ANTIMICROBIAL AND BIODEGRADABLE FOOD PACKAGING FILMS WITH CHITOSAN -BASED N -HALAMINE STRUCTURES TO PREVENT CONTAMINATION BY DRUG SUSCEPTIBLE AND RESISTANT STRAINS OF SALMONELLA TYPHIMURIUM By Vinni Thekkudan Novi A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Biosystems Engineering Œ Master of Science 2019 ABSTRACT ANTIMICROBIAL AND BIODEGRADABLE FOOD PACKAGING FILMS WITH CHITOSAN - BASED N - HALAMINE STRUCTURES TO PREVENT CONTAMINATION BY DRUG SUSCEPTIBLE AND RESISTANT STRAINS OF SALMONELLA TYPHIMURIUM By Vinni Thekkudan Novi Contamination of food samples with antibiotic resistant Salmonella Typhimurium has become a cause for concern due to difficulty in treating infections caused by this pathogen. In one approach, chitosan/PVA - based N - halamine (CPN) film was developed and test ed for its efficacy in inactivating drug susceptible and ampicillin resistant Salmonella Typhimurium strains. The CPN film significantly (100%) inactivated the growth of both strains during the antimicrobial sandwich assay when tested for five days since f ilm preparation , while the CH/PVA films showed around one log reduction ( p <0.05). CPN films reduced the drug resistant growth on cheddar cheese slices by 5 - 6 logs at 25°C and 3 - 4 logs at 4°C when packaged and stored over a period of five days unlike CH/PVA films that did not show significant reduction. The second approach involves the synthesis of a stronger chitosan N - ha lamine - based coating on p lasma treated polycaprolactone film (CH - NX/PCL film). The FTIR peaks obtained for chitosan coated PCL film (CH/PCL) showed characteristic peaks of both PCL and chitosan, specifically at 1720 cm - 1 and 3354 cm - 1 , respectively. The te nsile strength of the PCL modulus value was higher for CH/PCL . CH/PCL film showed better barrier against water and oxygen compared to PCL . The antimicrobial efficacy of the CH - NX/PCL film was 100% against both strains of Salmo nella Typhimurium when compared to PCL and CH/PCL, indicating that this fabricated film has promising applications in food safety. iv ACKNOWLEDGEMENTS The research was funded by The Axia Institute Michigan State University. This funding source was not involved in the actual research or writing of this report. Dr. Shan Shan is acknowledged for the original fabrication of the chitosan/PVA - N - halamine films and determination of optimal con ditions for the antimicrobial sandwich assay. Raymond Lesiyon is acknowledged as a valuable undergraduate contributor to this study. Ms. Xinyi Wang is acknowledged for providing consultation on plasma treatment and coating technique for film fabrication. The author would like to acknowledge Mr. Michael Rich and the Composite Materials and Structures Center for training and offering advice on characterization techniques such as thermogravimetric analyzer, water vapor transmission tester and oxygen transmiss ion tester. Dr. Per Askeland is acknowledged for his assistance in Plasma Reactor use and FTIR. Mr. Brian Rook is acknowledged for offering tensile strength testing services. Additionally, the author would like to thank Dr. Shannon Manning and the Manning Laboratory for providing the bacterial cultures and advice on microbiological methods and Dr. Jade Mitchell for providing advice on experimental design. Dr. Maria Rubino and Dr. Rafael Auras are acknowledged for offer ing consultation on fabrication of pac kaging films. The author would also like to acknowledge Dr. Evangeline Alocilja for providing guidance throughout the study and the NanoBiosensors Laboratory , specifically, Saad Sharief and Dr. Leann Lerie Matta for their help and support. Finally, the author would like to express gratitude to family and friends for being supportive and encouraging throughout the period of this study. v TABLE OF CONTENTS LIST OF TABLES ................................ ................................ ................................ ........................ v ii LIST OF FIGURES ................................ ................................ ................................ ....................... i x KEY TO ABBREVIATIONS ................................ ................................ ................................ ........ x i INTRODUCTION ................................ ................................ ................................ ........................... 1 HYPOTHESES AND OBJECTIVES ................................ ................................ ............................. 4 CHAPTER 1 ................................ ................................ ................................ ................................ .... 6 LITERATURE REVIEW ................................ ................................ ................................ ................ 6 1.1. Biodegradable polymers used for packaging ................................ ................................ 6 1.1.1. Polysaccharide - based polymers ................................ ................................ ............. 6 1.1.2. Protein - based polymers ................................ ................................ .......................... 8 1.1.3. Microbiological polymers ................................ ................................ ..................... 9 1.1.4. Synthetically produced biopolymers ................................ ................................ .... 10 1.2. Antimicrobial agents used in biodegradable packaging materials .............................. 11 1.2.1. Plant - based antimicrobial agents ................................ ................................ ......... 11 1.2.2. Metal ion - based antimicrobial agents ................................ ................................ .. 15 1.2.3. Other antimicrobial agents ................................ ................................ ................... 1 7 1.3. Antimicrobial packaging systems against Salmonella contamination ....................... 1 9 CHAPTER 2 ................................ ................................ ................................ ................................ .. 22 MATERIALS AND METHODS ................................ ................................ ................................ ... 22 2.1. Materials ................................ ................................ ................................ ..................... 22 2.2. M ethod s ................................ ................................ ................................ ....................... 22 2.2.1. Preparation of chitosan/PVA N - halamine - based (CPN) films ............................ 22 2.2.2. Determination of active chlorine content ................................ ............................. 23 2.2.3. Testing antimicrobial activity of CPN film against bacteria ............................... 24 2.2.4. Application of CPN films on packaging cheese ................................ .................. 2 5 2.2.5. Statistical Analysis ................................ ................................ ............................... 2 6 2.2.6. Preparation of chitosan - based N - halamine coated polycaprolactone films (CH - NX/PCL) ................................ ................................ ................................ ............. 2 6 2.2.7. Characterization of CH - NX/PCL films ................................ ............................... 28 2.2.8. Determination of active chlorine content ................................ ............................. 30 2.2.9. Testing antimicrobial activity of CH - NX/PCL film against bacteria .................. 31 2.2.10. Statistical analysis ................................ ................................ ................................ 31 CHAPTER 3 ................................ ................................ ................................ ................................ .. 3 2 TESTING ANTIBACTERIAL ACTIVITY OF CHITOSAN/PVA - BASED N - HALAMINE FILM AGAINST DRUG SUSCEPTIBLE AND AMPICILLIN RESISTANT SALMONELLA vi TYPHIMURIUM AND ITS APPLICATIONS IN PACKAGING CHEDDAR CHEESE SLICES ................................ ................................ ................................ ................................ ........................ 32 3.1. Introduction ................................ ................................ ................................ ................. 32 3.2. Methods ................................ ................................ ................................ ....................... 34 3.3. Results and Discussion ................................ ................................ ............................... 34 3.3.1. Determination of active chlorine content ................................ ............................. 34 3.3.2. Antimicrobial activity of CPN films ................................ ................................ .... 36 3.3.3. Application of CPN films on packaging cheese ................................ .................. 4 7 CHAPTER 4 ................................ ................................ ................................ ................................ .. 5 3 SYNTHESIS OF CHITOSAN - N - HALAMINE - COATED POLYCAPROLACTONE FILM AGAINST DRUG SUSCEPTIBLE AND AMPICILLIN RESISTANT SALMONELLA TYPHIMURIUM FOR FOOD PACKAGING APPLICATIONS ................................ ................ 5 3 4.1. Introduction ................................ ................................ ................................ ................. 5 3 4.2. Methods ................................ ................................ ................................ ....................... 5 4 4.3. Results and Discussion ................................ ................................ ............................... 5 4 4.3.1. Characterization of CH - NX/PCL films ................................ ............................... 5 4 4.3.2. Determination of active chlorine content ................................ ............................. 60 4.3.3. Testing antimicrobial activity of CH - NX/PCL film against bacteria .................. 62 CONCLUSION ................................ ................................ ................................ .............................. 6 6 REFERENCES ................................ ................................ ................................ .............................. 69 vii LIST OF TABLES T able 1: Examples of studies on antimicrobial packaging systems against Salmonella species ................................ ................................ ................................ ......................... 1 9 Table 2: ANOVA for S almonella Typhimurium treated with CH/PVA film compared with control ................................ ................................ ................................ ................................ ........... 38 Table 3: ANOVA for Salmonella Typhimurium treated with CPN film compared with control ................................ ................................ ................................ ................................ ........................ 3 9 Table 4: ANOVA for Ampicillin resistant S almonella Typhimurium treated with CH/PVA film compared with control ................................ ................................ ................................ .................. 3 9 Table 5: ANOVA for Ampicillin resistant S almonella Typhimurium treated with CPN film compared with control ................................ ................................ ................................ .................. 40 Table 6: Comparison of growth on day 0 between both strains when treated with CPN film along with control ................................ ................................ ................................ ................................ ... 4 4 Table 7: Comparison of growth on day 1 between both strains when treated with CPN film along with control ................................ ................................ ................................ ................................ ... 4 5 Table 8: ANOVA Cheese packed with CH/PVA film at 25 ° C compared with control ............ 50 Table 9: ANOVA Cheese packed with CPN film at 25 ° C compared with control ................... 51 Table 10: ANOVA Cheese packed with CH/PVA film at 4 ° C compared with control ............ 51 Table 11: ANOVA Cheese packed with CPN film at 4 ° C compared with control ................... 52 Table 1 2: Water vapor and oxygen permeability of pure and chitosan coated PCL ................................ ................................ ................................ ................................ ................ 60 Table 1 3 : ANOVA for Ampicillin resistant Salmonella Typhimurium treated with CH/PCL film and control group ................................ ................................ ................................ .......................... 6 4 viii Table 1 4 : ANOVA for Ampicillin resistant Salmonella Typhimurium treated with CH - NX/PCL film and control group ................................ ................................ ................................ .................. 6 4 Table 1 5 : ANOVA for S almonella Typhimurium treated with CH/PCL film and control group 6 4 Table 1 6 : ANOVA for Salmonella Typhimurium treated with CH - NX/PCL film and control group ................................ ................................ ................................ ................................ ............. 6 4 ix LIST OF FIGURES F igure 1: Chlorination of chitosan forms N - halamine structures that enhances the antimicrobial property of the film [69] ................................ ................................ ................................ ................ 33 Figure 2: The change in active chlorine content of CPN films over five days .............................. 36 Figure 3: Comparison of bacterial growth when treated with N - halamine CH/PVA (CPN) and CH/PVA films (contact time 30 minutes ; detection limit serial dilution of 10 - 3 ) ; (a) effect on drug susceptible Salmonella Typhimurium; (b) effect on ampicillin resistant Salmonella Typhimurium ................................ ................................ ................................ ................................ . 3 8 F igure 4: Comparison of bacterial growth under different conditions of the antimicrobial sandwich assay to the original bacterial concentration in the initial culture (contact time 30 minutes; detection limit serial dilution of 10 - 3 ) ; (a) drug susceptible Salmonella Typhimurium; (b) ampicillin resistant Salmonella Typhimurium ................................ ................................ ......... 41 Figure 5: Comparison of bacterial growth in broth culture treated with CH/PVA and CPN films over time; (a) drug susceptible Salmonella Typhimurium; (b) ampicillin resistant Salmonella Typhimurium ................................ ................................ ................................ ................................ 4 3 Figure 6: Remaining chlorine content (%) in CPN films after incubation in TSB against time (almost 60% reduction after 24 hours) ................................ ................................ ........................... 4 6 Figure 7: Comparison of bacterial growth (log CFU/ml of peptone water used) in cheese slices packaged with CH/PVA and CPN films over storage time (a) at 25°C; (b) at 4°C ....................... 4 7 Figure 8: Comparison of the FTIR spectra of pure PCL, chitosan coated PCL (CH/PCL) and chitosan ................................ ................................ ................................ ................................ .......... 5 6 Figure 9: TGA curves for (A) PCL; (B) CH/PCL; (C) CH - NX/PCL ................................ ............ 5 7 Figure 10: Mechanical properties of PCL and CH/PCL films (with statistically significant differences between each film type ( p <0.05)) (A) Tensile strength (GPa) ................................ ................................ ................................ ................................ .............. 5 8 Figure 11: Strips of films after mechanical testing (A) PCL; (B) CH/PCL ................................ ... 5 9 x Figure 12: Iodometric titration solutions with (A) chlorinated and uncoated PCL film; (B) CH/PCL film; (C) CH - NX/PCL film ................................ ................................ ............................. 61 Figure 13: Bacterial growth reduction for (A) ampicillin resistant Salmonella Typhimurium; (B) drug susceptible Salmonella Typhimurium (contact time 30 minutes; detection limit serial dilution of 10 - 3 ) ................................ ................................ ................................ .............................. 6 3 xi KEY TO ABBREVIATIONS CDC Centers for Disease Control and Prevention PCA Peanut Corporation of America ZnO Z inc oxide TiO 2 T itanium dioxide PVA P olyvinyl alcohol PCL Polycaprolactone ASTM American Society for Testing and Materials CH Chit osan PHB Polyhydroxybutyrate PHA P olyhydroxyalkanoate PHBV Poly(3 - hydroxybutyrate - co - 3 - hydroxyvalerate) PLA Polylactic acid FDA Food and Drug Administration EO Essential Oil GRAS Generally Recognized as Safe xii ZEO Zataria Essential Oil CEO Cinnamon E ssential O il CD Cyclodextrin EGO Eucalyptus globulus E ssential O il EEO E ucalyptus E ssential O i l TP Tea Polyphenols MgO Magnesium Oxide CaO Calcium Oxide UV Ultraviolet SEM Scanning Electron Microscopy EDTA Ethylenediaminet etraacetic acid BEO MIC Minimum Inhibitory Concentration MBC Minimum Bactericidal Concentration CFU Colony Forming Unit LAE Lauric arginate NaL Sodium Lactate xiii SA Sorbic acid AA Acet ic Acid CA Citric Acid LA Lactic Acid LevA Levulinic Acid OEO Oregano Essential Oil TSB Tryptic Soy Broth TSA Tryptic Soy Agar BPLS Brilliant Green Phenol Red Lactose Sucrose Agar PBS Phosphate Buffer Saline NaOH Sodium Hydroxide KI Potassium Iodide CPN C hitosan/ Polyvinyl Alcohol N - halamine CH/PVA C hitosan/ Polyvinyl Alcohol Na 2 S 2 O 3 Sodium Thiosulphate AATCC American Association of Textile Chemists and Colorists ANOVA A nalysis of V ariance xiv SS Sum of Squares DF Degrees of Freedom MS Mean of Squares CH - NX/PCL C hitosan - based N - halamine coated P olycaprolactone FTIR Fourier Transform Infrared TGA Thermogravimetric Analysis UTS United Testing Systems WVTR Water Vapor Transmission Rate RH Relative Humidity WVP Water Vapor Permeation OTR Oxygen Transmission Rate CH/PCL Chitosan/Polycaprolactone OP Oxygen Permeation 1 INTRODUCTION The Centers for Disease Control and Prevention (CDC) has reported several foodborne outbreaks in the U . S . over the past few years of which most of the contaminations are due to the bacteria Salmonella [1] . Every year, there are about 1.2 million illnesses caused by this bacterium, with 23,000 cases of hospitalizations and about 450 deaths just in the U.S. [1] . Almost a million of these illnesses have happened due to contamination in the food consumed [1] . There are different strains of Salmonella involved in each of these outbreaks and most recently there have also been reports of multi - drug resistant Salmonel la strains causing infections in individuals through contaminated food [1] . Other pathogens involved in such outbreaks are norovirus, Campylobacter spp., Shigella spp., Escherichia coli [2] and Listeria monocytogenes [3] . There have been 16 outbreaks reported in 2018 in a wide range of food products including raw meat, sprouts, dry cereals and coconut, fresh vegetables and chicken [1] . There have also been outbreaks in dairy products such as cheese in the past [4] . Most suc h issues occur during the food processing phase, where the food gets exposed to pathogens that lead to such large - scale outbreaks. There have been hospitalizations and death reported in extreme cases, and economic losses of affected consumers. In 2009, the estimated annual cost of illness caused by non - typhoidal Salmonella spp. is $3,309.3 million [5] . Apart from these, huge monetary losses for the food companies involved have also been reported, primarily due to food recalls and settlements made in lawsuit s against them. Some companies have also gone bankrupt due to this. For example, Peanut Corporation of America (PCA) was a relatively small company that went down due to one of the largest food recalls that happened in recent years, a result of failing to act against Salmonella contamination in their peanut products [6] . There are larger companies like [6] . 2 These critical issues make it necessary to find methods to prevent such foodborne contaminations in future. Food packaging is generally done to improve the shelf - life, quality and safety of food. Specifically, antimicrobial food packaging is designed to prevent spoilage through exposure to contaminating pathog ens in the environment [7] . However, most of the contaminations occur before the food is packaged and usually through its surface [8] . Therefore, an ideal way to tackle this issue would be to develop food packaging materials that can eliminate the microorg anism that invaded the food surface and prevent further contamination. Currently used commercial packaging materials are petroleum - based products and mostly synthetic in nature such as polystyrene [9] . Some of these films have been modified to incorporate antimicrobial property and tested on food samples [9] . The modification is usually in terms of antimicrobial coating applied on their surface [9] . However, there is now an increased trend towards identifying bio - based films due to their eco - friendly nature and flexibility in integrating antimicrobial agents. There are two main methods through which this can be done by embedding the antimicrobial agent within the film and by coating the agent on the film surface. Several biodegradable polymers have been st udied for this purpose including chitosan, starch, polylactic acid, etc. [10] . A wide range of antimicrobial agents have been explored for food safety, many of them being essential oils from plant sources. These essential oils are rich in active component s such as terpenoids and phenolic particles, which can affect bacterial growth through disruption of the cytoplasmic membrane, disruption of the transport system in their cell wall and inhibition of protein synthesis [11] . Silver with inherent antimicrobia l properties have been incorporated as antimicrobial agents in food packaging in the form of nanoparticles [11] . Inorganic nanoparticles such as zinc oxide (ZnO) and titanium dioxide (TiO 2 ) were studied for their ability to inactivate 3 microbial growth [11] . TiO 2 is photoreactive and generates hydroxyl radicals along with reactive oxygen species that can oxidize the phospholipids present in the bacterial cell membrane. Other antimicrobial agents include chitosan, enzymes such as lysozyme s and bacteriocins from lactic acid bacteria [11] . Some films have also included chemical agents that can preserve food and prevent contamination, especially quaternary ammonium salts [12] while some films also contain chitosan - based N - halamine structures [13] . 4 HYPOTHESES AND OBJECTIVES This study focusses on the use of the natural polymer, chitosan, to prepare a biodegradable packaging film, primarily because it has the added advantage of being antimicrobial in nature [13]. The objective is to enhance the antimicrobial activity of fabricated chitosan - based food packaging films using simpler methods and cost - effective raw materials. The novelty of the study is the simple fabrication of a chitosan - based N - halamine functionalized fi lm, that does not require additional processing steps unlike most studied in the literature. The antimicrobial activity of such films against drug resistant pathogens, especially Salmonella Typhimurium have not been studied in the past and this work aims to tackle this problem. Generally, biodegradable polymers are observed to have low mechanical strength compared to the synthetic polymers currently used in the packaging industry. However, b lending such polymers with another biodegradable polymer can improve their mechanical and barrier properties [ 14 ], which are essential for any food packaging material. In this study, previously synthesized chitosan and polyvinyl alcohol (PVA) blended film, with simple generation of N - halamine structures on their surface, was tested as a potential packaging material for cheddar cheese slices. The film was specifically tested for antimicrobial activity against the common foodborne pathogen Salmonella Typhimur ium, both drug - susceptible and drug - resistant strains. Another way to incorporate the enhanced antimicrobial chitosan activity in food packaging material is to coat them on a stronger film. There is a limited availability of biodegradable polymers bearing ideal physical properties for packaging applications. Polycaprolactone (PCL) is one such polymer obtained synthetically and having aliphatic ester linkages in its main chain. This polymer meets the American Society for Testing and Materials ( ASTM ) standard for biodegradability and has a melting point of 60°C. PCL with molecular weight higher than 40,000 5 g/mol form films that are water resistant and ideal for packaging applications [ 15 ]. This study also focusses on synthesizing ideal packaging films using PC L incorporated with the antimicrobial property of N - halamine through chitosan coating, followed by characterization of its physical properties. Antimicrobial activity of the synthesized film against drug susceptible and ampicillin resistant Salmonella Typh imurium was also tested. The following are the hypotheses tested: 1. N - halamine - based chitosan films can inactivate growth of both drug susceptible and drug resistant strains of the foodborne pathogen Salmonella Typhimurium. 2. The fabricated chitosan/PVA based N - halamine films can inactivate growth of both strains of bacteria in contaminated cheddar cheese matrix. 3. The fabricated chitosan - N - halamine ( CH - N - halamine ) coated PCL film can inactivate growth of both strains of bacteria apart from having better mechanical properties for packaging applications. 6 CHAPTER 1 LITERATURE REVIEW 1.1. Biodegradable polymers used for packaging 1.1.1. Polysaccharide - based polymers It is important to consider the w ater barrier properties of polymers while being considered for food packaging applications. In case of fresh produce, it is necessary to avoid de hydration while in case of dry food such as chips, it is important to prevent the food from turning soggy. Poly saccharide materials generally have good CO 2 and O 2 barriers . However, they are hydrophilic in nature and have poor water barrier properties. There are several polysaccharides found in nature that have been studied for food packaging application which incl udes starch, chitosan, cellulose, alginate and carrageenan [1 6 ] . Starch This is the most naturally abundant biodegradable polymer that is of low cost and easily available. It has good film forming properties due to the presence of amylose, which is its linear component and the other being amylopectin, which is highly branched. Genetically modified corn is a good source of starch with high amylose content and is generally used to make biodegradable films. However, starch with high amylose content has the disadvantage of being highly crystalline in nature which would need to be g elatinized at high temperatures (above 100°C and atmospheric pressure) [1 7 ] . This polysaccharide is considered to be a good matrix to incorporate antimicrobial materials for packaging applications [1 8 ] . 7 Cellulose Cellulose is the most abundantly available polymer in nature that ha s film forming proper ties , making it an ideal candidate for studies as a food packaging material. It can be isolated from several plant - based sources such as hemp, woo d and cotton. It has the advantage of being non - toxic and less expensive. Most often cellulose is used in the form of derivatives with improved properties. Hydroxypropyl cellulose, m ethylcellulose and cellophane are examples of such derivatives. Though they have good gas barrier properties, they are not resistant enough to water vapor [1 6 ] . Cellulose acetate is formed by the acetylation of cellulose and has advantages of being non - toxic , stable and odor less . It is water vapor permeable [1 9 ] , which can be an advantage or a disadvantage depending on the type of food being packaged . Alginate Alginate is extracte d from an algal source which is the brown seaweed, Phaeophyceae . It is a linear copolymer made of 1 - - d - - l - guluronic acid (G) and are salts of alginic acid. They have the ability to interact with cations , which enables them to form films that can be used in packaging application. More commonly they interact with calcium to form calcium alginate. The cations serve as a gelling agent for the alginate material [1 6 ] . Pectin Pectin consists of linear and branched regions of poly - 1,4 - galacturonic acids and has a complex hetero - polysaccharide structure. This polymer, however, do not have good physical properties that can be applied as ideal food packaging material and may require modifications before being used for that purpose [ 20 ] . 8 Chitosan Chitosan is a derivative of the naturally occurring polymer, chitin. This is generally obtained from shells of crustaceans and insect cuticles . The removal of N - acetyl groups o n chitin through al kali treatment at high temperatures produce chitosan [ 21 ] . Both chitin a nd chitosan polymers exhibit antimicrobial properties which makes them desirable for several applications that involve prevention of contamination. Chitosan is linear in structure and consists of (1,4) - linked 2 - amino - deoxy - - d - glucan , is non - toxic and biocompatible [1 6 ] . 1.1.2. Protein - based polymers Protein - based polymers are made of amino acid monomer units. Whey, soy and corn zein proteins have been commonly studied for the fabrication of ideal food packaging materials [1 8 ] . Whey protein Whey protein isolate is a by - product of cheese or casein manufacture and is abundantly available in milk. It has polymeric properties and forms films with good gas barrier properties. However, these films have low tensile strength and poor water barrier properties , which necessitates its modification through blending or coating with other polymers and plasticizers [2 2 ] . Soy protein A by - product from soy oil production is used to extract soy protein isolate that has shown film - forming properties. These films, like most other naturally occurring polymers , have excellent oxygen barrier properties and high water vapor permeability due to their hydrophilic nature [2 3 ] . 9 Corn zein Zein is a protein found in corn and maize that is hydrophobic in nature. This protein can form films that have been explored for food packaging applications. Though they have excellent gas barrier properties, they do not have desirable physical propertie s that limit their use as films for long term food storage [2 4 ] . Unlike films obtained from other protein sources, zein shows good thermoplastic behavior [ 2 5 ] that is useful for packaging applications, if modified to have better mechanical characteristics . 1.1.3. Microbiological polymers Polyhydroxybutyrate (PHB) This is a synthetically produced biodegradable polymer that has been studied in food packaging application. It belongs to the polyhydroxyalkanoates (PHAs) family and is produced using microorganisms such as Ralstonia eutrophus, Bacillus megaterium, etc. th rough the process of fermentation. This polymer has poor mechanical properties compared to most other biodegradable polymers, is brittle and have low gas barrier properties which may not be ideal for food storage [2 6 ] . Poly(3 - hydroxybutyrate - co - 3 - hydroxyv alerate) (PHBV) To improve the properties of PHB, poly(3 - hydroxybutyrate - co - 3 - hydroxyvalerate) (PHBV) was created with three units of PHB and one unit of hydroxyvalerate. This polymer shows lower melting temperature and crystallinity with respect to PHB, m aking it more suitable for commercial application. However, this modification has lowered the mechanical properties along with making it thermally less stable [2 7 ] . 10 1.1.4. Synthetically produced biopolymers Polycaprolactone (PCL) Polycaprolactone is a synthetic polymer fabricated by the ring - opening polymerization of the - caprolactone [1 9 ] . This material shows good thermoplastic behavior , is biocompatible, and has good mechanical properties. Its biocompatibility mak es it an ideal polymer for use in biomedical applications [2 8 ] . Polylactic acid (PLA) Polylactic acid has been derived from natural sources and is one of the most promising biodegradable polymers for food packaging applications. It is usually obtained by the fermentation of renewable starch - rich products like corn, wheat, sugar beet, etc. this polymer can be synthesized either through condensation or ring - opening polymerization process of lactic acid. The advantages of this polymer include good mechanical properties, high barrier and low toxicity [1 9 ] . Polyvinyl alcohol (PVA) Polyvinyl alcohol is another synthetic polymer with biodegradable properties . It has a linear structure and has good film forming properties, is non - toxic and compatible with other materials and has good adhesive properties as well. This polymer has good mechanical properties, with chemical resistance and low moisture absorption properties compared to most other hydrophilic polymers. This material has been approved by the Food and Drug A dministration ( FDA ) to be used as food packaging material because of additional advantages such as barrier to oxygen and aromatic compounds [1 7 ] . 11 1.2. Antimicrobial agents used in biodegradable packaging materials 1.2.1. Plant - based antimicrobial agents Essential oils (EO) Essential oils are generally produced by plants to protect themselves from microbial and insect attacks. They are colored li quids with volatile compounds that have antimicrobial activity. These compounds are commonly plant secondary metabolites with aromatic functional groups that give out strong odor . EOs are known to have larvicidal, antifungal, antioxidant, antitumor and anti - inflammatory activities. Due to the emergence of multidrug resistant bacterial pathogens, more research is being carried out with essential oils to identify ideal antimicrobial d oses that can replace ineffective antibiotics against bacterial infections or contaminations [2 9 ] . E O s can be extracted from different parts of a plant including leaves, seeds, herbs, wood, fruits, roots and flowers. Zataria multiflora Boiss consists of EOs with antimicrobial activity such as carvacrol and thymol . Both these phenolic compounds have been approved by the FDA as Generally Recognized as Safe (GRAS) and so, the antimicrobial efficacy of the Zataria EO (ZEO) was studied again st pathogens such as L. monocytogenes, E. coliO157:H7, S. aureus and S. Typhimurium, and some kinds of fungi in the past. Specifically , it has been incorporated in zein based films and tested for antimicrobial efficacy against L. monocytogenes and E. coli. Two concentrations of ZEO were teste d and it was found that 5% of the EO caused around one log reduction, whereas, 10% of EO applied showed around two log reductio n of both bacterial strains [2 5 ] . Cinnamon essential oil (CEO) is one of the most studied E Os for antimicrobial food packaging applications. It is a natural EO that possesses antifungal and antimicrobial activity against other 12 pathogens. However, CEO easily decomposes at temperatures higher than 60°C and forms benzaldehyde, losing its desirable antimicrobial properties in the process [ 30 ] . A previous stu dy tested the activity of CEO against Escherichia coli and Staphylococcus aureus by its incorporation into a PVA based film. This antimicrobial film was fabricated using electrospinning method and consists of - cyclodextrin - CD) to encapsulate CEO . The encapsulation is done to improve its stability and mask its undesirable flavor in the film. The - CD nanofibrous film was able to inhibit both bacteria but the addition of CEO and - CD increased the hydrophilicity of the fil m [ 31 ] , which could potentially reduce its water barrier properties. Yet another study incorporated CEO into PVA films with the aid of P ickering emulsion to stabilize this essential oil. Due to the low thermal stability, susceptibility to oxidation and light [3 2 ] , the CEO requires systems that could stabilize them while being applied as an antimicrobial agent in a potential packaging film. Eucalyptus globulus essential oil (EGO) is another compound studied for its antimicrobial activity in food packaging films. One study used this EO in a chitosan film and tested for its antimicrobial efficacy against Gram - negative bacteria - Salmonella enter i tidis and Escherichia coli ; Gram - positive bacteria - Bacillus cereus and Staphylococcus aureus . In all the cases the films were able to show around 4 log reductions compared to the co ntrols when EGO was applied in the liquid form. In its vapor form, the reduction was lesser, around one log . The active films exhibited morphological changes compared to the control , which included reduction in its tensile strength due to increase in porosity of the chitosan film with the incorporation of EGO [3 3 ] . Another study describes the fabrication of chitosan films incorporated with cumin (CEO) and eucalyptus essential oils (EEO) , which was tested for increasing the refrigerated storage life of fresh chicken meat . The inhibition of Listeria monocytogenes , Salmonella typhi , 13 Streptococcus pyogenes and Shigella dysenteriae by chitosan films with cumin and eucalyptus EO were tested . The results indicated potential applica tion of these films for enhancing the storage life of packaged food [3 4 ] . Though EO based films exhibit good antimicrobial activity , there are certain drawbacks such as high cost, low water solubility, low stability and they contain volatile compounds that produce off - odors . These properties limit their practical application as a food packaging material [3 5 ] . Plant polyphenols Polyphenols obtained from plants show potential antimicrobial activity against pathogenic microorganisms apart from being antioxidant. They also contribute to nutritional value apart from color and taste to the packaged food [3 5 ] . Gallic acid (3,4,5 - trihydroxybenzoic acid) [3 6 ] has shown potential antimicrobial activities in past studies against bacteria such as Salmonella typhi and Staphylococcus aureus. This phenolic compound was extracted from Caesalpinia mimosoides Lamk (Leguminosae). There are other sources of gallic acid such as the flower of Rosa chinensis Jacq. That has shown potential antimicrobial activity against Vibrios species. This compound has also shown to modify the physical properties of films such as elasticity and reduced its brittleness. One study specifically tested the incorporation of various Escherichia coli, Salmonella T yphimurium , Listeria innocua and Bacillus subtilis . The results indicated that in addition to enhanced antimicrobia l activity, water and oxygen barrier properties of the film also increased with the addition of this phenolic compound [3 7 ] . 14 In one study, apple peel was used as a source of polyphenols and was incorporated into chitosan film matrix . Apple peel consists of bioactive compounds such as phenolic acids, flavonols, flavon - 3 - ols, anthocyanins , dihydrochalcones , p rocyanidins, (+) - - epicatechin, chlorogenic acid, phloridzin and quercetin conjugates . The results for this study indicated that t he antimicrobial activity of chitosan film was enhanced with the addition of apple peel extracts containing polyphenols. The mechanical properties of the film w ere also modified , where its thickness and density increase d along with decrease in the water vapor permeability. However, the tensile strength and elongation at break also reduced from that of regular chitosan films. The antimicrobial activity of these films depended on the concentration of the extract incorporated into the chitosan film [3 8 ] . Tea is a source of a class of phenols called tea polyphenols (TP) . They are known to have excellent antimicrobial and antioxidant properties and t heir antimicrobial mechanism is to prevent microbial attachment through direct disruption of cells. Specif ically, TPs can inhibit Gram - negative and Gram - positive bacteria along with fungal growth. In one study four polylactic acid/tea polyphenol composite nanofibers were prepared using electrospinning and studied for its application in food packaging. It was found that the incorporation of TP into the nanofiber reduced its tensile strength and elongation at break. Its antimicrobial activities agai nst Escherichia coli and Staphylococcus aureus showed over 90% growth inhibition , showing that the films could potentially be used to improve the shelf life of food [3 9 ] . In most cases involving the incorporation of plant - based secondary metabolites into biodegradable polymer matrix, the mechanical properties of the film are being compromised. Though their antimicrobial efficacy is promising, it is equally important to prod uce packaging films that have physical properties that make them ideal for long - term storage. Therefore, further 15 studies are required to produce films with excellent antimicrobial properties that do not adversely 1.2.2. Meta l ion - based antimicrobial agents Metal ion - based antimicrobials are generally termed inorganic agents and mostly possess biocidal activity in their oxi dized nanoparticle forms. The reason for this activity is due to the ir resistance to harsh treatment conditions under which they are prepared. Some of the metal oxides that were studied for food packaging applications include TiO 2 , ZnO, mag nesium oxide ( MgO ) and calcium oxide ( CaO ) [ 40 ] . TiO 2 nanoparticles TiO 2 can absorb ultraviolet (UV) light from the sun or artificial light sources that leads to a photochemical reaction involving oxidation - reduction, making the metal ion more biocidal against microorganisms due to the production of free radicals . Antimicrobial PVA films based on the incorporation of TiO 2 nanoparticles were fabricated and their mechanical and antimicrobial properties were tested for packaging Macrobrachium rosenbergii. The results indicated that the tensile strength and oxygen barri er propert y of the PVA film increased with the incorporation of the nanoparticle . The fabricated films were thermally stable compared to pure PVA films and exhibited good antimicrobial activity against Escherichia coli and Staphylococcus aureus [ 41 ] . Another study reported the fabrication of chitosan films incorporated with TiO 2 nano - powder and its antimicrobial activity against Escherichia coli , Staphylococcus aureus , and the fungal strains, Candida albicans , and Aspergillus niger . The metal ion in the film enhanced the antimicrobial efficacy and showed 100% sterilization in 12 h [4 2 ] . Though the antimicrobial and mechanical properties exhibited by films incorporated with this metal ion are excellent, due to 16 their potential toxicit y to human cells [ 40 ] , the application of TiO 2 in food packaging systems is not yet fully practical . Silver nanoparticles Silver nanoparticles have been incorporated into chitosan and starch - based films for enhanc ed antimicrobial properties. These films showed improved tensile strength and oxygen barrier properties. However, the water barrier properties were diminished . Their antimicrobial activity against E. coli , S. aureus and B. cereus was tested, and the results indicated that the fabricated film can potentially be applied for food packaging. The silver nanoparticles produced in this - ray irradiation unlike the conventional method and did not require additional steps in its production. Silver nanopart icles are conventionally produced by the reduction of silver salts by a reducing agent in the presence of a stabilizer. This means that the production of these nanoparticles would also require removal of these additional agents at the end of the process making it costly and time consuming. Additionally, the residues could be toxic [4 3 ] . ZnO nanoparticles ZnO nanoparticles have exhibited antimicrobial activity against bacteria, including some resistant strains and is approved by the FDA as GRAS. These nanoparticles were mixed with sodium carboxymethyl cellulose to form antimicrobial packaging films and their efficacy to inhibit Staphylococcus aureus was tested. Specifically, the study involved testing the film as a package for pork meat for 14 days in cold storage. The scanning electron micro scopy (SEM) showed that the ZnO nanoparticles were able to rupture the bacterial cells in cold storage [4 4 ] . Another study used ZnO nanoparticles in poly(3 - hydroxybutyrate - co - 3 - hydroxyvalerate) films. 17 The thermal stability of the film s improved with its addition and the films showed promise in inhibiting the growth of the foodborne pathogen Listeria monocytogenes . Generally metal based materials are rarely recognized as safe [4 5 ] and are usually toxic for food - based applications . Therefore, there is a need to look for sources and doses of such material s that reduces toxicity and enhances antimicrobial activity against just the pathogenic microorganism cells. 1.2.3. Other antimicrobial agents Lysozyme Lysozyme is a naturally occurring enzyme that has demonstrated its use as a bio - preservat ive for food packaging studies. One study used lysozyme obtained from hen egg white as an antimicrobial agent and was blended into cellulose acetate films. The study investigated the morphology of the film with respect to the method of lysozyme incorporation and it was found the immobilization of lysozyme improved antimicrobial activities and tensile strength of the film [4 6 ] . The antimicrobial mechanism of t his lytic enzyme - 1 - 4 - glycosidic linkage between the N - acetylmuramic acid and the N - acetylglucosamine groups of the peptidoglycan layer in the bacterial cell wall. For this reason, Gram positive bacteria are highly susceptibl e to the effects of lysozyme than Gram negative bacteria [4 7 ] . There have been studies incorporating lysozyme in zein films along with the inclusion of EDTA to enhance its antimicrobial activity against Gram negative bacteria [4 8 ] . Nisin Nisin is a bacteriocin produced by certain strains of Lactococcus lactis and is thermally stable. This antimicrobial agent is effective in inhibiting the growth of Gram - positive bacterial species 18 such as Clostridium , Bacillus , Staphylococcus and Listeria. One study explains the fabrication of nisin embedded PLA films for antimicrobial food packaging. The fabricated film was tested against Listeria monocytogenes , Escherichia coli O157:H7, and Salmonella Enteritidis and it was fou nd that film had reduced the growth of L. monocytogenes by around 4 logs in both culture medium and liquid egg white. In the case of the E. coli strain, the antimicrobial effect was more prominent in orange juice samples than in the culture medium. Growth of Salmonella Enteritidis reduced by two logs, much lower than that seen for the other two strains. However, at lower temperature, the nisin inco rporated film could reduce around 3 logs of this bacteria after 21 days in liquid egg white compared to the control [4 9 ] . Another study use chitosan /PVA films with nisin to test its activity against Staphylococcus aureus [ 50 ] . N - halamine - based antimicrobial agents N - halamine consists of nitrogen - halogen covalent bonds with potential antimicrobial activity against pathogens and have been exploited in past studies for their use as an antimicrobial agent in packaging materials [ 51 ] . There are sever al compounds that have the N - halamine structure and they usually involve complex preparation processes with various chemicals. One of the compounds prepared for this purpose is 1 - chloro - 2,2,5,5 - tetramethyl - 4 - imidazolidinon , which was used in absorbent pads for beef [ 51 ] and chicken [5 2 ] . The preparation of N - halamine structures with the food packaging polymer usually involves the chlorination of available or synthesized N - halamine precursors and these include polymethacrylami de [5 3 ] , [5 4 ] , m [5 5 ] , etc. Studies involving N - halamine based antimicrobial films have shown excellent antimicrobial efficacy against pathogenic microorganisms. However, their complex preparation process may be time consuming and expensive and this necessitates a need to look for simpler methods to 19 prepare such films with minimal damage to the mechanical properties of the biodegradable film used. 1.3. Antimicrobial packaging systems against Salmonella contamination The following table gives a list of antimicrobial packaging systems that have been specifically tested against the foodborne pathogen, Salmonella. Table 1 : Examples of studies on antimicrobial packaging systems against Salmonella species Salmonella strain Biodegra dable poly mer Antimicro bial agent Film fabricati on method Addition al materials for fabricati on Antimicrobi al efficacy (MIC, MBC, log /CFU reductions) Reference s Salmonella sp p. PLA Silver - based nanoclay Solvent casting method with homogen eous dispersio n of silver nanoclay Montmor illonite for silver immobili zation 99.99% CFU reduction [5 6 ] Salmonella enterica Serovar Typhimurium ( S . Typhimurium ) PLA Lauric arginate (LAE) PLA film surface activated with corona discharge and coated with LAE Silicone used as surfactant 2 to 3 log CFU/tested film with 0.07% of the agent [5 7 ] 20 Table 1 : Salmonella T yphimurium PLA Edible chitosan - acid solutions incorporati ng lauric arginate ester (LAE), sodium lactate (NaL), and sorbic acid (SA) alone or in combinati on Commerc ial PLA films with antimicro bial coating 2 % of acetic acid (AA), citric acid (CA), lactic acid (LA), and levulinic acid (LevA) for chitosan coating preparati on <0.69 log CFU/ ml with 1.94 mg/cm 2 of chitosan and 1.94 2 of LAE in PLA film [7] Salmonella enterica PVA Oregano e ssential oil (OEO) PVA solution casted with OEO in emulsion Glycerol (0.5 wt%) and Tween 20 (0.5 wt%) for emulsion Around 4.7 log CFU/tomato on first day [58] Salmonella Typhimurium Chitosan; polyamide 6/66 based blend; coated polyamide 6/66 Chitosan Solvent evaporati on technique ; incorpora tion of chitosan powder in extruded polyamid e 6/66; regular coating on polyamid e 6/66 - Contact inhibition [59] 21 As can be seen, limited studies have focused on this pathogen, though it is one of the most common pathogens seen in foodborne outbreaks. Past literature has concentrated on inhibiting the growth of drug susceptible bacterial pathogens and have not explored the activities of antimicrobial packaging systems for drug resistant bacterial strains. Antimicrobial resistance in b acteria is usually acquired through mu tations in genes that are responsible for preventing antibiotics from entering the cell transport system and inactivat ing the pathogen [ 60 ] . Strains that are already resistant to a biocidal agent have the potential to develop resistance to other antimicrobial agents upon continuous exposure to them through a phenomenon called selection pressure. Therefore, studying the eff icacy of antimicrobi al packaging material in inactivating drug resistant pathogen warrants special attention and the present study aims to achieve that. 22 CHAPTER 2 MATERIALS AND METHODS 2.1. Materials Chitosan (CH, low molecular weight, deacetylated chitin), polyvinyl alcohol (PVA, Mw 85,000 - 124,000, 99+% hydrolyzed), polycaprolactone (PCL, average Mn 80,000), sodium hypochlorite, tryptic soy broth (TSB), tryptic soy agar (TSA), brilliant green phenol r ed lactose sucrose agar (BPLS), sodium thiosulfate penta - were purchased from Sigma Aldrich (St. Louis, MO). Acetic acid was purchased from EMD Millipore (Burlington, MA) and sodium hydroxide (NaOH ) pellets were purchased from J.T. Baker (Phillipsburg, NJ). Potassium iodide (KI) (ACS grade, granular) was purchased from Columbus Chemical Industries (CCI, Columbus, WI). Sliced sharp cheddar cheese was purchased from a local grocery store. Bacterial cu ltures of Salmonella Typhimurium, and ampicillin resistant Salmonella Typhimurium (TW16633) were maintained in TSB. 2.2. Methods 2.2.1. Preparation of chitosan/PVA N - halamine - based (CPN) films The preparation of the film is a modified procedure following previous literatures [6 1 , 6 2 ] . Briefly, chitosan solution (2 %) was prepared by dissolving in 2 % acetic acid at 65°C for 60 minutes and PVA solution (2 %) was prepared by dissolving in deionize d water at 275°C for 30 minutes. The two solutions were then mixed together to form a homogeneous blend of 50 ml in the ratio of 2 CH:3 PVA for another 30 minutes. This solution was centrifuged at 8000 rpm for 10 minutes to separate impurities and air bubb les before being casted on glass containers of uniform size (11 x 16 cm 2 ). The solution was dried at room temperature overnight and the dried film was treated with 1 N NaOH solution to neutralize the acidic pH caused by acetic acid. The 23 films were washed repeatedly with deionized water, dried and treated with 0.65 % sodium hypochlorite solution to form N - halamine structures on the film surface. They were further dried after washing repeatedly with deionized water and stored in a desiccato r before they were used for further experiments. 2.2.2. Determination of active chlorine content The active chlorine content of the film is proportional to the N - halamine structures present on the surface of the CPN films. Therefore, detecting the presence of ch lorine and quantifying it is important to confirm the presence of N - halamine, which is the bioactive agent. Iodometric titration method previously used in the literature was used to quantify the chlorine content [6 1 ] . In this study, 50 mg of the CPN films were suspended in 40 ml of 2% acetic acid solution. To this, 1 g of potassium iodide was added, and the mixture was stirred vigorously. This was then titrated against sodium thiosulphate solution until the blue - black color of the film turns colorless. Unch lorinated CH/PVA films were used as controls for this experiment. The following equation is used to calculate the chlorine content of the CPN film, (1) Where V Cl and V 0 are the volumes of Na 2 S 2 O 3 used up during the titration of CPN and unchlorinated CH/PVA films, respectively and W Cl is the weight of the film sample used for titration, which is 50 mg in this case. CPN films 0, 1, 2, 3, 4 and 5 days old after preparation were tested to detect changes in their chlorine content over time. 24 2.2.3. Testing antimicrobial activity of CPN film against bacteria The antimicrobial test against Salmonella Typhimurium and ampicillin resistant Salmonella Typhimurium strains were conducted t o compare the activity of the fabricated films in eliminating both drug susceptible and drug resistant strains of food contaminants. This test is based on modified American Association of Textile Chemists and Colorists (AATCC) Test Method 100 [6 1 ] . The bac terial strains were cultured in TSB for 24 hours at 37°C. They were cultured again from this broth for 4 hours in TSB at 37°C to attain log phase. The bacterial cells were harvested by centrifugation at 8000 rpm for 10 minutes in 4°C, washed and resuspende d in PBS. The density of the harvested culture was between 10 8 - 10 9 colony forming units per milliliter (CFU/ml). 10 µl of this was sandwiched between 1 X 1 cm 2 pieces of the CPN films for 30 minutes. This was then transferred to 5 ml of Na 2 S 2 O 3 solution and ultrasonicated for 10 minutes. The samples were then serially diluted ( 10 - 3 ) and surface - plated on TSA plates and incubated for 24 hours at 37°C. The number of colonies were counted to check the reduction in bacterial growth. Samples without treatment with the film were used as negative control and samples treated with regular plastic film were used as positive control. C H /PVA films without treatment with sodi um hypochlorite were also tested for their antimicrobial activity and compared with the activity of CPN films. CPN and CH/PVA films that were 0, 1, 2, 3, 4 and 5 days old were tested to check for any changes in their antimicrobial activity over time. The a ntimicrobial activity of CPN films against the studied bacterial strains in liquid culture were also tested based on a method previously described in the literature [7] . CPN films cut into 1 x 3 cm 2 pieces were introduced into 10 ml TSB culture tubes. Thes e tubes were immediately inoculated with S. Typhimurium and ampicillin resistant S. Typhimurium strains which were previously prepared to have a final concentration of 10 3 - 10 4 CFU/ml. These tubes were 25 incubated with shaking at 100 rpm for 0 and 24 hours. The samples were surface plated on TSA plates after serial dilutions and incubated for 24 hours at 37°C to determine the bacterial growth after treatment with the film. Bacterial culture tubes without the films were used as controls. The effect of C H/PVA film against the growth of bacteria was also tested the same way for comparison. Samples without bacterial inoculation were used to check the chlorine content of the films at 0 and 24 hours after interaction with TSB. 2.2.4. Application of CPN films on pack aging cheese Cheese slices purchased from the store were aseptically cut into four equal pieces and weighed before starting the experiment. Each cut piece of slice was transferred to a petri dish and inoculated with ampicillin resistant S. Typhimurium stra in prepared to a final concentration of 10 8 - 10 9 CFU/ml. The bacterial culture was harvested following the procedure in the antimicrobial test before being used for these experiments. The cheese slices were covered with CPN and CH/PVA films and the petri di shes were sealed with paraffin films to make sure that air is kept out. Samples without inoculation with bacteria were also maintained to quantify the presence of previously existing S. Typhimurium growth in the store - bought cheese slices. Ampicillin resis tant S. Typhimurium inoculated cheese pieces without treatment with any film were used as controls to quantify bacterial growth without the effect of films. Triplicates were maintained for this experiment at two different temperatures, 25°C and 4°C, respec tively, to study the temperature effect on the activity of films in preventing bacterial contamination. The effect of storage time was also quantified by maintaining packaged cheese slices under the same conditions for up to five days. The bacterial growt h in cheese samples was quantified by transferring the cheese to sterile stomacher bags containing 5 ml of 0.1 % peptone water and homogenized using a stomacher for 26 2 minutes. The juice from the sample was serially diluted and plated on BPLS agar and incub ated for 24 hours at 37°C. The color of the plate remains red in the presence of S. Typhimurium strains and the number of colonies counted will help quantify the bacterial growth on the cheese samples. 2.2.5. Statistical Analysis All experiments were conducted wi th samples maintained as triplicates. The CFU/ml units obtained from the plate counting were converted to log CFU/ml to get the results, which were 2.2.6. Preparation of chitosan - based N - halamine coated polycaprolactone films (CH - NX/PCL) Preparation of PCL films Homogeneous solution of PCL was prepared by dissolving 10% PCL pellets in chloroform solution and stirring for 4 - 5 hours in room temperature. This s olution was casted on glass containers of uniform size (11 x 16 cm 2 ) and heated on a hot plate at 70°C for an hour before continuing to dry the films in a fume hood for another 15 - 20 minutes. This is done to obtain smooth PCL films of uniform thickness. Th e overall film preparation process takes 5 - 6 hours. Preparation of chitosan coating solution Chitosan solution (2 %) was prepared by dissolving in 2 % acetic acid at 65°C for 60 minutes. This solution was centrifuged at 8000 rpm for 10 minutes to separate impurities and air bubbles before being poured into glass containers. 27 Coating chitosan on PCL PCL being hydrophobic in nature, has low surface adhesivity and wettability [6 3 ] to natural materials such as chitosan that is hydrophilic. It is, therefore, necesary to modify the surface of PCL to improve its wettability and adhesion to the chitosan coat by increasing its hydrophilicity. Plasma treatment has been used in the past on hydrophobic materials to improve their adhesion characteristics and give them desirable properties for various applications [6 4 , 6 5 ] . In this case, the PCL films are subjected to plasma treatment in the presence of oxygen gas for a process time of one min ute. The radio frequency level was set 50% of the total, that is 300 W power. The plasma modified PCL films were then immediately dip - coated with the previously prepared chitosan solution and left to dry at room temperature overnight. Modification of chit osan coating to give N - halamine structures The dried chitosan coated film was treated with 1 N NaOH solution to neutralize the acidic pH caused by the acetic acid used in preparing the coating. The films were washed repeatedly with deionized water, dried a nd treated with 0.65 % sodium hypochlorite solution to form N - halamine structures on the chitosan coated film surface. They were dried again after copiously washing with deionized water and stored before being used for further experiments. The treatment of chitosan in the above - mentioned steps is a modification of the procedure followed in previous literature [6 1 ] . 28 2.2.7. Characterization of CH - NX/PCL films Fourier Transform Infrared (FTIR) analysis FTIR analysis for PCL, chitosan, CH/PCL and CH - NX/PCL was carried out using FTIR spectrometer (FT/IR - 4600typeA, JASCO, Maryland, USA). The spectra were obtained at room temperature for the range of 400 to 4000 cm - 1 with a resolution of 4 cm - 1 . Thermogravimetric analysis (TGA) The thermogravimetric analysis of PCL (9.4 mg), CH/PCL (14 mg) and CH - NX/PCL (20 mg) was carried out using TGA 500 (TA instruments, DE, USA) to check the thermal degradation rate of these films at high temperatures. The materials were heated in aluminium pans from 25 to 500°C and the weight l oss of samples as a function of temperature was recorded in oxygen atmosphere. Mechanical properties The tensile strength of PCL and CH/PCL films were tested using United Testing Systems (UTS) model SFM - 20 load frame by following the ASTM D882 - 12 method [ 6 6 ] . Samples were cut into six - inch strips of one - inch width for the tests. The thickness of the samples was noted before the tests began. Triplicates for each film was maintained and the tests were conducted at room temperature. The tensile strength and y determine the mechanical properties of PCL films before and after coating with chitosan. The following equations were used for the calculations: ( 2 ) ( 3 ) 29 Where, the slope is obtained from the linear portion of the plot of force versus extension. Barrier properties Water vapor transmission rate (WVTR): The moisture barrier properties of PCL films before and after coating with chitosan was tested using MOCON PERMATRAN - W® 3/33 (Modern Controls Inc., USA). The films were sandwiched between aluminium foils wit h an uncovered area of 5 cm 2 for the mositure to pass through. The relative humidity (RH) was maintained at 100% and the temperature at 23°C. In the case CH/PCL film, the coated chitosan surface was placed facing the source of moisture in the cell. The fil m thickness was noted, and the films were conditioned for one hour before the measurements began. The system generated WVTR values for the films over hours and this was used to calculate the permeation of moisture in each film. Equation 4 was used to calcu late the water vapor permeation (WVP): ( 4 ) vapor pressure at the given temperature and RH. Oxygen transmission rate (OTR): Oxygen transmission rates through PCL films before and after chitosan coating was quantified using MOCON OX - TRAN® 2/21. Conditioning was done for one hour before the measurements began. The films were sandwiched between aluminium foils placed inside the cells with 5 cm 2 open area through which the oxygen gas (100%) was passed. The film thickness was noted before the samples were placed in the cells. The surface of chitosan coating in the CH/PCL film 30 was placed facing the source of oxygen gas. A mixtu re of nitrogen and hydrogen gases were passed through the other side. The experiments were carried out at 0% RH and 23°C. The oxygen permeability values were calculated from the OTR values obtained from the system. Equation 5 was used to do the calculation s: ( 5 ) 2.2.8. Determination of active chlorine content N - halamine structures on the CH - NX/PCL film surface can be quantified by quantifying the chlorine ions attached to the amino groups of the chitosan coating. Since the active antimicrobial agent of the film is N - halamine, determining the active chlorine con tent of the film is crucial to understanding the efficacy of the film in preventing bacterial contamination. Previously in the literature, an iodometric titration method was applied to quantify chlorine content in treated chitosan films [6 1 ] . This study follows the same procedure for the CH - NX/PCL films. Around 50 mg of the CH - NX/PCL film was suspended in 40 ml of 2% acetic acid solution. To this, 1 g of potassium iodide powder was added, and the mixture was stirred vigorously. This solution turns yellow and the film turns blue black in the presence of chlorine. It was then titrated against sodium thiosulphate solution until the film and solution turns colorless. Unchlorinated CH/PCL and chlorinated PCL films without the chitosan coating were used as controls for this experiment and equation 1 was used to calculate the chlorine content of the CH - NX/PCL film, The chlorinated PCL films were subjected to copious washing using deionized water to remove excess chlorine on its surface, the same way C H - NX/PCL films were treated. 31 2.2.9. Testing antimicrobial activity of CH - NX/PCL film against bacteria The antimicrobial activity of the fabricated CH - NX/PCL film was tested against drug susceptible and ampicillin resistant strains of Salmonella Typhimurium to com pare its efficacy in eliminating both drug susceptible and drug resistant strains of this foodborne pathogen. A modified version of the American Association of Textile Chemists and Colorists (AATCC) Test Method 100 [6 1 ] was used for the antimicrobial activ ity tests. TSB was used to culture the bacterial strains for 24 hours at 37°C. They were re - cultured from this broth for 4 hours at 37°C to attain log phase. The cells were then harvested by centrifugation at 8000 rpm for 10 minutes in 4°C, washed using PB S and resuspended in the same. This was done to achieve a culture density of 10 8 - 10 9 colony forming units per milliliter (CFU/ml). Out of this culture, 10 µl was sandwiched between 1 X 1 cm 2 pieces of the CH - NX/PCL films (sterilized before use) for a perio d of 30 minutes. The sandwich was later transferred to 5 ml of Na 2 S 2 O 3 solution and ultrasonicated for 10 minutes. Following this the samples were serially diluted using PBS and surface - plated on TSA plates. These plates were incubated for 24 hours at 37°C. Bacterial growth reduction was quantified by counting the number of colonies grown on the plates. Negative and positive controls were samples without treatment with the film and samples treated with plain PCL film. CH/PCL films without sodium hypochlorite treatment were also tested for their antimicrobial efficacy and comp ared. 2.2.10. Statistical analysis The tensile strength tests and barrier tests were done with replicates for PCL and CH/PCL films. The antimicrobial efficacy tests were done for PCL, CH/PCL and CH - NX/PCL films with triplicates. In all the cases, the calculated p roperties were compared for each of the films and 32 CHAPTER 3 TESTING ANTIBACTERIAL ACTIVITY OF CHITOSAN/PVA - BASED N - HALAMINE FILM AGAINST DRUG SUSCEPTIBLE AND AMPICILLIN RESISTA NT SALMONELLA TYPHIMURIUM AND ITS APPLICATIONS IN PACKAGING CHEDDAR CHEESE SLICES 3.1. Introduction This study focusses on the use of the natural polymer, chitosan, to prepare a biodegradable packaging film, primarily because it has the added advantage of being antimicrobial in nature [13] - - linked D - glucosamine (deacetylat ed unit) and N - acetyl - D - glucosamine (acetylated unit) [ 21 ] and the positive charges of its amine group is considered the reason for the antimicrobial activity. This positive charge reacts with the negative charge of the bacterial cell wall that leads to ce ll lysis [6 7 ] . The charge on chitosan is induced by the protonation of the amino groups in the presence of an acidic environment or through modifications introduced in their structure [6 8 ] . However, the killing efficiency of this material is highly depende nt on the pH of the system and is seen to increase with acidic conditions [11] . This may not be ideal for most food packaging conditions as they do not necessarily have to be acidic in nature. Methods to improve its antimicrobial activity in non - acidic con ditions have also been investigated. One way to do this is to exploit the presence of amine groups in chitosan, where the amine group, upon reaction with a chlorine - based compound can form N - halamine structures that enhances the antibacterial activity at m ost pH conditions [6 1 ] . 33 Fig ure 1 : Chlorination of chitosan forms N - h alamine structures that enhances the antimicrobial property of the film [6 9 ] Generally, biodegradable polymers are observed to have low mechanical strength compared to the synthetic polymers currently used in the packaging industry [ 14 ] . However, blending such polymers with another biodegradable polymer can improve their mechanical and barrier properties [ 14 ] , which are essential for any food packaging material. In this study, previously synthesized chitosan and polyvinyl alcohol (PVA) blended film, with simple generation of N - halamine structures on their surface, was tested as a po tential packaging material for cheddar cheese slices. The film was specifically tested for antimicrobial activity against the common foodborne pathogen Salmonella Typhimurium, both drug - susceptible and drug - resistant strains. Salmonella contamination of cheese products have been reported in the past, which is often accompanied with food recalls. Flat Creek Farms recalled three lots of cheese due to potential Salmonella contamination. Two of the lots involved cheddar cheese [ 70 ] . Another c ompany, contaminations by Salmonella [7 1 ] . It is, therefore, necessary to check the application of the fabricated antimicrobial film in preventing bacterial contam ination in cheddar cheese. This study specifically uses cheddar cheese slices to test the application of the film as a packaging material. Testing the antimicrobial activity of the fabricated film, particularly in packaging cheese slices 34 contaminated with drug resistant strain of Salmonella Typhimurium can give information on the possibility of using the film for commercial purposes. 3.2. Methods Chitosan (CH)/PVA - based N - halamine film was prepared by blending chitosan solution prepared in acetic acid with PVA s olution prepared in deionized water. The blended solution were casted glass plates and left to dry overnight. The dried films were treated with NaOH solution to neutralize the acidic pH from the acetic acid, was dried and chlorinated using sodium hypochlor ite solution to form N - halamine structures. The chlorine content of the film was tested using iodometric titration method and compared with the control, which was unchlorinated CH/PVA films. The antimicrobial activity of these films was tested using a modi fied version of the American Association of Textile Chemists and Colorists (AATCC) Test Method 100 against drug susceptible and ampicillin resistant strains of Salmonella Typhimurium. Their biocidal activity against these pathogens were also tested in liqu id culture. The efficiency of the fabricated film as an antimicrobial food packaging material was also tested by packing cheddar cheese slices and storing over a period of five days at 25°C and 4°C. 3.3. Results and Discussion 3.3.1. Determination of active chlorine content The chlorination of CH/PVA films involves an interaction between the amino groups of chitosan and chlorine molecules of sodium hypochlorite solution, which leads to the formation of N - halamine structures on the film surface. The hydrophili c nature of chitosan possibly caused the film to swell when introduced into the diluted sodium hypochlorite solution that lead to the 35 exposure of its amino groups to the chlorine molecules for interaction [6 1 ] . Copious washing of the film after chlorinatio that the chlorine remaining on the film are bound to the amino groups of the chitosan and quantifying them would give a measure of the N - halamine structures on the film. In this study, t he change in active chlorine content of the film during its storage over five days after preparation have been quantified. Figure 2 shows that the chlorine content decreases linearly over time as the shelf life increases. This is significant in determining how long the film can remain active against specific bacterial contaminants when used for packaging. The loss of chlorine molecules from the film could be due to the hydrophilic nature of chitosan, which causes it to interact with the moisture in the surr ounding environment that promotes dissociation of chlorine molecules [6 1 ] . The chlorine content was found using equation 1 and expressed as a percentage. The initial p reparation, the chlorine content was found to be 1.44% and the reduction from initial content was 0.37%, which may not cause significant changes in the N - halamine content on the CPN films. 36 Fig ure 2 : The change in active chlorine content of CPN films over five days 3.3.2. Antimicrobial activity of CPN films The antimicrobial activities of CPN and CH/PVA films were tested against ampicillin resistant and drug susceptible strains of S. Typhimurium. Figures 3a and 3b compares the initial bacterial concentration in broth culture to bacterial growth obtained after the antimicrobial sandwich assay. The data shows the effect of the CPN and CH/PVA films against the two strains of bacteria as their storage period inc reases, along with the control, which were not treated with any films but were subjected to similar conditions of the assay. As seen in the figures, the antimicrobial activity of CPN films is 100 % and there is no bacterial growth for both the drug resista nt and drug susceptible strains , irrespective of the age of the film ( p <0.05). Tables 3 and 5 show p values , 2.85E - 50 for S. Typhimurium and 9.13E - 65 for ampicillin resistant S. Typhimurium , which are less than 0.05, showing that there is a significant reduction in bacterial growth when treated with CPN film compared to the original bacterial concentration. This means that the loss of chlorine over a period of five days , as seen in F igure 2, has not significantly 37 affected the antimicrobial efficacy of the CPN film against both drug susceptible and ampicillin resistant strains. The original bacterial concentration used for the assay each day for both strains of bacteria was 9.5±0.2 log CFU/ml. From the figures it is evident that bacterial growth for samples treated with CH/PVA films and the control is not reduced to a greater extent when compared to those treated with CPN film . The little to no bacteria reduction in the antimicrobial assay control samples when compar ed to the original concentration gives evidence that the ultrasonication treatment with sodium thiosulphate solution in the assay do not influence the bacterial growth. However, almost one log reduction in bacterial growth w as noted for samples that were t reated with CH/PVA films compared to the original samples on almost all days ( p <0.05) , which is evidenced by the statistical analysis shown in Tables 2 and 4 , where the p values are 1.71E - 11 for S . Typhimurium and 2.3E - 09 for ampicillin resistant S. Typhimurium . antimicrobial activity [13] . The initial concentration of bacteria used in the test was between 10 8 - 10 9 and this might be too high for the given amount of nutrients in TSB. This could mean that the bacterial cells could have already reached steady state and not growing enough due to lack of nutrients by this point. Therefore, the effect of the CPN film coul d have been enhanced leading to a 100% bacterial reduction in this condition. 38 Fig ure 3 : Comparison of bacterial growth when treated with N - halamine CH/PVA (CPN) and CH/PVA films (contact time 30 minutes ; detection limit serial dilution of 10 - 3 ) ; (a) effect on drug susceptible Salmonella Typhimurium; (b) effect on ampicillin resistant Salmonella Typhimurium Table 2 : ANOVA for S almonella Typhimurium treated with CH/PVA film compared with control Source of Variation Sum of squares ( SS ) Degrees of freedom ( df ) (Mean of squares) MS F P - value F crit ical Sample (between treatment s ) 7.970476 1 7.970476 139.6642 1.71E - 11 4.259677 Columns (between film age ) 0.349923 5 0.069985 1.22632 0.327448 2.620654 Interaction (between treatment s and film age ) 0.850945 5 0.170189 2.98217 0.03119 2.620654 Within 1.369652 24 0.057069 Total 10.541 35 39 Table 3 : ANOVA for S almonella Typhimurium treated with CPN film compared with control Source of Variation SS df MS F P - value F crit ical Sample (between treatment s ) 820.8478 1 820.8478 277272.8 2.85E - 50 4.259677 Columns (between film age) 0.066821 5 0.013364 4.514298 0.004839 2.620654 Interaction (between treatment s and film age) 0.066821 5 0.013364 4.514298 0.004839 2.620654 Within 0.07105 24 0.00296 Total 821.0525 35 Table 4 : ANOVA for A mpicillin resistant S almonella Typhimurium treated with CH/PVA film compared with control Source of Variation SS df MS F P - value F crit ical Sample (between treatment s ) 4.915809 1 4.915809 85.18153 2.3E - 09 4.259677 Columns (between film age) 1.492281 5 0.298456 5.171674 0.002328 2.620654 Interaction (between treatment s and film age) 0.864321 5 0.172864 2.995404 0.030663 2.620654 Within 1.385035 24 0.05771 Total 8.657446 35 40 Table 5 : ANOVA for A mpicillin resistant S almonella Typhimurium treated with CPN film compared with control Source of Variation SS df MS F P - value F crit ical Sample (between treatment s ) 803.5983 1 803.5983 4474809 9.13E - 65 4.259677 Columns (between film age) 0.091207 5 0.018241 101.5762 2.42E - 15 2.620654 Interaction (between treatment s and film age) 0.091207 5 0.018241 101.5762 2.42E - 15 2.620654 Within 0.00431 24 0.00018 Total 803.785 35 To ensure that the reduction in bacterial growth on the agar plates during the assay is not due to its inaccessibility while being sandwiched between the films, the assay was conducted using a regular plastic film without any antimicrobial activity of its own. The results were compared with the original growth, control and those treated with CPN and CH/PVA films. Figures 4a and 4b indicate that the treatment with regular plastic film has reduced the growth of bacteria only by 0.5±0.1 logs, which means that sandwiching the bacteria between the films did not significantly influence the bacterial concentration. 41 Fig ure 4 : Comparison of bacterial growth under different conditions of the antimicrobial sandwich assay to the original bacterial concentration in the initial culture (contact time 30 minutes; detection limit serial dilution of 10 - 3 ) ; (a) drug susceptible Salmo nella Typhimurium; (b) ampicillin resistant Salmonella Typhimurium N - halamines are a combination of halogens with nitrogen based functional groups that have potential antimicrobial activities. In this study, the CPN films contain N - halamines on its surfac e through the interaction of the amino group of chitosan with the chorine molecules from the sodium hypochlorite treatment. The advantages of N - halamine - based antimicrobial agents is that they have oxidative halogens in their structures that inherently hav e strong antimicrobial potential in them. Since the halogens present in these structures are bound to the amino groups, they are highly stable unlike the free halogens and are comparatively safer to use. Once the oxidative chlorine group is consumed during its activity against a microorganism, they can be recharged again by exposing the amino groups on the surface of CPN films to sodium hypochlorite solution [7 2 ] . It is noted that the antimicrobial mechanism of N - halamines depend on the transfer of its oxid ative halogen group to the bacterial cell wall. The interactions between this halogen ion and the bacterial cell wall is seen to cause destruction of the cells irrespective of whether the pathogen has drug resistant genes. Therefore, using N - halamine based antimicrobial agents have an advantage over using antibiotics in combating drug resistant strains of bacteria as well [7 3 ] . In this study, the results obtained for the sandwich assay evidently suggests that the 42 growth of both the non - drug resistant and ampicillin resistant S. Typhimurium strains have been significantly reduced when treated with CPN films containing N - halamine structures on their surface. It has also been noted that there is no significant variation in the effect of CPN films over the two bacterial strains. The antimicrobial mechanism of N - halamine has shown to require direct contact with bacterial cells for t heir effective elimination. However, it is hypothesized that the antimicrobial action of N - halamines can also be initiated with the release of their halogen molecules into an aqueous environment that can be exposed to the pathogens [7 2 ] . The sandwich assay conducted in this study gives evidence of the biocidal activity of N - halamine upon direct contact with the bacterial cells. The oxidative chlorine ions (Cl + ) present in the N - halamine structure interacts with the charges on the bacterial cell wall and pen etrates the cell through its transport system. This interaction is followed by the generation of reactive oxidation species that can interrupt regular cellular functions, thereby, causing cell necrosis [6 9 , 7 2 , 7 4 ] . From Figure 3, it can be inferred that t here is no significant difference in the activity of the CPN eliminate 100 % of bacterial cells when treated on both the non - drug resistant and ampicillin resis tant S. Typhimurium strains. From the chlorine titration experiments conducted previously, it can be said that the decrease in the chlorine content of CPN films each day has not significantly affected its biocidal activity. As noted in the previous section , the reduction in the - halamine content. This means that there could potentially be more N - not yet saturated by interactions with bact eria and can still potentially be used after longer storage periods. The effect of CH/PVA films on bacterial growth also have not shown significant 43 difference with respect to its storage period and remains similar to the results obtained on the first day o f test. As mentioned previously, N - halamine could potentially fight bacteria by first releasing their halogens into an aqueous environment. To check whether this would be applicable in the case of the CPN film, its antimicrobial activity was also tested against ba cterial broth cultures of both non - drug resistant and ampicillin resistant S. Typhimurium. The tests were conducted for two days since the preparation of the CH/PVA and the CPN films and the results are shown in Figure 5. Bacterial growth in broth samples treated with CH/PVA and CPN films were compared with the control, which was not exposed to any film. From the results it can be noted that there is no apparent significant antimicrobial effect of CH/PVA or CPN films on the bacterial growth of either of the strains used in this study. The initial bacterial growth was found to be around 9 logs for both strains, which was the same for the cultures treated with the films as well. After 24 hours, the growth was higher, reaching around 12 logs in all the cases in dicating that there is no reduction of growth over time. Fig ure 5 : Comparison of bacterial growth in broth culture treated with CH/PVA and CPN films over time; (a) drug susceptible Salmonella Typhimurium; (b) ampicillin resistant Salmonella Typhimurium 44 Table 6 : Comparison of growth on d ay 0 between both strains when tre ated with CPN film along with control Source of Variation SS df MS F P - value F critical Sample ( between treatment condition) 0.006537 1 0.006537 1.318368 0.284058 5.317655 Columns (between the two strains) 0.947679 1 0.947679 191.1332 7.24E - 07 5.317655 Interaction (between treatment condition and strains) 0.000985 1 0.000985 0.198744 0.667555 5.317655 Within 0.039666 8 0.004958 Total 0.994867 11 There is a significant difference between the growth of both the strains at 3 hours ( p value = 7.24E - 07 ) , where the drug resistant strain shows almost one log higher growth than the drug susceptible strain. This indicates that both strains show differences in growth and tolerance to the antimicrobial agent, which is the N - halamine structures on the CPN film . The p value for sample and intera ction exceeds 0.05, suggesting that there is no significant difference between the bacterial growth observed for control samples and those treated with CPN film . The two strains seem to behave similarly when under the two different treatment conditions, su ggesting that the CPN film has little effect in reducing bacterial growth in the broth maintained in this experiment irrespective of the type of strain . A fter 24 hours, t he growth of both strains has increased considerably and have possibly reached steady state condition due to which a significant difference between bacterial growth is not evident in both the strains . Table 7 shows p value s exceeding 0.05, which means that there is no significant difference between the bacterial growth in both strain s when treated with the CPN film in broth and compared to the control. 45 Table 7 : Comparison of growth on d ay 1 between both strains when treated with CPN film along with control Source of Variation SS df MS F P - value F crit ical Sample ( between treatment condition) 0.001393 1 0.001393 0.019253 0.893074 5.317655 Columns (between the two strains) 0.083855 1 0.083855 1.159018 0.31306 5.317655 Interaction (between treatment condition and strains) 0.002587 1 0.002587 0.035755 0.854731 5.317655 Within 0.578804 8 0.07235 Total 0.666639 11 The antimicrobial efficacy of CPN films could have been insignificant in this case due to the saturation of chlorine ions present on the 1 x 3 cm 2 strips of films by the bacterial cells that encounter the film. Even if the chorine ions were released into the broth, they may not have been enough to attack the bulk of the bacterial cells that remained and continued to replicate in the broth over time. The broth was also shaken at 100 rpm, causing an aerobic environment which was ideal to improve the growth rate of the bacteria. Tests could be done without shaking to test the effect of the film in eliminating the pathogen. Another reason could be its ex posure to TSB, which largely contains water molecules that can film with the surrounding aqueous environment could cause a reverse reaction where the chlorin e gets released into the broth. As mentioned previously, this could happen due to the hydrophilic nature of chitosan that interacts with the surrounding moisture [6 1 ] . The reduction in 46 the chlorine percentage on CPN films means that the number of N - halamin e groups on its surface has reduced as well. This in turn leads to its decreased antimicrobial activity when interacting with bacteria in broth culture. Figure 6 shows the residual chlorine content on CPN films of size 1 x 3 cm 2 when placed in tubes contai ning TSB for 3 and 24 hours. The results initial chlorine content of the film, as found previously, was 1.81%, and about 78% of this was lost from the film withi n the first three hours. At the end of the 24 hours, the final chlorine content on the film treated with TSB was around 0.16%, which is close to 91% reduction from the initial chlorine content. This indicates that chlorine ions have been released into the broth. Fig ure 6 : Remaining chlorine content (%) in CPN films after incubation in TSB against time (almost 60% reduction after 24 hours) 47 3.3.3. Application of CPN films on packaging cheese Fig ure 7 : Comparison of bacterial growth (log CFU/ml of peptone water used) in cheese slices packaged with CH/PVA and CPN films over storage time (a) at 25°C; (b) at 4°C The effect of temperature on the antimicrobial activity of the CPN films as packages for cheddar cheese slices was tested at 25°C and 4°C to compare the quality of the packaged food in room temperature and when stored in refrigerators. The samples were pla ted on BPLS agar, which is a selective medium for Salmonella species. The colonies grow in red because they do not ferment the lactose or sucrose present in the agar. Escherichia coli also grows on this agar. However, it turns the medium yellow due to the fermentation of the sugars present in the agar. This medium was used in these experiments to ensure that only the inoculated ampicillin resistant S. Typhimurium strain is quantified. A negative control was used in these experiments where, cheese samples were treated the same way but without bacterial inoculation. This will be useful to quantify any existing Salmonella contamination in the cheese. From Figure 7, it can be observed that the bacterial growth on days 0 and 1 after packaging are lower than that of days 2, 3, 4 and 5. This is more prominent in cheese samples maintained at 25°C than at 4°C . The statistical analys e s shown in Table s 8 and 10 give p value s equal to 3.22E - 23 and 1 .16E - 06 , respectively, which are less than 0.0 5. This proves that there is a significant 48 difference in bacterial growth over the days when stored at these two different temperatures . It is also evident that cheese slices packaged with CH/PVA has not shown significant reduction in bacterial growth from day 2 onwards when compared to the controls, which were samples not packaged with either films when stored at 4 ° C ( p value = 0.83 ) . However, the statistical analysis in Table 8 shows significant difference ( p value = 3.14E - 05 ) between the control and the samples stored using CH/PVA film at 25 ° C, though it is not as effective as CPN film in eliminating the pathogen based on the log re ductions seen in F igure 7. This means that CH/PVA films had little effect on reducing bacterial contamination on cheese. On the other hand, CPN films showed around 3 - 6 log reductions in bacterial growth compared to the controls ( p <0.05) irrespective of the temperatures at which the samples were stored as evidenced by the p values for interaction in Tables 9 and 11 . At 4 ° C, the samples stored in CPN film showed similar growth pattern as the control over the days ( p value = 0.01 08 ) , though there is a significant bacterial reduction in CPN treated samples compared to the control ( p value = 7.91E - 20 ) , which is evident from the p value of interaction in T able 11 being greater than 0.05, which is 0.397 . At both temperatures, the bacterial log reduction on days 0 and 1 is lower than that observed for the remaining storage period. At 25°C, around 5 logs and at 4°C, around 3 logs of bacterial growth reduction were observed, while the remaining period shows a round 6 and 4 log reductions at 25°C and 4°C, respectively. Every cheese sample was inoculated with the same concentration of ampicillin resistant S. Typhimurium and its growth is seen to increase until day 2 in samples stored at 25°C followed by a stable bacterial concentration until day 5. However, this trend is not prominent at 4°C, possibly due to the inhibition of S. Typhimurium strains at lower temperatures. At refrigerated temperatures Salmonella strains slows growth and continues to stay dormant. Th e results obtained in this case are consistent with previous studies on Salmonella growth in food 49 samples [7 5 , 7 6 ] . Studies conducted on cooked ham resulted in Salmonella population remaining the same when stored at lower temperatures such as 5°C, while th ere was an increase in growth at higher temperatures such as 25°C [7 5 ] . S. Typhimurium growth in inoculated buffalo mozzarella cheese was shown to reduce at 4°C and increased at 20°C when tested over a period of 12 days [7 6 ] . From the results above, it is evident that the CPN films can reduce the growth of ampicillin resistant S. Typhimurium in cheese at temperatures 25°C and 4°C. However, a complete elimination of contamination could not be achieved, as shown by the 2 - 3 log bacterial growth during the ent ire storage period, irrespective of the temperature. This could be due to the inaccessibility of the N - halamine structures on the CPN films to the bacterial cells that could have diffused into the food matrix. While inactivation of bacterial cells in conta ct with the film surface is happening , the remaining bacteria l cells in the food matrix may not be affected and can continue to grow. The cheese matrix provides for a nutrient rich substrate for bacteria to grow. However, the growth of ampicillin resistant Salmonella Typhimurium has not increased from day 3 to day 5 and this could possibly be due to the inhibition of bacterial growth accompanied with the inactivation effect of the chitosan - based packaging film , causing the surviving bacteria to grow at a slower pace. Also, t he concentration of bacteria used to inoculate the cheese samples i s around 10 8 , which co uld be too high compared to actual bacterial concentration contaminating food products. This could possibly b e the reason for bacteria remaining in the food matrix and further studies at lower concentrations can give insight into the efficacy of the proposed antimicrobial film in eliminating bacteria in practical situation. Different food samples have varying texture and physical properties that make them more susceptible to contamination than others. In this study, the food sample selected for antimicrobial 50 efficacy test, was porous in nature and could have allowed the diffu sion of bacterial culture into the cheese matrix more easily. For less porous food materials, this process may have happened slower , in which case the CPN film could have closely interacted with more bacterial cells on the surface and eliminated those as w ell. Therefore, the texture of the food sample tested may also play a role in affecting the capability of the proposed packaging film in reducing contamination. As discussed in the previous section, direct contact of films is required for effective elim ination of all bacterial cells. Therefore, the film was able to eliminate only those bacterial cells that remained on the cheese surface, while those that diffused into the cheese matrix survived. Table 8 : ANOVA C heese packed with CH/PVA film at 25 ° C compared with control Source of Variation SS df MS F P - value F crit ical Sample (between treatment s ) 0.656662 1 0.656662 26.11706 3.14E - 05 4.259677 Columns (between days) 60.27707 5 12.05541 479.4734 3.22E - 23 2.620654 Interaction (between treatment s and days) 0.906137 5 0.181227 7.207861 0.000303 2.620654 Within 0.603433 24 0.025143 Total 62.4433 35 51 Table 9 : ANOVA C heese packed with CPN film at 25 ° C compared with control Source of Variation SS df MS F P - value F crit ical Sample (between treatment s ) 274.4269 1 274.4269 9503.589 1.05E - 32 4.259677 Columns (between days) 26.27412 5 5.254824 181.9781 2.9E - 18 2.620654 Interaction (between treatment s and days) 4.008775 5 0.801755 27.76532 3.06E - 09 2.620654 Within 0.693027 24 0.028876 Total 305.4028 35 Table 10 : ANOVA Cheese packed with CH/PVA film at 4 ° C compared with control Source of Variation SS df MS F P - value F crit ical Sample (between treatment s ) 0.004158 1 0.004158 0.0461 0.831809 4.259677 Columns (between days) 6.665219 5 1.333044 14.77923 1.16E - 06 2.620654 Interaction (between treatment s and days) 0.797638 5 0.159528 1.768656 0.157556 2.620654 Within 2.16473 24 0.090197 Total 9.631745 35 52 Table 11 : ANOVA Cheese packed with CPN film at 4 ° C compared with control Source of Variation SS df MS F P - value F crit ical Sample (between treatment s ) 129.0651 1 129.0651 782.2672 7.91E - 20 4.259677 Columns (between days) 3.158917 5 0.631783 3.829257 0.010825 2.620654 Interaction (between treatment s and days) 0.88893 5 0.177786 1.077565 0.397571 2.620654 Within 3.959724 24 0.164989 Total 137.0727 35 53 CHAPTER 4 SYNTHESIS OF CHITOSAN - N - HALAMINE - COATED POLYCAPROLACTONE FILM AGAINST DRUG SUSCEPTIBLE AND AMPICILLIN RESISTANT S ALMONELLA TYPHIMURIUM FOR FOOD PACKAGING APPLICATIONS 4.1. Introduction Chitosan is a natural polymer, a derivative of the compound chitin, that is commonly foun d on the shells of crustaceans. It is formed from the alkaline treatment of chitin and is reported to have antimicrobial properties [7 7 ] , apart from being biodegradable. However, chitosan derivatives have been proven to show much higher efficacy against pa thogens than chitosan itself. This is especially proven for the N - halamine derivative of this natural polymer [6 1 ] . Chitosan is - - linked D - glucosamine (deacetylated unit) and N - acetyl - D - glucosamine (acetylated unit) [ 21 ] . The amine group of this polymer can interact with halogens to form N - halamine structures. The halogen ions of this derivative, then, upon interaction with any pathogen, gets transferred into the cells and disrupt their metabolic activity [ 6 9 ] . The use o f chitosan as a biodegradable food packaging material has been investigated in the past. However, due to its poor physical properties, that often do not meet the requirements of an ideal food packaging system, it is combined with other physically stronger polymers [7 8 ] . This chapter discusses the fabrication of plasma treated PCL films coated with chitosan and chlorinated to form N - halamine structures. The physical properties of the film were tested and its antimicrobial efficacy against drug susceptible an d ampicillin resistant Salmonella Typhimurium strains were quantified. 54 4.2. Methods Chitosan - N - halamine - coated PCL films were fabricated beginning with the preparation of smooth PCL films in chloroform. The dried films are plasma treated in oxygen environment and immediately coated with chitosan solution prepared in acetic acid. The coated PCL film was left to dry overnight before being treated with NaOH solution to neutralize the acidic pH of the acetic acid. This was dried and treated with sodium hypochlorite solution to form N - halamine structures on the chitosan coated surface of PCL films. The chlorine content of the fabricated antimicrobial film was quantified using iodometric titration with chlorinated PCL and unchlorinated chitosan - coated PCL films as controls. Characterization of the films were done using FTIR and TGA. The mechanical properties such as - coated PCL films. The barrier properties of the film against mo isture and oxygen were also quantified. The antimicrobial efficacy of the fabricated films against drug susceptible and ampicillin resistant Salmonella Typhimurium was tested using the modified version of American Association of Textile Chemists and Colori sts (AATCC) Test Method 100. 4.3. Results and Discussion 4.3.1. Characterization of CH - NX/PCL films Fourier Transform Infrared (FTIR) analysis Figure 8 shows the FTIR spectra for pure PCL, CH/PCL and pure chitosan films. As can be noted from the figure, the spectra for CH/PCL films include characteristic peaks of both PCL and chitosan. The characteristic peaks of PCL are visible in the spectra, which inc lude peaks at 1720.19 cm - 1 attributing to the carbonyl group ( - C=O), ~2941 cm - 1 and ~2863 cm - 1 attributing to 55 the asymmetric and symmetric CH 2 stretching, respectively [7 9 ] . Similar peaks can be noted in the spectra for CH/PCL at 2932.23, 2862.81 and 1719. 23 cm - 1 showing evidence that the modified film retains the chemical structure of PCL. The spectra for chitosan show its characteristic peaks at ~1571 cm - 1 associated with the N - H band of the primary amines or amide II [ 80 ] , 3354 cm - 1 for the O - H stretch t hat overlap with the N - H stretch, 2914.88 cm - 1 and 2869.55 cm - 1 for the C - H stretch, 1641.12 cm - 1 C - O stretch of the acetyl group and the amide II band, ~1571 cm - 1 for N - H stretch, 1374.99 cm - 1 for the asymmetric C - H stretch bending of CH 2 group and 1063.5 4 cm - 1 for the skeletal vibration with the bridge C - O stretch of the glucosamine residue [8 1 ] . The CH/PCL films also show same peaks at 3354 cm - 1 and 1374.99 cm - 1 and similar peaks for the other functional groups at ~1654, ~1560, 1169.69 and 1064.51 cm - 1 . This is evidence of CH/PCL film retaining the chemical structure of chitosan, when it is coated on the plasma treated PCL films. Considering there are no overlap other than those observed for pure PCL and pure chitosan, it can be concluded that there are no covalent interactions or chemical reactions between the PCL film and the chitosan coating [8 2 ] . This m eans that the plasma treatment assisted in adhesion of the chitosan coating on PCL film, but did not significantly alter its chemical structure. FTIR spectrum for CH - NX/PCL film was found to be similar to the spectrum for CH/PCL film with no significant ch anges. 56 Fig ure 8 : Comparison of the FTIR spectra of pure PCL, chitosan coated PCL (CH/PCL) and chitosan Thermogravimetric analysis (TGA) The TGA curves for PCL, CH/PCL and CH - NX/PCL films were obtained in oxygen atmosphere to account for their degradation properties in the presence of air at high temperatures. The TGA curve in F igure 9 (A) for PCL shows degradation beginning at 283.68°C. Th e thermal degradation of this polymer was accelerated during its reaction with the oxygen gas present in the system, which is the reason for the lower degradation temperature compared to the usual ~400°C as seen in nitrogen environment. The decrease in wei ght happens in three steps (283.68°C, 361.18°C and 392.13°C) which is in accordance with past literature [8 3 ] . The experiment was carried out until 500°C and at 474.29°C, only 6.73% of the original weight of PCL remains. 57 In the case of CH/PCL, the degrada tion begins slowly beyond 100°C and rapidly occurs around 275°C. The curve is not as sharp as was observed in the case of PCL. However, this is still in accordance with the degradation that would be observed for PCL and chitosan. Chitosan begins degradatio n around 300°C in oxygen atmosphere just as in the case of PCL [8 4 ] . This could be the reason why there is little difference in the initial degradation temperatures of both PCL and CH / PCL films. This also means that the plasma treatment and chitosan coatin g of the PCL film did not significantly affect its thermal properties. The TGA curve for CH - NX/PCL indicates that degradation began at 302.36°C and continues to decrease beyond 500°C. The weight loss occurs in steps just as in the case of PCL. The CH - [8 4 ] , due to the presence of the chitosan coating. The degradation around 300°C in the cases of CH/PCL and CH - NX/PCL goes well with the degradation of PCL and the potential polysaccharide degradation of the chitosan coating [8 3 ] . The TGA curves for CH/PCL and CH - NX/PCL films vary to some extent, and this could be the effect of the chlorination of the latter. Fig ure 9 : TGA curves for (A) PCL; (B) CH/PCL; (C) CH - NX/PCL 58 Mechanical properties force and extension data obtained and is displayed in F igure 10 . Fig ure 10 : Mechanical properties of PCL and CH/PCL films (with statistically significant difference s between each film type ( p (GPa) The tensile strength of a material is a measure of the maximum strain that it can withstand before breaking. The tensile strength of CH/PCL was found to be around 8.907 MPa, while that of PCL film was found to be 11.302 Mpa, showing that the PCL film can withstand higher stress compared to its modified counterpart ( p <0.05). This could be due to the changes induced in the physical properties of the PCL film wh ile it was subjected to plasma treatment and the subsequent coating with chitosan, known to have undesirable mechanical properties [7 8 ] . The value is higher for materials that are stiff and less flexible. They do not show any difference in (~0.37 GPa) compared to PCL (~0.13 GPa), indicating that former is stiffer compar ed to the 59 latter ( p <0.05). This was also visible in the way the two films broke, which is shown in F igure 11 . Fig ure 11 : Strips of films after mechanical testing (A) PCL; (B) CH/PCL Barrier properties Water vapor transmission rate (WVTR): The water vapor transmission rates of pure PCL and CH/PCL films were quantified and used to calculate the water vapor permeability. The thicknesses of the films play an important role in the barrier properties of the film and was used for these calculation s. The vapor pressure at 23°C and 100% RH is 2810 Pa. From T able 1 2 , it can be noted that water vapor permeability of PCL film is higher than that of CH/PCL ( p <0.05). The thicknesses of both the films are similar (~0.2 mm) , indicating that the variations i n the permeability values for both films depend on the modifications made on the PCL film. The lower permeability of water for CH/PCL film indicates that the plasma treated PCL film coated with a layer of chitosan has increased its barrier against moisture . This may be desirable in food packaging applications that require the packaging 60 material to protect the food from moisture and subsequent contaminations from microbial sources. Oxygen transmission rate (OTR): The oxygen transmission rates were obtained for PCL and CH/PCL films at 23°C, 0% RH and 1 atm. Based on the noted thicknesses for these films (~0.2 mm) , the oxygen permeability (OP) values were calculated. Table 1 2 shows the values OP values and the thicknesses of both the films. It is observed that the OP value for CH/PCL film is lower than that of PCL ( p <0.05). This is possibly due to the chitosan coating which has high oxygen barrier properties [8 5 ] . Generally, s ynthetic polymers like PCL have high OTR values. This is undesirable for food packaging since oxygen can easily pass through such films and oxidize the food [8 6 ] . The presence of oxygen can also promote the growth of biological contaminants and reduce the shelf - life of the food. The presence of chitosan has increased the oxygen barrier properties of the PCL film, making it desirable for food packaging applications. Here the thicknesses of both the films are similar and so, any variation the barrier properti es depend on the modification of PCL. Table 12 : Water vapor and oxygen permeability of pure and chitosan coated PCL WVP (g m/m 2 s Pa) OP (cc cm/cm 2 s Pa) PCL 12.82E - 10 9.16E - 14 CH/PCL 0.63E - 10 3.54E - 14 4.3.2. Determination of active chlorine content To understand the antimicrobial efficacy of the CH - NX/PCL films against the selected strains of the pathogen, the active chlorine content of the film was quantified using an iodometric titration method. The amount of chlorine attached to the surface of the film was found to be 1.5%, when 61 tested on the same day it was synthesized. Chlorinated PCL film without chitosan coating and unchlorinated CH/PCL film were used as controls and it was found that neither of the films had chlorine on them. The absence of ch lorine on the chlorinated PCL film is because of the absence of chitosan coating on the film. There were no amino groups present for the chlorine ions to attach to and so, N - halamine structures could not be formed. This also indicates that the presence of chlorine on the CH - NX/PCL films can directly be correlated to the presence of the antimicrobial N - halamine structures on the film. The absence of chlorine ions on the CH/PCL films indicates that there are no chlorine ions previously associated with the chi tosan coating and all the chlorine ions come from the sodium hypochlorite treatment of the coated PCL films. Figure 12 s hows the titration solutions for chlorinated and uncoated PCL, unchlorinated CH/PCL and CH - NX/PCL films. It can be observed that only th e CH - NX/PCL turns blue black in the presence of potassium iodide in the solution, indicating the presence of chlorine ions on its surface. Fig ure 12 : Iodometric titration solutions with (A) chlorinated and uncoated PCL film; (B) CH/PCL film; (C) CH - NX/PCL film 62 4.3.3. Testing antimicrobial activity of CH - NX/PCL film against bacteria The antimicrobial activity of CH - NX/PCL film was tested against the drug susceptible and ampicillin resistant strains of Salmonella Typhimurium to check its efficacy against the pathogens irrespective of whether they are resistant to antibiotics. The effic acy of CH - PCL in eliminating these pathogens was also studied and compared with that of CH - NX/PCL film. Figure 13 shows the bacterial growth in log CFU/ml for both the strains when treated with these films. From the graph, it is clearly observed that 100% of each strain was eliminated in the antimicrobial assay, when treated with the CH - NX/PCL film ( p <0.05). Table 1 4 shows p value to be 2.29E - 11 for ampicillin resistant S. Typhimurium strain, while the p value could not be determined in Table 16 for S. Typ h imurium since there was absolutely no difference in growth for within the control group and treatment group . Though not significant enough for eliminating contamination, the CH/PCL films also show bacterial growth reduction of around one log for ampicillin resistant Salmonella Typhimurium, which is 8.5 logs, and around two logs for drug susceptible Salmonella Typhimuri um, which is 8 logs, as opposed to the original culture growth of 9.5 and 10 logs obtained without any treatment for each strain, respectively ( p <0.05). Tables 13 and 1 5 show p values to be 0.004 7 and 1.3E - 04 for the drug resistant and drug susceptible str ains, respectively. This is possibly due to the inherent antimicrobial activity of the chitosan eliminate contamination completely. While drug susceptible Salmo nella Typhimurium was reduced by two logs, ampicillin resistant Salmonella Typhimurium was reduced by one log. The difference in the activity of CH/PCL on both the strains could be due to the difficulty of chitosan molecules to interact with the drug resis tant strain causing lower bacterial reduction in this case. 63 PCL film did not show significant growth reduction of either strains when compared to the original bacterial concentration for ampicillin resistant and drug susceptible Salmonella Typhimurium, respectively. This indicates the absence of any antimicrobial a ctivity associated with the film. Samples without treatment with films also did not show significant growth reduction when compared to the original. This indicates that the treatment steps followed in the assay do not significantly play a role in reducing bacterial growth on their own and any bacterial elimination occurs due to the antimicrobial film that is being tested. The absence of bacterial growth for samples treated with CH - NX/PCL film indicate that the N - halamine structures ace play a crucial role in eliminating the pathogens. The results from the iodometric titration also confirms the presence of chlorine ions on this film, that interacts with the amino groups of the chitosan coating to form the antimicrobial N - halamine stru ctures. Fig ure 13 : Bacterial growth reduction for (A) ampicillin resistant Salmonella Typhimurium; (B) drug susceptible Salmonella Typhimurium (contact time 30 minutes; detection limit serial dilution of 10 - 3 ) 64 Table 1 3 : ANOVA for A mpicillin resistant S almonella Typhimurium treated with CH/PCL film and control group Source of Variation SS df MS F P - value F crit ical Between Groups 1.276671 1 1.276671 32.38964 0.004708 7.708647 Within Groups 0.157664 4 0.039416 Total 1.434335 5 Table 1 4 : ANOVA for A mpicillin resistant S almonella Typhimurium treated with CH - NX/PCL film and control group Source of Variation SS df MS F P - value F crit ical Between Groups 134.0797 1 134.0797 512186.9 2.29E - 11 7.708647 Within Groups 0.001047 4 0.000262 Total 134.0808 5 Table 1 5 : ANOVA for S almonella Typhimurium treated with CH/PCL film and control group Source of Variation SS df MS F P - value F crit ical Between Groups 5.450014 1 5.450014 212.5153 0.000129 7.708647 Within Groups 0.102581 4 0.025645 Total 5.552595 5 Table 1 6 : ANOVA for S almonella Typhimurium treated with CH - NX/PCL film and control group SUMMARY Groups Count Sum Average Variance Control 3 30 10 0 CH - NX/PCL 3 0 0 0 ANOVA Source of Variation SS df MS F P - value F crit ical Between Groups 150 1 150 65535 _ 7.708647 Within Groups 0 4 0 Total 150 5 65 The chlorine ions on CH - NX/PCL are stable due to their attachment to the amino groups of the chitosan coating. This makes them much safer compared to free chlorine ions, which is essential when considering food - based applications [ 7 2 ] . The chlorine ions in the N - halamine structure are oxidative in nature and upon contact with bacterial cells, get transferred to the cell wall. This interaction causes cell destruction due to the interference of the ions in the metabolic functions of the cell. Since this proce ss happens irrespective of whether the bacterial strain has drug resistant strains, the application of N - halamine for eliminating drug resistant pathogen contaminations is promising [ 7 3 ] . The results of the sandwich assay confirm this activity, wherein, th e CH - NX/PCL film was efficient in eliminating both the drug susceptible and ampicillin resistant Salmonella Typhimurium. The chlorine ions consumed in the elimination of such pathogens can be replenished by sodium hypochlorite treatment of the used film an d reapplied for food packaging purposes [ 7 2 ] . 66 CONCLUSION The antimicrobial activity of CPN films against both drug susceptible and ampicillin resistant S. Typhimurium strains was evident in the sandwich assay and in ampicillin resistant bacteria inoculated cheese samples. The correlation of chlorine percentage of the N - halamine structures on the CPN films with its antibacterial activity was specifically evi dent from the fact that the CH/PVA films without these ions did not show significant bacterial inactivation . However, all tests were conducted for five days. Further testing beyond those five days could possibly give an insight into how long the CPN films could be used effectively to eliminate pathogens. This can also help in identifying the critical chlorine content at which its biocidal efficacy is intact. This will help in designing films with lower chlorine content that meets the required standards and still be able to retain its biocidal activity. Although total bacterial elimination was not possible in cheese samples using the CPN films, there was a significant bacterial reduction when compared to the samples that were not treated with these films, sho wing that CPN films are effective in eliminating ampicillin resistant S. Typhimurium contamination in food samples upon direct contact. Further tests would need to be conducted to create a more effective film design that targets the bacteria that are not i n contact with the film. T he fabrication of N - halamine based antimicrobial film using plasma treated and chitosan coated polycaprolactone polymer was done to obtain an ideal packaging material with strong physical properties. This film along with pure PCL and chitosan coated PCL were characterized using FTIR and TGA and the fabricated films showed properties similar to PCL and chitosan. Their physi cal properties in terms of mechanical strength, water vapor and oxygen barrier were also analyzed, and it was found that the modified PCL films showed promise as food packaging materials with higher strength and barrier compared to pure PCL film. The chlor ine content of 67 the CH - NX/PCL film was found to be 1.5% and correlates to its antimicrobial activity. The sandwich assay tests showed that the fabricated CH - NX/PCL film can eliminate both drug susceptible and ampicillin resistant Salmonella Typhimurium stra ins with 100% efficacy, unlike the one to two log reductions obtained by treatment with CH/PCL film. The physical properties of the film, together with its biodegradability and antimicrobial activity makes the CH - NX/PCL film a promising food packaging mate rial. Though the antimicrobial efficacy of CPN and CH - NX/PCL films depended on the chitosan - based N - halamine structures, their mechanical properties depended on the polymers used for fabrication. Both films showed excellent antimicrobial property against the two strains of bacteria. However, PCL based films were prepared keeping in mind the necessity to apply the films in practical packaging systems. The PVA based films, though improved the mechanical properties of chitosan, did not perform well under mois t environment due to absorption of water, making them unfit for practical applications. The PCL based films on the other hand were able to overcome this problem due to their high barrier to water and resemblance to synthetic polymers currently used in food packaging. Future studies: The fabrication of chitosan - based N - halamine films proposed in this study requires the use of diluted sodium hypochlorite for the chlorination process. Though the preparation process uses diluted quantities of this chemical , t here is a set standard for allowable chlorine that can come in contact with food products consumed by humans , according to FDA regulations. For this reason, further studies must be done to determine the minimum chlorine percentage that remain within FDA limit and can form N - halamine structures to effective ly eliminat e pathogens. Toxicity tests 68 on human cells may also be required to ensure safety in using these films for food - base d applications. This study specifically used drug susceptible and ampicillin resistant strains of S. Typhimurium. However, there has been outbreaks due to multidrug resistant strains of this bacteria associated with meat products [1] . Further studies will be required to study the antimicrobial efficacy of CPN and CH - NX/PCL film s against other strains of Salmonella . There are other foodborne pathogens that top the list in causing outbreaks as mentioned previously and studies to test the efficacy of these films against Gram positive, other Gram negative and fungal contaminants must be done to check the applicability of these films in preventing contamination in food matrix in a broad sense. Cheddar cheese slices were used for testing the application of CPN films in packaging and eliminating contamination. Based on the results it is evident that the properties of the food also application in a wide range of food products such as meat, vegetables, dry food such as cereals, etc. will have to be tested to check if the antimicrobial activity is retained while packaging a broad range of food products. 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