Antimicrobial and Physicochemical Characterization of Biodegradable

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Antimicrobial and Physicochemical Characterization of Biodegradable, Nitric Oxide-Releasing Nanocellulose-Chitosan Packaging Membranes Jaya Sundaram, Jitendra Pant, Marcus James Goudie, Sudhagar Mani, and Hitesh Handa J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01936 • Publication Date (Web): 03 Jun 2016 Downloaded from http://pubs.acs.org on June 8, 2016

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Journal of Agricultural and Food Chemistry

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Antimicrobial and Physicochemical Characterization of Biodegradable, Nitric Oxide-

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Releasing Nanocellulose-Chitosan Packaging Membranes

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Jaya Sundaram*, Jitendra Pant, Marcus J. Goudie, Sudhagar Mani, Hitesh Handa*

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School of Biological and Biochemical Engineering, College of Engineering, University of

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Georgia, Athens, GA, USA

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*Corresponding Authors: Jaya Sundaram

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College of Engineering

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University of Georgia

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597 D W Brooks Dr

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Athens, GA 30602

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Telephone: (706) 542-3815

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E-mail: jayas@engr,uga.edu

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Hitesh Handa

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College of Engineering

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University of Georgia

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220 Riverbend Road

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Athens, GA 30602

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Telephone: (706) 542-8109

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E-mail: [email protected]

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Abstract

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Biodegradable composite membranes with antimicrobial properties consisting of nanocellulose

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fibrils, chitosan, and S-Nitroso-N-acetylpenicillamine (SNAP) were developed and tested for

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food packaging applications. Nitric oxide donor, SNAP was encapsulated into completely

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dispersed chitosan in 100 mL, 0.1N acetic acid and was thoroughly mixed with nanocellulose

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fibrils (CNF) to produce a composite membrane. The fabricated membranes had a uniform

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dispersion of chitosan and SNAP within the nanocellulose fibrils, which was confirmed through

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Scanning Electron Microscopy (SEM) micrographs and chemiluminescence nitric oxide

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analyzer. The membranes prepared without SNAP showed low water vapor permeability than

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that of the membranes with SNAP. The addition of SNAP resulted in a decrease in the Young’s

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modulus for both 2-layer and 3-layer membrane configurations.

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evaluation of SNAP incorporated membranes showed an effective zone of inhibition against

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bacterial strains of Enterococcus faecalis, Staphylococcus aureus, and Listeria monocytogenes

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and demonstrated its potential applications for food packaging.

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Keywords: nanocellulose fibrils; nitric oxide; SNAP; chitosan; antimicrobial; biodegradable;

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packaging membranes; zone of inhibition

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Antimicrobial property

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Introduction

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The Centers for Disease Control and Prevention (CDC), estimates that one in every six

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Americans gets sick from foodborne illness each year, results in 128,000 hospitalizations and

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3,000 deaths. Foodborne pathogens deteriorate the quality of food, causing increased food

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wastage. Recent foodborne microbial outbreaks are driving a search for innovative ways to

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inhibit microbial growth in foods while maintaining quality, freshness, and safety and extend the

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shelf-life of fresh produces. The Federal government advises and encourages healthy eating

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habits, which includes consumption of a variety of fresh fruits and vegetables1, 2. As a result, the

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per capita consumption of eating fresh produce has increased to $12 billion annual sales in the

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past few years3,4 and the fresh-cut food industry sector becomes the fastest growing segment of

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food industries. As the fresh-cut produce market continues to grow, such producers face the

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challenge of an increase in microbial safety for longer shelf life. From 1996 to 2006, 22

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foodborne illness outbreaks were associated with the consumption of fresh produce. Of these

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outbreaks, according to Food and Drug Administration (FDA), 18 outbreaks were implicated by

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fresh-cut produce. Foodborne illness outbreaks also impact the fresh produce trade leading to

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economic losses4. Food related epidemic can be prevented by means of improved surveillance

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and detection of contaminations, enhanced epidemiological investigation, safe packaging, and

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effective methods to identify pathogens5-10.

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The packaging of fresh fruits and vegetables is one of the most important steps in the long

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supply chain from the producer to the consumer. Protecting the fresh produce by packing them in

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antimicrobial (AM) films can extend their shelf life of food11-14. They also effectively control the

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foodborne pathogens and food-spoiling microorganisms.

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manufactured by incorporating antimicrobial agents, immobilized or coated on the surface of the


These packages are typically


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packaging material. Even though the AM packaging films and number of antimicrobial agents

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have been studied for many years, commercial successes of these packaging materials are very

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limited due to many constrains in large scale production15. Selection of packaging system and the

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antimicrobial agents are very critical as they would influence the inherent physicochemical

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properties of food. Now there is also an increasing demand for green labeling and environmental

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safety, leading to an increasing number of R & D efforts in the field of biodegradable food

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packaging materials. Instead of using polymer materials derived from petroleum products,

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biopolymers derived from renewable sources (starch, cellulose, protein etc.) are more favorable

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in developing an eco-friendly packaging system for food. When the antimicrobial agents are

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combined with biodegradable packaging materials, it features the merits of the packaging system

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in terms of food safety, shelf-life, and environmental friendliness.

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Cellulose is one of the most abundantly available biopolymers. The cellulose pulp derived from

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plants and trees are either mechanically or chemically fibrillated into nanocellulose fibrils (CNF)

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having 5-20 nm diameter. These nanocellulose fibrils highly influence the properties and

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functionality of the final products16-19. George et al made food packaging membrane using

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nanocellulose derived from bacterial cellulose and demonstrated its relevance as a packaging

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material for the food industry20. The prepared membrane possessed minimal oxygen

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permeability, good mechanical stability, and controlled water permeability, which are critical for

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food packaging materials to maintain the quality of packed food. Reinforcing nanocellulose with

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other long chain polymers such as chitosan to develop biodegradable nanocomposite food

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packaging materials can further improve the quality of the packaging material as well as long

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storage life21. Chitosan is a linear polysaccharide, deacetylated derivative of chitin, which is the

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second most abundant polysaccharide found in nature after cellulose. Chitosan is nontoxic,


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biodegradable, and biocompatible with antimicrobial characteristics as well and it could be used

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as a packaging material for the quality preservation of food22, 23, 24.

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Antimicrobial films with controlled release of antimicrobial agents are more advantageous

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than dipping or spraying the food with antimicrobial agents containing edible polymers25-27. In

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these types of coatings, the antimicrobial activity is lost due to its inactivation by the food

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components leading to concentrations dropping below active levels28. There are several

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antimicrobial agents available to incorporate into packaging films. However, each one has its

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own disadvantages apart from their noted advantages. Sodium nitrite salt has been used for

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centuries in meat curing. Nitric oxide (NO) released from this salt terminates free radicals

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present in lipid oxidation and provide the typical property for the cured meat29. There are many

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NO-donating compounds such as nitrates and nitrites that have been used for several years in

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curing and preserving meats, fish, and certain cheeses30, 31. Nitric oxide inhibits the growth of

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wide varieties of bacteria (both gram positive and gram negative), viruses, fungi, and yeast32-34.

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The biggest advantage of NO as an antimicrobial agent is its antimicrobial activity against

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antibiotic-resistant strains35-37.

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nitrosothiols in the packaging materials for food has not been yet studied. In this study, we

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investigated the effects of incorporating the NO donor, S-Nitroso-N-acetylpenicillamine (SNAP)

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into biodegradable nanocellulose-chitosan composite membranes for increased antimicrobial

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activity for potential food packaging application.

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Materials and Methods

However, incorporating NO donor compounds such as S-

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Chitosan (85% deacetylated) derived from shrimp shell was purchased from Thermo- Fischer

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(Waltham, MA USA), glacial acetic acid, glycerol, N-acetylpenicillamine, sulfuric acid,

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hydrochloric acid, sodium nitrite, potassium chloride, sodium chloride, potassium phosphate 5 ACS Paragon Plus Environment


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monobasic and sodium phosphate dibasic were purchased from Sigma-Aldrich (St. Louis, MO,

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USA). Nanocellulose fibrils were bought from the University of Maine, MI. Luria broth (LB)-

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Lennox and Luria Agar (LA) - Miller were obtained from Fisher Bioreagents (Fair Lawn, NJ).

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Synthesis of S-Nitroso-N-acetyl-D-penicillamine (SNAP)

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S-Nitroso- acetyl penicillamine was synthesized using a modified version of a previously

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reported method by Clough and Thrush (1967). Equimolar ratios of N-acetylpenicillamine (NAP)

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and sodium nitrite were added to a reaction vessel, which had 1:1 mixture of water and methanol

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containing 2 M HCl and 2 M H2SO4, and stirred for 30 minutes. The reaction vessel was cooled

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in an ice bath to precipitate the SNAP crystals. After precipitation of SNAP crystals, they were

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collected by filtration and washed with water to remove unreacted salts, and air dried. The entire

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process of SNAP synthesis was protected from light. The purity level of synthesized SNAP was

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tested using the Sievers Chemiluminescence Nitric Oxide Analyzer and recorded greater than

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95%.

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Fabrication of composite membrane

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Biodegradable antimicrobial packaging membranes were prepared in 2-layer and 3-layer

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configurations. The 2-layer membranes contained one layer with 2 wt% SNAP (antimicrobial

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agent) in nanocellulose-chitosan with a top layer of nanocellulose-chitosan mix without SNAP.

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Similarly, the 3-layer membranes contained the middle layer with 2 wt% SNAP in

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nanocellulose-chitosan with a top and bottom layers of nanocellulose-chitosan mix without

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SNAP. The control membranes of 2-layer and 3-layer configurations were prepared separately

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using nanocellulose-chitosan mix, without SNAP antimicrobial agent. About 2 wt % of chitosan

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was completely dispersed in 100 mL of 0.1N acetic acid at room temperature (21±2oC) using

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magnetic stirrer plate. In another beaker 2 wt% of nanocellulose fibrils (CNF) was thoroughly


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dispersed in 100 mL of distilled (DI) water using magnetic stirrer plate. Completely dispersed

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CNF and chitosan were combined together and mixed homogeneously; 3 mL of 80% glycerol

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was added as a plasticizing agent while mixing them together. The resulting mixture was

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degassed and poured into a clean glass petri dish of 14 cm diameter and air dried. While the

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drying was incomplete, another set of membrane solution was made as mentioned above and

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poured on the partly dried membrane as a second layer after 24 hours. On average it took 48

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hours to dry for 2-layer control membrane. Similarly, 3-layer control membrane was also made

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as 2-layer control membrane with additional layer of CNF-chitosan mix and additional drying

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time of 24 h; in total approximately 72 h to complete the processing of 3-layer configuration.

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To prepare NO-releasing antimicrobial membranes, 2 wt % of chitosan was completely

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dispersed in 100 mL of 0.1N acetic acid as said above. 2 wt% SNAP was dissolved in 80%

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ethanol and 3 mL of the resulting solution was added into the completely dispersed chitosan and

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the mixing process was continued to obtain complete encapsulation of SNAP. In another beaker

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2 wt% of nanocellulose fibrils (CNF) was thoroughly dispersed in 100 mL of distilled (DI) water

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as mentioned in control membrane fabrication. Completely dispersed CNF and chitosan with

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SNAP were then homogeneously mixed together; while mixing them, 3 mL of 80% glycerol was

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added as a plasticizing agent. This mix was degassed before casting the membrane. The 2-layer

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antimicrobial membranes were prepared by casting membrane without SNAP first and after 24

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hours of drying, SNAP mixed chitosan and CNF homogeneous mix was poured on top of the

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partially dried membrane of without SNAP and air dried completely. For 3-layer membranes, the

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middle layer was made with SNAP and top and bottom were made without SNAP in an order

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such as CNF-chitosan mix without SNAP first, CNF-chitosan mix with SNAP second and again

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CNF-chitosan mix without SNAP third.



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Physicochemical characterization

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Water permeability

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To determine water vapor permeability of the composite membranes the procedure followed

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by Jaya and Das38 was used. Approximately 5 g of dehydrated silica gel was filled separately in 6

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glass vials of uniform volume. The lid was replaced by one of the 6 membranes (2-layer with

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SNAP, 3-layer with SNAP, 2-layer control, 3-layer control, control chitosan and control CNF).

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All the vials were weighed and then placed in an environment maintained at 75% relative

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humidity (RH) and 22±2o C established with saturated sodium chloride solution in a desiccator39.

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The weight of each vial containing the silica gel was recorded at 24 h intervals for 7 days, and

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the mean weight gained by the silica gel was calculated for each day. The water vapor

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permeability, K (kg. m/ m2.day.Pa), of the composite membrane was calculated using the

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following equation. 𝐾=

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𝑑𝑤/𝑑𝜃𝑃 × 𝑡 𝐴𝑃 𝑝 ∗

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where 𝑑𝑤/𝑑𝜃𝑃 is the slope of curve plotted between the time 𝜃𝑃 (day) and cumulative moisture

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gain; w (kg) is the weight of the silica gel packed in the vials; t is the thickness of the film (m);

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𝐴𝑃 (m2) is the surface area of the composite membranes; 𝑝∗ (Pa) is the saturation vapor pressure

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of water at 22oC; the temperature of the environment chosen for the experiment and the relative

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humidity (75%).

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Tensile strength

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Mechanical attributes of the composite membranes were characterized in terms of tensile

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force using an Instron material testing machine (Instron model 5545, USA) with 1kN load cell.

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Test specimens were cut according to the IPC-TM-650 standards with a 6:1 length to width ratio.

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Gauge length and thickness values were recorded individually for each sample, and carefully 8 ACS Paragon Plus Environment

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aligned to ensure minimal torsional forces were applied, and held by pneumatic jaws with 1”x1”

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against 1”x3” rubber faces. Specimens were tested at a constant extension rate of the cross head

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speed of 1 mm/s. The tests were done at 23 ± 2ºC and 50 ± 5% RH. Data points of force,

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distance and time were collected and analyzed for stress and strain relationships. Young’s moduli

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of the composite membranes were derived from the stress and strain relationships. Tensile

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strength and Young’s modulus were then calculated for each sample and averaged for each

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material. All materials were tested in triplicate.

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Morphology study using scanning electron microscopy

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Film surface morphology and microstructure were examined using scanning electron

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microscopy (SEM) (FEI Inspect F FEG-SEM). Dried film samples were mounted on a metal stub

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with double-sided carbon tape and sputter coated with 10 nm gold-palladium using a Leica EM

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ACE200 sputter coater. Images were taken at accelerating voltage 20 kV and a magnification of

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2000X.

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NO release measurements

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Nitric oxide release from the chitosan-nanocellulose composites were measured using a

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Sievers Chemiluminescence Nitric Oxide Analyzer (NOA) model 280i (Boulder, CO). The NOA

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has the ability to selectively measure NO through the reaction of NO with oxygen plasma, giving

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it the ability to reduce interference from molecules such as nitrates and nitrites40. The ability of

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NOA to selectively measure NO has made this technique a gold standard in the field of NO-

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releasing materials41-43. Films were punched to 5/16’’ diameter and threaded with silk surgical

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suture to be suspended in an amber NOA cell. The NOA cell with the membrane is lowered into

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a 37oC water bath while the amber cell protects the sample from light. Deionized water (3 mL)

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was added to the NOA cell and allowed to heat to 37°C. Nitrogen was bubbled into the DI at 100



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mL/min to provide a humid environment for the film. Both 2-layer and 3-layer membranes

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containing SNAP were tested for initial release, as well as after 24-hour storage in humid 37°C

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conditions in water jacketed incubator (Thermo Fisher Scientific, Waltham, MA USA). A

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representation of the measurement cell is shown in Figure 1.

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Zone of inhibition (ZOI) studies

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Antibacterial properties of SNAP incorporated chitosan-nanocellulose based packaging

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membranes were tested against three common bacterial strains using zone of inhibition (ZOI)

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assay. The bacteria used in the study were Staphylococcus aureus (S. aureus), Listeria

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monocytogenes (L. monocytogenes), and Enterococcus faecalis (E. faecalis).

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version of agar diffusion standard protocol was followed to carry out this study aseptically44, 45.

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A single isolated colony of each bacterium was suspended individually in Luria broth (LB)

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medium and allowed to grow at 37ᴼC for 14 hours at a rotating speed of 150 rpm using a shaker

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incubator. The optical density (OD) of the liquid suspension of each of the strains was measured

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by UV-Vis spectrophotometer (Genesis 10S-Thermo Scientific) at 600 nm (OD600) using LB

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medium as blank and was adjusted to 1x107 colony forming units per mL (CFUs/mL) based on

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the calibration curve between CFUs and OD. A sterile swab was placed into the bacterial

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culture, gently pressed and rotated against the inside of the petridish (14 cm) to spread the

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bacteria uniformly and aseptically. The circular pieces (dia= 14 mm) of packaging membranes

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(control and test) were placed over the bacterial culture and pressed gently. The resulting plates

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with bacterial strain and membranes were incubated overnight at 37ᴼC for 20h. The diameters of

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the ZOI of the membranes were compared with each other and among the bacteria strains to

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evaluate the antimicrobial effectiveness of the membranes.

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A modified

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Results and Discussion

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Water vapor permeability characteristics analysis

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Table 1 shows the water vapor permeability of the control and SNAP incorporated packaging

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membranes developed in this study. Control membranes prepared with a single material such as

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chitosan or CNF showed less water vapor permeability compared to the other membranes.

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Chitosan and CNF combined control membranes (2-layer and 3-layer) had slightly higher

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permeability than the membranes with single material even though their thickness is slightly

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higher than the single material control membranes. In ideal polymeric structures, gas and vapor

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permeability are independent of film thickness46. However, the result from chitosan and CNF

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membrane showed that it behaves like an ideal polymer. It is possible that during the

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permeability test, the side exposed to high relative humidity absorbed more water and developed

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desorption rate independent of the thickness-resistance for water vapor diffusion. As a result of

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this, the side exposed at low relative humidity became responsible for the vapor transfer. Under

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this condition, the diffusion flux could become independent of thickness and resulted in higher

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permeability than the single component control film47. SNAP incorporated membranes had

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higher water vapor permeability than the membranes prepared without SNAP. Even though the

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thickness of SNAP incorporated membranes with 2-layer and 3-layer were significantly higher

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than other control membranes, they demonstrated the highest permeability indicating that the

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membranes containing SNAP had a role in increasing the water vapor permeability. However,

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the water permeability values obtained in this study were lower than the methyl cellulose based

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biodegradable membranes developed by Turhan and Sahbaz48 and other starch-based

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biodegradable films made by Para et al49 and Bertizzi et al47. The addition of SNAP to the

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chitosan cellulose matrix likely decreases the attractive forces between the chain networks hence



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increasing the free volume and segmental motions, which could result in easy diffusion of water

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molecules and thereby increase the water vapor permeability.

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Tensile strength analysis

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Figure 2 shows the Young’s modulus measured for control and NO-releasing membranes.

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The mechanical strength of the membranes was studied by measuring their Young's modulus.

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Since nanocellulose possess high mechanical strength, the membrane prepared with only

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nanocellulose showed highest Young's modulus (1587.6 ± 282 MPa) as compared to the other

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membranes. Chitosan is structurally similar to cellulose except it contains NH2 group in the

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position of the C2 hydroxyl group and mechanical properties of chitosan vary depending on the

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percentage of deacetylation50. Chitosan used in this study was 85% deacetylated and its Young’s

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modulus was measured at 245.4 ± 89 MPa. The 2-layer and 3-layer control films showed higher

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Young's modulus than control chitosan. As expected, chitosan and nanocellulose together

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increased the mechanical strength due to increased hydrogen bonding between the polymer

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chains of nanocellulose and chitosan51. However, incorporation of SNAP into the 2-layer and 3-

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layer membranes reduced the mechanical strength much lower than the control chitosan films.

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One of our previous studies showed similar reductions in strength upon incorporation of SNAP

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into medical grade polymers, which can arise due to the solubility of the SNAP molecule within

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the polymer matrix52. As the concentration of SNAP within the matrix surpasses the solubility

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limit, localized regions of SNAP crystallization occur53. The localized crystalline SNAP might

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create gaps between the nanocellulose network chains for its mobility and reduce inter-chain

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interactions leading to significant reduction in tensile strength of the membrane. Similar to the

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effect of SNAP on water vapor permeability, the addition of SNAP to the nanocellulose-chitosan


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matrix might decrease the attractive forces between the network chains and increase the free

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volume and segmental motions, which could cause a reduction in mechanical strength as well.

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NO release characteristics

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Nitric oxide released from the 2-layer and-3-layer chitosan compositions were measured

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using the Sieves Chemiluminescence Nitric Oxide Analyzer (model 280i, Boulder, CO). The

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release of nitric oxide (NO) from SNAP is highly sensitive to heat, light (340 and 590 nm), and

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moisture, as these catalyze the spontaneous decomposition reaction54, 55. The 2-layer and 3-layer

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designs were implemented to see if the top layer of chitosan-nanocellulose would provide a

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barrier to help selectively deliver NO to one side of the film. This selective release of NO would

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be accomplished by limiting the exposure of the SNAP layer to both light and moisture.

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However, no significant difference was observed between the 2 and 3-layer configurations (p =

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0.36) as measured using a two-tailed Student's t-test. Release of NO from the chitosan-

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nanocellulose composites were measured for initial release, as well as a release after 24 hours to

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determine the level of NO that is being delivered during zone of inhibition studies (Figure 3). All

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measurements were conducted at 37°C and the samples were protected from light at all times.

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Initial release of NO from 2-layer and 3-layer composites were found to be 0.1±0.03x10-10

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mol. cm-2. min-1 and 0.18 ± 0.07 x 10-10 mol. cm-2. min-1 respectively. In both cases, release of

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NO decreased after 24 hours at 37°C to 0.07 ± 0.01x10-10 mol. cm-2. min-1 and 0.05 ± 0.01x10-10

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mol. cm-2. min-1. While the NO release appears to be higher for the 3-layer configuration, it is

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possible the NO release from the 2-layer configuration is asymmetric and is providing a larger

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flux of NO to the exposed SNAP layer than that of the chitosan-nanocellulose layer. This could

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result in an underestimation of the NO release.

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Antibacterial characteristics evaluation

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Table 2 shows the zone of inhibition (ZOI) of each selected bacterium strain for both 2 and 3

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layer membranes with NO-releasing (SNAP) component as an antimicrobial agent. The chitosan-

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nanocellulose films with incorporated antimicrobial component resulted in the varying level of

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NO release, which resulted in ZOI (mm) with varied diameters (Figure 4) depending on the level

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of NO release. As expected all bacterial strains (S. aureus, L. monocytogenes, and E. faecalis)

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were found susceptible to the packaging material owing to the bactericidal effect of NO. The

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antimicrobial activity of the membranes resulted in similar ZOI between 2-layer and 3-layer

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membranes against E. faecalis, and S. aureus. However, L. monocytogenes showed a significant

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difference in the ZOI between the 2-layer and 3-layer membranes. Among all the bacteria, L.

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monocytogenes was most susceptible to both the 3-layer and 2-layer membranes. Overall, S.

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aurues exhibited the smallest ZOI (as compared to E. faecalis, and L. monocytogenes). The

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higher antimicrobial activity of 3-layer membrane as compared to 2-layer membrane can directly

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be correlated to the NO flux exhibited by these films. As shown in Figure 3, the 3-layer

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membranes have higher NO flux in the beginning which might have resulted in higher bacteria

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killing in the initial few hours. However, over an incubation period of 24 hours during ZOI

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testing, the NO flux reached almost the same value for both 2-layer and 3-layer membranes

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hence resulted in similar diameter of ZOI. The difference in the antimicrobial activity among the

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bacterial strains could be attributed to their cell membrane properties56.

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Morphology of membranes

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Figure 5 shows the surface morphology of 2-layer and 3-layer control (A and B) membrane

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and the membrane with SNAP material (C and D). Membranes were showed smooth surface

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without any pores or cracks and with good structural integrity. The membranes obtained after


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drying were flat and compact. All of them show the nanocellulose fibrils network structure. It

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could be assumed that within the fibrils network chitosan was smoothly dispersed, because

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chitosan disperses within the nanocellulose matrix with relatively good interfacial adhesion

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between the two components50. These results could be attributed to the strong interactions

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between nanocellulose fibrils and chitosan and SNAP, which are caused by the hydrogen

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bonding between the hydroxyl groups of nanocellulose and carbonyl group in the chitosan.

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In conclusion, S-nitroso-N-acetylpenicillamine incorporated antimicrobial membranes mixed

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with nanocellulose fibrils and chitosan were successfully fabricated. Developed membranes

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exhibited good film forming properties due to the presence of high density of amino groups and

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hydroxyl groups and inter and intramolecular hydrogen bonding. The chitosan-CNF-NO-

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releasing composites showed antimicrobial characteristics in the packaging membranes. The

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addition of chitosan and SNAP into nanocellulose to develop composite biodegradable

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membrane with antimicrobial activity showed clear effects towards inhibition of E. faecalis,

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S.aureus, and L.monocytogenes as shown using ZOI. The membranes developed in this study

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showed excellent water barrier property with a low value of water vapor permeability. Surface

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morphology showed the strong interactions between nanocellulose fibrils, chitosan, and SNAP

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materials. Tensile strength measurements showed decreased Young’s modulus for SNAP

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incorporated membranes, which would be further studied to improve its mechanical properties.

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Acknowledgement

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Authors acknowledge the Poultry processing research unit in US poultry research center at Athens, GA for proving the bacteria strains for this study.

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520 521 522 523 524 525 526 527 528 Nitrogen Bubble flow

Suspension of NO releasing film

Nitrogen Sweep flow

NO rich gas to NOA

Water bath (37°C)

529 23 ACS Paragon Plus Environment


Page 24 of 29

530

Figure 1 Representation of test configuration for NOA cell for measurement of NO released

531

from chitosan films in a humid environment

532 533 534 535 2000 1800 1600

Modulus (MPa)

1400 1200 1000 800 600 400 200 0 2 layer control 3 layer control

2 layer NO

3 layer NO

536 537

Cellulose control

Figure 2 Young’s modulus of nanocellulose-chitosan membranes.

538 539 540 541 542 543 24 ACS Paragon Plus Environment

Chitosan control

Page 25 of 29


544 545 0.30

NO flux (x10-10 mol cm-2 min-1)

0.25

0.20

0.15

0.10

0.05

0.00

546

Chitosan 2 Layer (Initial)

Chitosan 3 Layer (Initial)

Chitosan 2 Layer (24 h)

Chitosan 3 Layer (24 h)

547

Figure 3 Nitric oxide release rates of 2-layer and 3-layer chitosan-nanocellulose composites as

548

measured by chemiluminescence

549 550 551 552 553 554



555 556

Figure 4 Images of the zone of inhibition comparison of 2-layer chitosan-CNF control, 2-layer

557

SNAP incorporated chitosan-CNF film, 3-layer SNAP incorporated chitosan-CNF film and 3-

558

layer control (clockwise from top left) for (A) Listeria monocytogenes (B) Staphylococcus

559

aureus (C) Enterococcus faecalis.

560 561 562 563 564 565


Page 26 of 29

Page 27 of 29


566 567

Figure 5 Scanning electron microscope images of the chitosan-nanocellulose film before and

568

after addition of SNAP. A) 3-layer chitosan-nanocellulose, B) 2-layer chitosan-nanocellulose,

569

C) 3-layer chitosan-nanocellulose with SNAP, and D) 2-layer chitosan-nanocellulose with

570

SNAP.

571 572 573 574 575 576 577 578 579 580 581 27 ACS Paragon Plus Environment


582

Page 28 of 29

Table 1. Water permeability of different types of packaging membranes Membrane type

Water vapor permeability Membrane (kg.m/ m2.day.Pa)

thickness (mm)

Chitosan

7.01x10-6

0.058

Nano Cellulose Fibrils (CNF)

6.88x10-6

0.060

2-layer CNF + Chitosan with SNAP

1.56x10-5

0.126

2-layer CNF +Chitosan without SNAP

8.81x10-6

0.068

3-layer CNF + Chitosan with SNAP

1.11x10-5

0.124

3-layer CNF + Chitosan without SNAP

8.31x10-6

0.096

583 584

Table 2. Comparative analysis of zone of inhibition among different bacterial strains using

585

developed antimicrobial packaging membranes Film type

Bacterial strain

ZOI (mm)

2-layer SNAP incorporated chitosan-CNF films

S. aureus

30


S. aureus

30


E. faecalis

34


E. faecalis

35


L. monocytogenes

37


L. monocytogenes

45

586 587 588 589


Page 29 of 29

590


Abstract Graphics

591 592

For table of contents only


Antimicrobial and Physicochemical Characterization of Biodegradable

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