the artificial seawater case. The leachate of the treated cotton textile might not have effect on the bacteria in the artificial seawater, which was the case in our study as indicated in Figure 4 6 (d). The surface bound AEM 5700 still contributed approximately one log reduction antibacterial activity regardless of the concentration of AEM 5700 for textile treatment. In contrast, twolog reduction remained in

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86 the deionized water case. Salinity significantly affected the antibacterial activity of AEM 5700 leachate and the surface -bound AEM 5700. No antibacterial activities of unbound trialkoxysilyl aldehyde compounds against E.coli were observed during one -hour exposure. The leachates from the treated-cotton textiles by anchoring these compounds were not evaluated for their antibacterial activities. Antibacterial activities of 1% aldehdyes treated -cotton textiles are presented in Figure 4 7. No antibacterial activities were observed for the cotton textiles tr eated by trialkoxysilyl aldehdye compounds even for 48hr exposure. Characterization of the treated surface was not conducted. The exact surface composition is unknown. Based on the antibacterial data obtained for the cotton textiles treated by various trialkoxysilyl compounds, we did not continue to study the anchoring approach to design antibacterial surface. We switched to study antimicrobial surface from dialdehyde polysaccharides. 4.3.2 Characterization of Dialdehyde Cellulose (DAC) Filter Paper One of the methods to determine the oxidation extent of DAC filter paper was to measure sodium periodate consumption during oxidation. Absorbance at 221nm by UV can be used to measure the consumption of sodium periodate (Maekawa and Koshijima 1984) Calibration curve for the determination of sodium periodates consumption is shown in Figure 4 8 (a). A blank control of sodium periodate was also conducted. In the blank control, sodi um periodate solution without adding cellulose filter paper was kept the same condition as periodate oxidation of cellulose filter paper. The concentration change of periodate in the blank control was measured as shown in Figure 4 8 (b). The concentration change of sodium periodate in the blank control was negligible. The absorbance of sodium periodate at 221nm was directly converted to the concentration based on the calibration curve.

PAGE 87

87 Another method to determine the oxidation extent of the cellulose filter was using elemental analysis. The measured oxidation extent by these two methods is shown in Figure 4 9. The values obtained in these two methods were in good agreement with each other. It has been shown that periodate attacks both the amorphous and the c rystalline regions (Guthrie 1961) Loss of crystallinity would be anticipated with increasing oxidation extent and is confirmed by XRD shown in Figure 410. XRD intensity profiles collected for cellulose filter papers exhibited three characteristic reflections (101), (10 1) and (002) (Princi et al. 2004) of cellulos e I. The first two are of medium strong intensity in the range between 13 and 18 degree of 2 he third is very sharp with an extremely strong intensity at 22.8 degree of 2 he well defined cellulose I pattern diminished with the increase in oxidation e xtent. The loss of crystallinity has been considered to result from the opening of glucopyranose rings and destruction of their ordered packing (Kim et al. 2000) Crystallinity index (Xc) of cellulose was evaluated according to the Segal method (Thygesen et al. 2005) using the following relationship: Xc=100* (I002Iam)/I002 (4 3) Where I002 is the intensity of 002 peak (2 =22.8 ) and Iam is the intensity of amorphous r egion (2 =18 ). Segal method is straightforward and simple to use, but it often overestimates the crystallinity. The relationship between the crystallinity and oxidation extent (using value obtained from elemental analysis) is presented in Figure 4 11. It clearly indicated the destruction of crystal structure with increasing oxidation extent. The changes in chemical structure by periodate oxidation were examined by FTIR and UV-vis. In the FTIR spectra of DAC filter papers as indicated in Figure 4 12, two c haracteristic

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88 bands of DAC appeared in the 1730 and 880cm1. The sharp peak at 1730cm1 is a characteristic band of carbonyl groups. The broad band at 880cm1 was assigned to the hemiacetal and hydrated form of the DAC filter paper (Kim et al. 2000) In general, these two bands increased with increasing oxidation level. A weak absorbance at 1693cm1 was also observed. The intensity of this band also increased with increasing oxidation level. This peak was probably the conjugate aldehyde stretching. Degradation of cellulose during periodate oxidation has been reported (Vicini et al. 2004) Periodate oxidation modified cellulose to make it more stiff and brittle. In a s trong oxidation condition, cellulose showed a yellow color. In our study, cellulose filter paper shrank with increasing oxidation time. For example, the diameter of as received filter paper was approximately 5.5cm. After 12 -hour oxidation, it was around 3. 5cm. FTIR analysis allows semi quantitative measurement of the oxidation degree by comparison of the absorbance of two bands (C=O stretching at 1730cm1 and CH2 stretching at 2900cm1). This evaluation does not allow one to exactly calculate the degree of oxidation (Vicini et al. 2004) The intensity ratio of these two bands is pres ented (Figure 4 11). UV-vis spectra of the DAC cellulose filter paper after different periodate oxidation time are presented (Figure 4 12). Distinct absorbance at 300 and 245 nm were observed in the DAC filter paper. In general, absorbance of these two ban ds increased with increasing oxidation time. These two peaks at 300 and 245 nm were assigned to the non-conjugated aldehyde and conjugate aldehyde respectively (Margel and Rembaum 1980) Similar results of DAS were discussed in Chapter 3. UV -vis spectra supported the FTIR analysis. Aldehyde function was introduced to the cellulose structure after periodate oxidation. However, the formation mechanism of conjugated

PAGE 89

89 aldehyde during periodate oxidation is unknown. It was probably caus ed by the -elimination degradation mechanism described in Chapter 3. Furthermore, periodate absorbance at 221nm was not observed in the UV -vis spectra of the treated cellulose filter paper, another indication of no residual periodate. 4.3.3 Destruction of Microorganisms by Dialdehyde Polysaccharide Surface Contact Antimicrobial activity of the DAS granules against microorganisms demonstrated in Figure 3 14 was an example of using the ASTM E 2149 standard. Instead of textile described before, the insoluble powder wa s employed. From Figure 3 14, the antimicrobial activity of DAS granules was very limited against test microorganisms in one hour exposure. It was difficult to spread bacterial suspension onto loose DAS powders. A modified ATTC100 method was only used to t est the antibacterial activity of the DAS film (Figure 4 14). The control was a polystyrene Petri -dish. OS was the oxidized starch film deposited by 2 ml 5% aqueous suspension. The DAS film was prepared by deposition of 2 ml 3% DAS aqueous suspension. Bac terial inactivation by the OS film was insignificant, but the DAS film completely inactivated the test bacteria in one -hour exposure. The DAS film against bacteria was also compared at zero time and one -hour exposure. No activation was observed in the DAS film against E.coli at zero time. This observation demonstrated the inactivation of DAS film against bacteria was not from the interaction between DAS and TTC. It was noted even at zero time, DAS film significantly inactivated S.aureus The live S.aureus r eacts with TTC to form the red colonies, but the numbers of red colonies were very limited. The inactivation of bacteriophages (MS2 and PRD1) was conducted in the same way. The concentration of bacteriophage was chosen to be 0.1*104 PFU (plaque forming unit). In this concentration, the number of survival bacteriophage can still be counted on a surface. At zero

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90 time, we found no activation of phages or host bacteria on DAS film. The one -hour antiviral result is shown in Figure 4 15. One black dot in the pic ture represented one live virus. The DAS film exhibited a significant antiviral activity against MS2 and PRD1 viruses No antiviral activity was observed in the controlled oxidized starch film. Antimicrobial activity of DAS film is summarized in Table 4 2. Destruction of microorganism by a DAS surface contact is believed from its dialdehyde function. In solid form, the pH effect on the antimicrobial activity is expected to be diminished. Another polymer with dialdehyde functions, i.e., DAC was evaluated for its antimicrobial activity. Figure 4 16 shows the antibacterial activity of 4hr treated DAC compared to the untreated cellulose filter. 0.1ml 107 CFU/ml bacterial suspension was pipetted onto the cellulose filter. The untreated cellulose filter can retai n particle size around 2.7m. This pore size is very close to the bacterial size to keep the bacteria on the filter surface. Four -hour treated cellulose filter completely inactivated E.coli and S.aureus in one -hour exposure. At the zero time, an inhibitio n zone was observed in the DAC against S.aureus Some studies were conducted to understand the inhibition zone observed in the S.aureus at zero time for 4 -hr treated DAC filter paper. As described in Figure 4 13, no absorbance at 221nm of periodate was de tected in UV -vis. We were still worried about residual periodate remaining. Four pieces of the 4 hr treated DAC filter paper were immersed again in 200 ml 0.5% (w/v) aqueous sodium metabisulfite solution for four hours in a shaker to destroy any possible residual periodate. These post treated filter papers repeated the wash/rinse process as described before. After they dried at room temperature, they were test for bioactivity against S.aureus as shown in Figure 4 17. An inhibition zone was still observed a t zero time.

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91 Another experimental setup was designed to test the bioactivity of the leachates from the 4 hr treated and the post -treated 4 hr treated DAC filter papers. Each filter paper was cut into small pieces. They were soaked in 5ml deionized water fo r 24-hr in a shaker at 37 C. The leachates were collected for antimicrobial activity. A low volume of bacterial suspension (0.1ml 105 CFU/ml) was mixed into the leachate. The purpose to choose this low volume and low concentration of bacteria suspension was to minimize the decrease of the concentration of leachate. Antimicrobial activities of these two leachates against E.coli and S.aureus in 1 -hr test were not found. A parallel experiment was conducted in the same condition to collect the leachates. Thes e leachates were analyzed (without dilution) by UV -vis as indicated in Figure 4 18. No absorbance at 221 nm was observed for either leachate, indicating that there was no periodate in the leachate. An absorbance at 235 nm was observed for the leachate from 4 hr treated DAC. Based on the discussion presented before, this absorbance was assigned to the conjugated aldehyde. If one still applied the relationship between this absorbance and the DAS concentration obtained before, the concentration of the DAC in l eachate was approximately 0.03%. This concentration was too low to inactivate bacteria in one hour aqueous test. But some dialdehyde cellulose might diffuse out in the agar media to inhibit S.aureus to form the inhibition zone as observed in Figure 416. N o absorbance was recorded for the leachate from the post treated DAC filter paper. Its spectrum was very similar to the untreated cellulose filter paper, even though the later was recorded in the reflectance mode. The concentration of leaching DAC from the post treated DAC filter paper might be too low to be detected. However, a narrower inhibition zone was still observed for the post treated DAC filter paper. Trace amount dialdehyde cellulose might still diffuse out.

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92 Based on the studies described above, the inhibition zone in the S.aureu s was probably caused by the diffusion of small molecular DAC after the periodate oxidation. Antiviral activity of the DAC filter paper was examined against MS2 as shown in Figure 4 19. At zero time, no activation of hos t E.coli was found for the DAC filter papers. An MS2 aliquot (0.1* 104 PFU/ml) was spread onto surface. Xgal was added into nutrient agar to facilitate the virus counting. Xgal had no effect on the viability of MS2 virus on polystyrene Petri -dish (Figure 4 19 (a)). Compared to the number of virus on plain Petri -dish, the number of virus on the untreated cellulose filter was significantly reduced in one hour test (Figure 4 19 (c)). One possible reason for this observation is the pore size of the untreated c ellulose filter paper. The pore size of the untreated filter paper is around 2.7 m and the size of MS2 is ca. 28nm. MS2 virus may be trapped inside the pore of filter paper. However, some virus was still observed on the untreated filter paper surface as well as other locations. The four -hour treated DAC filter paper showed some antivi ral activity compared to the untreated filter paper. This antiviral activity was significantly improved for the 60 hour treated DAC filter paper. It was expected because of the aldehyde contents of these two DAC filters. However, in each case, virus on the surface of DAC filter paper was not observed. Figure 4 19 (b) demonstrated that DAC had no effect on the Xgar staining MS2 virus. It was noted even at zero time, no virus was shown on the surface of DAC filter paper. The antimicrobial activity of the DAC filter paper is summarized (Figure 4 20). 4.3.4 Preliminary Results of DAC Filter Paper for MS2 Virus Aerosols Evaluation of DAC filter paper for filtration of MS2 virus aerosols was conducted in Dr. C.Y.Wus lab in the Department of Environmental Engineer ing Sciences at the University of

PAGE 93

93 Florida. Preliminary results from Dr. Wu including pressure drop, PRE, VRE and extracted fraction are summarized in Table 4 3. The pressure drop was lower for the DAC filter papers compared to the untreated cellulose filte r paper, especially in the 12hr treated DAC filter paper. The porosity of the cellulose filter paper may have changed during periodate oxidation. No improvement of filtration was observed for the 4 -hr and 8 -hr treated DAC filter paper. Analysis was focused on the 12 hr treated DAC filter paper. In the high humidity case (RT/HRH), PRE of the 12 -hr treated DAC filter paper was the lowest among all test filter paper; meanwhile the pressure drop was also the lowest. This indicated the porosity had increased si gnificantly in the 12 -hr treated filter paper. It was anticipated that longer oxidation times might significantly affect the porosity of the cellulose filter paper. With the higher porosity, the PRE was expected to be lower. The performance of cellulose filters no matter whether treated or not, was better in the high humidity than that in the medium humidity. This was probably related to the wetting of virus aerosol onto the cellulose filter paper. The 12 -hr treated DAC filter paper had the highest VRE; eve n its PRE was the lowest. This suggested that more viruses remained onto the 12 -hr treated DAC filter paper, or that some of the passingthrough viruses were inactivated. The extracted fraction of the 12 hr treated DAC filter paper was significantly lower than that of the untreated filter paper, indicating antiviral effect of the DAC filter paper. The DAC filter paper demonstrated encouraging result for its application in filtration in the preliminary study. More experiments are currently being worked on to understand the humidity effect on the filtration of DAC filter paper. However, these experiments are not covered by this thesis.

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94 4.4 Conclusions Destructions of microorganisms by surface contact of dialdehyde polysaccharides were confirmed in this study. Dialdehyde polysaccharides demonstrated broad-spectrum antimicrobial action by surface contact. Antimicrobial actions of dialdehyde polysaccharides were from their dialdehyde functions. Analogous dicarboxyl oxidized starch had no antimicrobial action in s olid surface contact. Anchoring small molecular compounds containing antimicrobial function including monoaldehyde and QAC onto surface was also carried out. No antimicrobial actions were observed when monoaldehyde compounds were anchored to a surface or not. When QAC compound was bound to a surface, antimicrobial activity of the surface was observed. The real surface-bound antimicrobial action was distinguished from the unbound QAC action. Salinity of the media significantly reduced the antimicrobial acti on of the selected QAC compound, i.e., AEM 5700, regardless of bound or unbound to a surface.

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95 Table 4 1 Antibacterial activity of free AEM 5700 compound MLC( g/g) DI water AEM 5700/silane <84 AEM 5700/silanol 1 Artificial seawater AEM 5700/silane 80 00 Note: AEM 5700/silane: as received AEM 5700, AEM 5700/silanol: 24 h-hydrolysis product of as received AEM 5700, artificial seawater: density 1.024g/cm3 Table 4 2 Antimicrobial activity of DAS films Microorganism E.coli S. aureus MS2 PRD1 Log reductio n 6 6 2 3 1 2 Table 4 3 Preliminary results of DAC filter paper for MS2 virus aerosol Environmental Condition Test filter Pressure drop at 5.3cm/s (Pa) PRE (%) VRE (Average SD)* (%) Extracted fraction RT/MRH Untreated 5978 NA 70.0 1.5 NA 8 hr treate d 4733 NA 59.5 2.9 NA 12hr treated 2989 NA 52.0 7.9 NA RT/HRH Untreated 5978 86 88.1 5.4 60 4 hr treated 4484 88 79.9 4.4 NA 8 hr treated 4733 88 NA NA 12hr treated 2989 66 95.8 2.0 4 The average measurements in triplicate, Not available

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96 O=CH(CH2)3Si(OC2H5)3 O=CH(CH2)10Si(OC2H5)3 triethoxysilylbutyraldehyde triethoxysilylundecanal Figure 4 1 Chemical structures of trialkoxysilyl compounds Figure 4 2 Schematic illustration of anchoring trialkoxysilyl compounds onto cotton textile, A presents antimicrobial functional group such as QAC or aldehyde Textile (0.75 0.05g)+45 ml DI water + 5 ml E.coli suspension (106107CFU/ml) 1hr shaking Direct contact Log reduction determination Textile +50 ml DI water 1hr shaking 9 ml leachate 1ml E.coli suspension 1 hr mixing Log reduction determination Leachate Textile (0.75 0.05g)+45 ml DI water + 5 ml E.coli suspension (106107CFU/ml) 1hr shaking Direct contact Log reduction determination Textile +50 ml DI water 1hr shaking 9 ml leachate 1ml E.coli suspension 1 hr mixing Log reduction determination Leachate (a) (b) Figure 4 3 Antimicrobial experimental set up for tex tile, (a) ASTM E2149 01 shake flask method, (b) antimicrobial activity test of leachate Substrate OH OH OH +(OH)3Si(CH2)nA (CH2)nAO ( O Si O)x Substrate Substrate OH OH OH +(OH)3Si(CH2)nA (CH2)nAO ( O Si O)x Substrate Substrate OH OH OH +(OH)3Si(CH2)nA Substrate OH OH OH +(OH)3Si(CH2)nA (CH2)nAO ( O Si O)x Substrate (CH2)nAO ( O Si O)x Substrate (RO) 3 Si(CH 2 ) n A + H 2 O (OH) 3 Si(CH 2 ) n A + H 2 O Hydrolysis Condensation AEM 5700

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97 (a) (b) Figure 4 4 Experimental setup for viable removal efficiency (a) and physical removal efficiency of the test filters (from Dr. C.Y.Wu).

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98 0 1 2 3 4 5 6 0 4 8 12 16 20 24 Time (hr) Log reduction triethoxysilylbutylaldehyde triethoxysilylundecanal Figure 4 5 Antibacterial activities of aldehyde compounds against E.coli at differ ent time 0.00 1.00 2.00 3.00 4.00 5.00 6.00 0 5 10 15 20 25 30 Number of Cycle Log reduction Leachate Direct contact 0.00 1.00 2.00 3.00 4.00 5.00 6.00 0 5 10 15 20 25 30 Number of cycle Log reduction leachate direct contact 0.00 1.00 2.00 3.00 4.00 5.00 6.00 0 5 10 15 20 25 30 Number of cycle Log reduction leachate direct contact 0.00 0.50 1.00 1.50 2.00 0 1 2 3 4 5 Number of cycle Log reduction 1% direct contact 8% direct contact 1% leachate 8% leachate Figure 4 6 Antimicrobial activity of cotton textile treated by AEM 5700 with various concentrations against E.coli in deionized water (a) 0.1% (b) 0.5% (c) 1% and in artificial seawater (d) Each cycle: 24hrs continuous shaking then cleaning and repeating First stage Level off First stage Level off Level off First stage a b c d

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99 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0 12 24 36 48 Time (hr) Log reduction triethoxysilylbutyraldehyde triethoxysilylundecanal Figure 4 7 Antibacterial activities of 1% trialkoxysilyl aldehdyes treated cotton textiles at different time y = 9.1905x R2 = 0.997 0 0.5 1 1.5 2 0 0.05 0.1 0.15 0.2 Mole concentration (M*10-3) Absorbance at 221 nm 0.15 0.2 0.25 0 20 40 60 80 Time (hr) Mole concentration (M) Figure 4 8 Calibration curve for the determination of sodium periodate (a), concentration of sodium periodate in blank control (b) Figure 4 9 Effect of reaction time on the oxidation extent of cellulose filter a b 0 20 40 60 80 100 0 10 20 30 40 50 60 Reaction time (hr) Oxidation extent (%) Periodate Elemental analysis

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100 5 15 25 35 45 55 65 2 (degree) Intensity Figure 4 10 XRD patterns of cellulose filter paper at different oxidation time (hr). Arrow indicates the assignments of patterns to the oxidation tim e (up to bottom) 0 20 40 60 80 100 0 20 40 60 80 100 Oxidation extent (%) Crystallinity (%) 0 10 20 30 40 50 Intensity ratio of CH2/CHO (%) Figure 4 11 Relationship between oxidation extent and crystallinity of DAC 0 4 8 12 24 36 60

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101 1480 1580 1680 1780 Wavenumber (cm-1) Absorbance 580 680 780 880 980 1080 Wavenumber (cm-1) Absorbance 1700 2000 2300 2600 2900 3200 3500 3800 Wavenumber (cm-1) Absorbance Figure 4 12 FTIR spectra of DAC filter paper at different regions, arrow direction indicates increasing oxidation time 1693 60 36 24 12 8 4 880 60 36 24 12 8 4 60 36 24 12 8 4 1730

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102 200 250 300 350 400 Wavenumer (nm) Absorbance 200 250 300 350 400 Wavenumber (nm) Absorbance Fig ure 4 13 UV -vis spectra of cellulose filter paper (a), and after subtraction of untreated cellulose filter paper (b) from bottom to up in different oxidation time: 4, 8, 12, 24, 36hr DAS film E.coli at 1 hr DAS film S.aureus at 1 -hr DAS film -E.coli at 0 -hr DAS film S.aureus at 0 -hr Figure 4 14 DAS film against 0.1ml*107CFU/ml bacterial suspension untreated a b 300 245 300 245 DAS Control OS OS DAS Control 0 hr 1 hr 1 hr 0 hr

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103 MS2 PRD1 Figure 4 15 DAS film against bacteriophages E.coli S.aureus Figure 4 16 Antibacterial of DAC filter paper after 4 -hr periodate oxidation Control OS DAS Control OS DAS 1 hr ctr 0 hr ctr 1 hr DAC 0 hr DAC 1 hr ctr 0 hr ctr 1 hr DAC 0 hr DAC

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104 Figure 4 17 Post -treated DAC filter paper (4 -hr periodate oxidation) against S.aureus 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 210 230 250 270 290 310 330 350 370 Wavelength (nm) Absorbance Figure 4 18 UV -vis spectra of leachates of 4 -hr treated DAC (B) and post treated DAC (C ). A is reference untreated filter paper in reflectance mode 1 h r ctr 0 hr ctr 0 hr DAC 1 hr DAC A B C 235

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105 Figure 4 19 DAC filter papers against MS2 1 hr, untreated 4 hr, treated 60 hr, treated c d e 1 hr w/o Xgal PS Petri -dish 0 hr w/o Xgal, 60hr treated

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106 0 0.5 1 1.5 2 2.5 3 0 15 30 45 60 Periodate oxidation time (hr) Log reduction 0 10 20 30 40 50 60 70 80 90 100 aldehyde content (%) Figure 4 20 Antiviral activity of cellulose filter paper

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107 CHAPTER 5 STUDY THE ANTIBACTERIAL MECHANISMS OF DIALDEHYDE STARCH 5.1 Introduction Several excellent reviews of antibacterial mechanism of glutaraldehdye have already been published (Gorman e t al. 1980; McDonnell and Russell 1999) Antibacterial mechanism of glutaraldehyde involves a strong association with the outer layers of bacterial cells, specifically with unprotonated amino groups on the cell surface. The crosslinking action of glutaral dehyde on bacterial cell wall results in the strengthening of the outer layers. Examination in the electron microscope of glutaraldehyde treated bacterial exposed to lysozyme for 5 18 hr showed that wall morphology had been extensively retained (Hughes and Thurman 1970) Very few artifacts have been observed in the electro microscope studies of glutaraldehyde -fixed cells and tissues with little structural alterations (Munton and Russell 1973b) Strengthening of cell wall by glutaraldehyde can also affect the cell enzyme activity. Inhibition of dehydrogenase activity has been reported in a concentration of glutaraldehyde which had little effect on cell viability (Munton and Russell 1973a) Ready access of substrate to enzyme was prevented because of the strengthening of the outer cell surface by glutaraldehyde. Surface properties of bacterial cells have been stu died by measurement of electrophoretic mobility (Douglas 1959; Munton and Russell 1972) Bacterial cells have different functions such as carboxylic acid and amino groups. Surface charges of bacterial cells are pH -depende nt. If some functions of bacterial cells are blocked, bacterial cells will exhibit different electrophoretic mobility. Recently, bacterial cell response to chemical and physical inactivation have been studied using spectroscopic methods such as Raman and F TIR (Al Qadiri et al. 2006; Escoriza et al. 2007) Bacteria can been r apid detected and identified by these spectroscopic analyses.

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108 Fluorescent dye staining of bacteria can provide information on membrane integrity of the bacterial cells (Kim et al. 2007) Bacteria with a damaged membrane will stain red, whe reas with an intact membrane will stain green. Various fluorescent analyses can be employed to study the stained bacteria. In this study, the action of dialdehyde starch on bacteria was studied. We postulated that the dialdehyde starch could modify the sur face of bacterial cell through crosslinking reaction. With this crosslinking action, the enzyme activity of bacteria will be inhibited. The integrity of bacterial membrane was also investigated by the fluorescent dye method. If the predominant action of di aldehyde starch is caused by the crosslinking action, the bacterial cell will retain its structure and become tight to prevent red dye penetration. In that case, the inactivated bacteria may not distinguish from the healthy bacteria by the fluorescent anal ysis. 5.2. Materials and Experiments 5.2.1 Antibacterial Experiments The supernatant of the as -prepared 3% DAS aqueous suspension after centrifugation was used in the antibacterial experiments. High concentration bacterial ( E.coli or S.aureus ) suspension was employed. A typical DAS antibacterial procedure was: 2 ml *109 CFU/ml bacterial suspension was mixed with 3 ml DAS supernatant for four -hour to completely inactivate bacteria. The reason to choose the DAS supernatant and high concentration of bacteria w as that pure bacterial suspension was used for further analysis. To obtain pure bacterial suspension, the process involved four cycles of centrifugation-wash to remove DAS from the mixture. Plate counting was employed to confirm the completely inactivation of treated -bacteria. 5.2.2 FTIR Spectra Aliquots of treated and untreated bacterial suspension were deposited onto Al2O3 substrate. They were dried at room temperature for two hours to form a bio-film. The samples were

PAGE 109

109 analyzed by FTIR using Micro ATR sta ge. A microscope was used to identify and focus interesting area on the surface. The spectra were the average of twenty different locations using 128 scan at 4cm1 resolution. 5.2.3 Electrophoretic Mobility A Brookhaven zata plus was used to measure the e lectrophoretic mobility of the treated and untreated bacterial suspensions with different pH values at room temperature. After completely remove the DAS in the final wash, the bacterial pellet was resuspended in a PBS buffer. The pH of the bacterial suspen sion was adjusted by HCl/NaOH. 5.2.4 Dehydrogenase Activity Experiments of dehydrogenase activity were set up as follows: 0.25ml bacterial suspension (ca.109 CFU/ml), PBS buffer 3ml (pH 7.4), glucose (0.01M) 1 ml, triphenyltetrazolium chloride (TTC) 5% (wt /vol) 20 l and 1 ml DAS supernatant or deionized water were mixed in a tube. In this formulation, the pH of the mixture was 7. After incubation for certain time at 37 C, the tube was transferred to an ice bath to terminate the metabolic activity. After 5 ml of but anol was added, the bacteria were removed by centrifugation. The upper butanol layer was pipetted off, and the absorbance was measured in a spectrophotometer at 525nm. Controls were made either without DAS or bacteria. 5.2.5 Fluorescent dye Studies Fluore scent dye, LIVE/DEAD BacLight Bacterial Viability Kit was purchased from Invitrogen, USA. This kit is composed of two components. SYTO 9 green-fluorescent nucleic acid stain and Propidium iodide (PI) red -fluorescent nucleic acid stain. The green stain can label bacteria with intact and damaged membranes. In contrast, PI penetrates only bacteria with damaged membranes. When both stains are present, a reduction in the green fluorescence and an

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110 increase of red fluorescence will be observed because of the membr ane damage of bacteria. Various fluorescent analytical instruments can be used to detect the green and red fluorescence. 5.2.5.1 Fluorescence microscopy After the final wash of the bacterial suspension to remove the biocide, the bacterial pellets were resu spended in deionized water. Equal volumes of the red and green dyes were mixed thoroughly in a microfuge tube for one minute. Three microliters of the dye mixture was added into 1 ml bacterial suspension. The stained suspension was mixed thoroughly and kep t in dark for 15 minutes. Five microliters of the stained bacterial suspension was placed between a slid and a cover slip for microscopy evaluation. A TE2000 fluorescent microscope from Nikon with Nikon Eclipse C1 confocal system in the Department of Chemi cal Engineering at the University of Florida was used for observation of the stained bacteria at 40 (magnification). Individual picture with red or green dye was acquired, and then, combined picture of these two dyes was also obtained. 5.2.5.2 Fluorescenc e spectroscopy Four milliliters of the stained bacterial suspension were placed in a four -side lightpath rectangular curvet. The fluorescence emission was measured at the emission spectrum (excitation 470nm, emission 500700nm) by a Molecular Devices Spect raMax M5 fluorescence spectroscopy at Interdisciplinary Center for Biotechnology Research (ICBR). 5.2.5.3 Flow cytometry Stained bacterial suspensions were also analyzed by LSR II flow cytometer at ICBR. 5.3 Results and Discussion 5.3.1 Bacterial Surface Properties We speculated one of the interactions between the DAS and bacteria was through the crosslinking reaction between the dialdehyde functions of DAS and the amino functions of

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111 bacteria (Figure 5 1). The mobility and charge density of bacteria surfac e is pH dependent (Douglas 1959) At low pH, some of the surface functions may exist as COOH and NH3 +, as COOand NH3 + at neutral and as COO and NH2 at alkaline condition. Based on the assumption illustrated in Figure 5 1, some of the amino functions would have been consumed for the treated bacteria. In the FTIR spectra, one would expect a new band (C=N) at 1637cm1 and an increase of the ratio of carboxyl over amino for the treated bacteria. The pH dependence of mobility of the treated bacteria would also be changed. The FTIR spectra of the bacteria and pure DAS are presented in Figure 5 2. The assignments of bands were according to liter ature (Al Qadiri et al. 2006) : C=O stretching vibration of amides associated with proteins (~1660 cm1), N H deformation of amides associated with proteins (~ 1550 cm1), CH3 and CH2 asymmetric and symmetric deformation of proteins (~1460 and 1400 cm1). P=O asymmetric stretching of the phosphodiester backbone of nucleic acids DNA and RNA (~1245 cm1), and C O C stretching vibrations of polysaccharides content of bacterial cells (~ 1090 cm1). No significant change between the DAS treated and untreated bacteria was observed in the FTIR spectra. The expected C=N band (~1637 cm1) could not be distinguished. O ne possible reason is the uptake of DAS for crosslinking reaction was very low. The characteristic absorbance of DAS was not detected in the DAS treated bacteria. Another explanation may be that the absorbance of C=N is overlapping with amide band of the protein. The intensity ratio of the C=O stretching vibration over the N H deformation was calculated as indicated in Table 5 1. The ratio of C=O over N H increased for the DAS treated bacteria compared to the untreated bacteria. This result may indicate som e consumption of amino groups of bacteria, but this information is not convincing enough to draw a sound conclusion.

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112 The mobilities of bacteria were measured (Figure 5 3). Electrophoretic study on E.coli treated by glutaraldehyde has been studied (Munton and Russell 1972) Increasing aldehyde conce ntration caused corresponding decrease in mobility of treated E.coli measured at pH 7.0. Treatment of E.coli by glutaraldehyde decreased the negative charge on the cells to give smaller mobility over a pH range of 3 12. Similar results were observed in our study. In general, mobilities of DAS -treated E.coli and S.aureus were lower than those of the untreated bacteria over the selected pH range. The reduction of the mobilities of the treated bacteria was caused by less negative charge on the bacterial surface. This indicated some amino groups were blocked by the DAS. In the selected pH range, the bacterial surface contained a net negative charge. Deprotonation of amino group in alkaline condition increases the negative charge of bacterial surface. When some amino groups are blocked, the negative charge of the bacterial surface was reduced to result in a lower mobility. In the acidic condition, the proportion of the protonation of amino functions was similar for the DAS -treated and untreated bacteria. Without the contribution from the blocked amino groups, the total negative charge was also reduced for the treated bacteria. Electrophoretic study and FTIR indicated the interaction between the DAS and protein. The mechanism of this interaction was not examined as part of this thesis. 5.3.2 Inhibition of Enzyme Activity of Bacteria The effect of DAS on dehydrogenase activity of bacteria was investigated. Glutaraldehyde was reported not to react with TTC (Munton and Russell 1973a) In our study, no absorbance reading could be recorded for the control without bacteria (DAS, TTC, glucose and PBS buffer). The results were expressed as an absorbance ratio, i.e., the ratio of the absorbance of the supernatant liquid from the DAS treated bacteria over the absorbance of the supernatant fluid from the control bacteria without DAS. Based on this definition, the maxim um dehydrogenase

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113 activity is 1. The viability ratio was defined as the number of survival bacteria from the DAS treated bacterial suspension over the controlled bacterial suspension from plate counting. No killing of the bacteria would equal to 1. Effect of the glutaraldehyde on the dehydrogense activity of E.coli was studied by Munton (Munton and Russell 1973a) Their experiments showed that this enzyme system was markedly inhibited at both 5 and 8.5 (glutaraldehyde absent). Furthermore, their results demonstrated this dehydrogenase system at pH 7 was extremely sensitive to the action of glutaral dehyde. A significant decrease in enzyme activity was observed, meanwhile, no inactivation of the bacteria. This phenomenon was explained to be the crosslinking of outer cell surface by glutaraldehyde. Crosslinking effectively prevented access of the subst rate to the enzyme. The same results were found in our DAS -bacteria system (pH=7) (Figure 5 -4). The incubation time was carefully selected to prevent the inactivation of bacteria, which could also decrease the absorbance ratio. Inhibition of dehydrogenas e activity was observed at the condition of no killing of the bacteria. However, this was a preliminary study on the inhibition of dehydrogenase activity of bacteria by DAS. This preliminary result indicated that crosslinking action between the DAS and ba cteria could occur to affect the dehydrogenase activity. 5.3.3 Fluorescent Dye Study Crosslinking and strengthening of bacterial cell wall by glutaraldehyde have been reported. With these actions, bacterial cell wall morphology had been extensively retain ed even after prolong exposure to lysozyme (Hughes and Thurman 1970) Our earlier studies mentioned above also suggested a crosslinking reaction between the DAS and bacteria. Initially, we posulated that the cell wall structure of bacteria was retained by DAS from the crosslinking action. If the DAS treated bacteria were stained by the dye kit for membrane da mage detection, the red dye (PI) might not penetrate into the bacteria. Fluorescent response of DAS -treated bacteria might be

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114 similar to that of the untreated bacteria. QEM 5700, a QAC type biocide was selected as another control, as the inactivation mecha nism of QAC is bacterial membrane damage (McDonnell and Russell 1999) Fluorescent microscopy pictures of studied systems are presented (Figure 5 5). As expected, control bacteria (live system) showed mostly green light. AEM 5700 treated bacteria (dead) showed mostly red light. However, DAS tre ated bacteria (dead) also displayed mostly red instead of green. Fluorescent microscopy pictures of AEM 5700 and DAS treated bacterial were very similar. This indicated that membrane damage also occured in the DAS treated bacteria. Further fluorescent dye studies on bacterial systems were analyzed by fluorescence spectroscopy. Fluorescence emission spectra were recorded for each bacterial suspension using excitation 470nm, emission 450700nm. The fluorescence emission at the range between 500540nm is the g reen emission; at the range between 600 650nm is the red emission. Integrated intensities of the green and red emission can be acquired to calculate the proportion of live/dead bacteria. In our study, the controlled bacteria system was 100% live, and the treated systems were 100% dead. Green emissions were observed in the controlled bacteria systems (Figure 56). For spectra of the DAS and AEM 5700treated bacteria, the green emissions disappeared. Only the red emissions were detected. This observation was expected for AEM 5700-treated bacteria, but not for the DAS treated bacteria. The DAS treated bacteria showed even stronger red dye penetration. Green emissions of the DAS treated bacteria were much weaker than those of the AEM5700treated bacteria, which a shoulder in the green emission was observed. A very weak red emission was detected for 0.1% AEM 5700. A strong fluorescent emission was detected for 0.3% DAS. This fluorescent emission located in the lower wavelength

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115 position compared to the DAS -treated bacteria. The maximum emission of the DAS itself was around 580nm, whereas 620nm for the DAS -treated bacteria. Furthermore, the biocide treated bacteria were centrifuged and washed for four times to remove the biocide. The residual biocide in the final b acterial suspension should be very low. After last centrifugation, the supernatant from the DAS treated bacteria was analyzed by UV -vis. No absorbance of aldehyde at 238nm was detected. These excluded the residual DAS caused the red emission in the DAS tre ated bacteria. The lack of characteristic fluorescent emission of DAS in the DAS treated bacteria was similar to the FTIR spectra. In the FTIR spectra of the DAS treated bacteria, the characteristic absorbance of DAS was not detected. Quantifications of th e reduction of the green emission and the increase of red emission in the treated bacteria systems were analyzed by a flow cytometer as shown in Table 5 2. Analysis of the treated bacterial systems was based on the controlled systems. Parameters of contr olled system analysis were selected to have negligible green reduction and red increase. Same parameters were applied to the treated bacteria system to analyze the intensity changes of green and red. For all the treated bacterial system, significant green reductions were observed. However, the DAS treated bacterial systems showed much stronger red increase. This indicated more PI penetrated into the DAS treated bacteria than AEM 5700 treated bacteria. Various fluorescent analyses were performed on the bioci de treated bacteria. The fluorescent dye studies found that the DAS and AEM 5700treated bacteria exhibited similar fluorescent response. More red increase was observed in the DAS -treated bacteria than that in AEM 5700 system. These findings suggested memb rane damage also occurred in the DAS bacteria system; the extent of membrane damage might be even higher than the AEM 5700

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116 system. This finding is contradictory to the crosslinking action of DAS. Direct observations of bacterial structure by TEM are underw ay but are not part of this thesis. 5.4 Conclusions The mechanism of DAS action on bacteria was investigated. Bacterial surface was modified by DAS, probably from the consumption of amino groups through crosslinking reaction. Preliminary result indicated t hat DAS might inhibit the enzyme activity of bacteria by the crosslinking reaction to affect the transport process. Membrane damage of the DAS treated bacteria was suggested from the fluorescent studies. However, a direct observation of bacterial cell stru cture by TEM needs to confirm this speculation.

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117 Table 5.1 Intensity ratio of C=O stretching vibration over N H deformation Bacteria Untreated E.coli E.coli DAS Untreated S.aureus S.aureus DAS C=O/NH 1.22 1.41 1.53 1.64 Table 5 2 Flow cytometer analys is of the treated -bacterial systems Bacterial System Green reduction (%) Red increase (%) AEM5700 E.coli 66 9 DAS E.coli 87 95 AEM5700 S.aureus 79 8 DAS S.aureus 99 24

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118 DAS Bacterial surface groups Figure 5 1 Schematic illustration of the reaction between DAS and bacterium 700 1200 1700 2200 2700 3200 3700 Wavenumber (cm-1) Absorbance 700 1200 1700 2200 2700 3200 3700 Wavenumber (cm-1) Absorbance S.aureus E.coli 700 1200 1700 2200 2700 3200 3700 Wavenumber (cm-1) Absorbance Pure DAS Figure 5 2 FTIR spectra of bacteria and pure DAS O=CH HC=O O=CH HC=O NH2COOH NH2COOH NH2COOH NH2COOH + C=N COOH C=N COOH C=N COOH C=N COOH Untreated DAS Untreated DAS

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119 -2.50 -2.00 -1.50 -1.00 -0.50 0.00 2 4 6 8 10 12 pH Mobility EC EC_das SA SA_das Figur e 5 3 pH -mobility behaviors of bacteria 0 0.2 0.4 0.6 0.8 1 S.aureus E.coli Absorbance ratio Viability ratio Figure 5 4 Effect of DAS at 37 C on viability and dehydrogenase activity of bacteria Control DAS E.coli 2hr incubation S.aureus 0.5hr incubation Control DAS

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120 S.aureus Control_combine Control_green Control_red S.aureus AEM 5700_comb ine AEM 5700_green AEM 5700_red S.aureus DAS_combine DAS_green DAS_red Continued

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121 Ecoli Control_combine Control_green Control_red E.coli AEM 57 00_combine AEM 5700_green AEM 5700_red E.coli DAS_combine DAS_green DAS_red Figure 5 5 Fluorescent microscopy pictures of S.aureus and E.coli. Green means for green light, red means for red light, combine means combined picture of green light and red light

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122 0 1000 2000 3000 4000 5000 6000 480 530 580 630 680 Wavelength (nm) Emission 0 100 200 300 400 Emission E.coli control E.coli-DAS E.coli-AEM 5700 0 500 1000 1500 2000 480 530 580 630 680 Wavelength (nm) Emission 0 50 100 150 200 Emission S.aurues control S.aureus-DAS S.aureus-AEM 5700 0 500 1000 1500 2000 480 530 580 630 680 Wavelength (nm) Emission 0 50 100 150 200 Emission 0.3% DAS 0.1% AEM 5700 Figure 5 6 Fluorescence spectroscopy of bacterial samples (A E.coli B S.aureus ), pure 0.3% DAS and 0.1% AEM 5700 (C) A B C

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123 CHAPTER 6 CONCLUSIONS AND FUTURE WORK 6.1 Conclusions Broad -spectrum antimicrobial activities of dialdehyde polysaccharides (dialdehyde starch and dialdehyde cellulose) have been confirmed in this study. Antimicrobial action of dialdehdye polysaccharides can be achieved by aqueous suspension or by surface cont act. Antimicrobial activity of dialdehyde starch in the aqueous suspension was contributed to its dialdehyde functions. This activity was found to be pH -dependent. The pH -dependent antimicrobial activity was related to the pH -dependence of reactivity of al dehyde, structure of dialdehyde polysaccharide, and the cell wall structure of bacteria. Because of the complex system, the decisive conclusion has not been obtained in this study. Linear bacterial inactivation kinetics of dialdehyde starch was found. This linear inactivation kinetics was derived from the model of pseudo-first order chemical reaction kinetics. The established model successfully predicated the bacterial inactivation response with time. Degradation of dialdehyde starch was observed during coo king. Formation of conjugated aldehyde functions was confirmed by different analytical methods. The extent of conjugated aldehydes was affected by pH. Higher content of conjugated aldehyde was found in the alkaline condition. -elimination was suggested to be the mechanism of the formation of conjugated aldehyde. Acidity of dialdehdye starch was increased after the cooking. This might be caused by Cannizzaro reaction. Periodate oxidation of cellulose filter paper introduced the aldehyde functions to cellul ose structure. With increasing oxidation time, the oxidation extent increased, accompanying with decreasing the crystallinity. Conjugated aldehdye was also found in the strong oxidation condition.

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124 Destruction of bacteria by surface contact was evaluated i n two different approaches, i.e., anchoring of trialkoxysilyl compounds to a substrate and dialdehdye polysaccharides surface contact. Dialdehdye polysaccharides demonstrated effective antimicrobial action against bacteria and bacteriophages. Dialdehyde ce llulose filter paper was further evaluated in the virus aerosol. Preliminary results indicated dialdehyde cellulose filter paper to be a promising candidate. When AEM 5700 was anchored to cotton textile, the treated textile showed significant antibacterial activity in the beginning. However, this antibacterial activity was the combined action of the surface bound and leached AEM 5700. After certain cycles, ca. 2 log reduction remained, which was caused by the surface -bound AEM 5700. When trialkoxysilyl mono aldehdye was anchored to cotton textile, no inactivation of bacteria was observed. Free trialkoxysilyl monoaldehyde also did not show any inactivation against bacteria, even though valeraldehyde demonstrated strong antibacterial action. The bacterial surfa ce was modified by the dialdehyde starch. This modification was speculated to be the crosslinking reaction between the dialdehdye functions and functional groups from bacterial surface. Preliminary result indicated the inhibition of bacterial enzyme activi ty. This inhibition might be caused by the crosslinking action to affect the transport process. Fluorescent studies indicated higher membrane damage occurred in the dialdehdye strach treated bacteria compared to the AEM 5700 treated bacteria. 6.2 Future W ork Antimicrobial action of dialdehyde starch is related to the contact between dialdehyde functions and microorganisms. In an aqueous suspension, improvement of dispersion of dialdehyde starch enhances its antibacterial activity. Nano particle size dialde hyde starch or water -soluble dialdehyde starch are candidates to prepare easy -dispersed dialdehyde starch aqueous system. Better antimicrobial systems can be developed.

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125 Dialdehyde polysaccharides have been evaluated to destruction of microorganisms by dir ect solid surface contact. Other delivery methods of solid contact, for example, dialdehyde polysaccharides as filler in a substrate or graft of dialdehyde polysaccharides to other substrates, are recommended to study. The pH effect on the structures of d ialdehyde polysaccharides, the reactivity of dialdehyde functions and the corresponding antimicrobial activities need to be further studied to establish the relationship between the structure and antimicrobial activity.

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136 BIOGRAPHICAL SKETCH Le Song was born in 1972. He was raised in southern China u ntil he went to college. He obtained the degree of Bachelor of Engineering in rubber and plastic engineering from Qingdao Institute of Chemical Technology (Qingdao, China) in July 1993. He entered graduate school and obtained Master of Science in polymer chemistry and physics from Zhongshan University (Guangzhou, China) in July 1996. He worked as an R&D scientist to develop water -based waterproof construction materials in a local company in Guangzhou. Later, he switched his career to be a technical sales r epresentative in EJ chemical (a joint venture between Eckart, Germany and Jebsen, Hong Kong), Guangzhou office, to promote imported pigments and additives for coating, ink and plastic industry in southern China. He obtained another masters degree in the D epartment of Mechanical Engineering from the University of Houston, Houston, TX in spring 2003. After his graduation, he worked as a mechanical engineer intern in Schlumberger, Sugarland, TX. He began studying for the degree of Doctor of Philosophy in materials science and engineering at the University of Florida, Gainesville, in August 2004. During his graduate study, he published 4 papers and applied for one US patent.