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Author: Yang, Dao Title: Antimicrobial Packaging Films: Encapsulation of Allyl-Isothiocyanate in β-

Cyclodextrin The accompanying research report is submitted to the University of Wisconsin-Stout, Graduate School in partial

completion of the requirements for the

Graduate Degree/ Major: MS Manufacturing Engineering

Research Advisor: Joongmin Shin, Ph.D.

Submission Term/Year: Spring, 2013

Number of Pages: 63

Style Manual Used: American Psychological Association, 6th edition

I understand that this research report must be officially approved by the Graduate School and that an electronic copy of the approved version will be made available through the University Library website

I attest that the research report is my original work (that any copyrightable materials have been used with the permission of the original authors), and as such, it is automatically protected by the laws, rules, and regulations of the U.S. Copyright Office.

My research advisor has approved the content and quality of this paper. STUDENT:

NAME DATE:

ADVISOR: (Committee Chair if MS Plan A or EdS Thesis or Field Project/Problem):

NAME DATE:

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This section for MS Plan A Thesis or EdS Thesis/Field Project papers only Committee members (other than your advisor who is listed in the section above) 1. CMTE MEMBER’S NAME: DATE:

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--------------------------------------------------------------------------------------------------------------------------- ------ This section to be completed by the Graduate School This final research report has been approved by the Graduate School.

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Yang, Dao. Antimicrobial Packaging Films: Encapsulation of Allyl-Isothiocyanate in β-

Cyclodextrin

Abstract

The construction of antimicrobial packaging films was performed by means of

compression. In order to sustain the release of allyl-isothiocyanate (AITC), a pungent

antimicrobial agent, it was encapsulated in β-cyclodextrin (β-CD) prior to compression with low

density polyethylene (LPDE). Four different molar ratios of AITC-β-CD complex were prepared

at (0.5:1.0, 1.0:1.0, 2.0:1.0, and 3.0:1.0) respectively, and more than 90% of AITC was included

at the molar ratio of 2.0:1.0. Different concentrations of AITC-β-CD complex (0%, 1%, 2%,

4%, 6%, 8%, and 10%) were impregnated into LDPE resin. The release of AITC agent from

these films was determined in different conditions: relative humidity and temperature. Both the

humidity and temperature conditions had a significant influence on accelerating the release of

AITC. Their barrier properties (oxygen transmission rate and water vapor transmission rate) and

mechanical properties (tensile strength and elongation) were determined. Overall, results

showed that the complexation of AITC by β-CD can provide a significant effect on sustaining

and controlling the release of AITC. Although some changes in properties were observed,

impregnating AITC-β-CD complex into LPDE may still be useful as potential antimicrobial

components.

Key words: Allyl-isothiocyanate, Cyclodextrin, Antimicrobial Packaging

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Acknowledgments

I would like to thank my advisor, Dr. Joongmin Shin, for his encouragement, guidance

and support throughout this process. Initially, this project seemed daunting; however, with Dr.

Shin’s insight my learning curve was significantly reduced. Lastly, I offer my regards and

consecrations to all of those who supported and/or helped me in any respect during my time at

the University of Wisconsin Stout. Without them, my success at UW-Stout would not have been

possible. Thank you.

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Table of Contents

.................................................................................................................................................... Page

Abstract ............................................................................................................................................2

Acknowledgements ..........................................................................................................................3

List of Tables ...................................................................................................................................7

List of Figures ..................................................................................................................................8

Chapter I: Introduction .....................................................................................................................9

Food Packaging ....................................................................................................................9

Allyl-Isothiocyanate ...........................................................................................................11

Cyclodextrin .......................................................................................................................13

Low-Density Polyethylene ................................................................................................15

Statement of the Problem ...................................................................................................16

Purpose of the Study ..........................................................................................................16

Assumptions of the Study ..................................................................................................17

Definition of Terms............................................................................................................17

Limitations of the Study.....................................................................................................17

Methodology ......................................................................................................................18

Chapter II: Literature Review ........................................................................................................19

Progress in Food Packaging ...............................................................................................19

Active Packaging ...............................................................................................................19

Antimicrobial Packaging ...................................................................................................22

Critical Factors to Consider ...............................................................................................26

Gas Chromatography-Mass Spectrometry .........................................................................28

Solid Phase Microextraction ..............................................................................................29

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Chapter III: Methodology ..............................................................................................................31

Defined Molar Ratio ..........................................................................................................31

Encapsulation of AITC in β-CD ........................................................................................32

SPME Extraction of AITC .................................................................................................33

External Calibration with GC-MS .....................................................................................33

GC-MS analysis of released AITC from β-CD .................................................................34

Impregnation of AITC-β-CD with LDPE ..........................................................................35

GC-MS Analysis of Released AITC from Films ...............................................................35

Determination of AITC in the Microcapsules ...................................................................36

Optical Property .................................................................................................................36

Mechanical Properties ........................................................................................................36

Measurement of OTR and WVTR .....................................................................................37

Chapter IV: Results ........................................................................................................................38

Effect of Encapsulated of AITC in β-CD ..........................................................................38

Development of AITC-β-CD Complex Impregnated Film ................................................41

Mechanical Properties of Compressed Film ......................................................................41

Optical Property of Compressed Film ...............................................................................42

Measurement of Oxygen and Water Vapor Transmission Rate ........................................44

Release of AITC reagent from Antimicrobial Films .........................................................45

Chapter V: Discussion ...................................................................................................................47

Conclusions .........................................................................................................................47

Recommendations……….. .................................................................................................48

Future Studies .....................................................................................................................48

References ......................................................................................................................................49

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Appendix A: Collected Data for Degradation of AITC .................................................................52

Appendix B: Collected Data for Antimicrobial Films: Encapsulation of AITC in β-CD .............55

Appendix C: Chromatogram of Antimicrobial Films: Encapsulation of AITC in β-CD ..............63

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List of Tables Table 1: Schematic of different active packaging systems………………………………………21

Table 2: Effect of molar ratio of AITC- β-CD on encapsulation efficiency %……….…………40

Table 3: Mechanical strength of the antimicrobial film with different AITC-β-CD concentration …………..…………………….…………….………………42

Table 4: Barrier properties of the various concentrated compressed films .…………….………44

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List of Figures

Figure 1: The molecular formula for allyl-isothiocyanate ………………………...……….……13 Figure 2: Chemical structure of the three main types of cyclodextrins ……….……...…………14 Figure 3: Hurdle technology: Conventional vs. Antimicrobial ……….………………...………23 Figure 4: Complex layers of antimicrobial packaging ……….……….…………………………25 Figure 5: Schematic of different antimicrobial features ……………………......………….……25 Figure 6: Schematic of a GC-MS system ……….……………………………....………………28 Figure 7: Design and enlarged view of commercial SPME device ……….……….....…………30 Figure 8: Experimental setup for encapsulation of AITC in β-CD …………………...…………32 Figure 9: Schematic of sample preparation with SPME ………………………………...………33 Figure 10: Example of a GC-MS Instrument …………………………………….………...……34 Figure 11: Schematic of an antimicrobial film by mean of compression ……………….………35 Figure 12: Sample Film Preparation for Mechanical Properties ……….……….………………37 Figure 13: Instruments used to analysis OTR and WVTR in antimicrobial film ………….……37 Figure 14: Schematic of encapsulated AITC in β-CD ……….…………………………….……39 Figure 15: Degradation of AITC without β-CD encapsulation ……….………...………………39 Figure 16: External calibration graph of AITC-β-CD complex ……….………..………………40 Figure 17: Distribution of AITC-β-CD complex in film ……………………….………….……43 Figure 18: Release of AITC reagent from antimicrobial film for 30 days (@23°C)…….………46

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Chapter I: Introduction

Food Packaging

Throughout history, preservation of food has always been a concern for mankind. Prior

to modern food packaging/preservation methods, simple processes, such as smoking and salting,

were considered the main means to prevent food deterioration (Rhoades & Rastall, 2000). What

began as a simple method has evolved into a precise science, creating various active packaging

systems. Food packaging has already paid significant dividends, allowing consumers the

opportunity to preserve and enjoy quality food at their convenience.

In order to fully understand the influences that active packaging systems have on food

preservation, it is important to clarify their basic functions and discuss any important information

beforehand. (Suppakul, Miltz, Sonneveld, & Bigger, 2003) described active packaging as “an

innovative concept that can be defined as a mode of packaging in which the package, the

product, and the environment interact to prolong shelf-life or enhance safety or sensory

properties while maintaining the quality of the product” (p. 408). The term active packaging

refers to several smart systems in which their design can vary; for example, a packaging system

can be designed with or without a headspace. (Robertson, 2006 as cited in Huff, 2008) reported

that if the system contains a headspace feature, its composition can significantly influence the

prevention of deterioration. Most active packaging systems consist of a food preserving agent

that migrates through the package by means of interaction between the packaging material and

surrounding environment. In addition to food, components like a sachet or a pad are added to

absorb specific gases or water, respectively. As stated above, active packaging systems can vary

in design and/or method; however, they all share similar goals: enhancement of safety and

quality and extension of the shelf-life expectancy in foods.

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Active packaging systems have been studied worldwide for more than 40 years. They

have gained a significant amount of attention and have been the ideal packaging method to

prevent food deterioration for over a decade. In 2003, the market was estimated to be worth

approximately $50 million worldwide (Ozdemir & Floros, 2004). Despite their benefits and

development, commercial or practical applications have been limited.

Active packaging systems have a variety of applications, and antimicrobial is one of the

most promising applications (Suppakul et al., 2003). Antimicrobial packaging is designed to

inhibit the growth of spoilage and pathogenic microorganisms by enhancing the correlation

and/or interface between packaging material, food agent, and the surrounding environment

without damaging the integrity of the food product (Han, 2000; Suppakul et al., 2003).

Antimicrobial packaging has proven to be an efficient method when compared with conventional

systems (without food agent). Despite these advantages, commercial or practical applications of

antimicrobial packaging have been limited.

In the past, most packaging systems used artificial agents as the main inhibiting

mechanism. Butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) have been

used in food packaging as the preserving agent. Although they are effective in inhibiting

oxidation growth in cereal, BHT and BHA have also been suspected of causing liver damage and

carcinogenesis (Onyeneho & Hettiarachchy as cited in Siro et al., 2006). As a result, some

consumers have developed a negative perception about the quality and safety of packaged foods

when an artificial agent is used. Consequently, stricter regulations were put in-place, creating

another obstacle for commercial or practical applications.

Recently, artificial substances have been replaced by organic substances as the main

preserving food agent. Organic agents have become the catalyst in packaging because they are

safer, a simpler authorization process, and just as efficient when compared with artificial agents

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(Han, 2000; Rhoades & Rastall, 2000). Organic agents can be extracted from various plants,

such as grapefruit seed, clove, and horseradish, just to list a few. Although various organic

agents have been proven to be effective in food packaging, it should be noted that each agent has

a specify activity and may only be efficient against certain microorganism. For example, allyl-

isothiocyanate (AITC) may be most effective if used to inhibit escherichia coli, salmonella

enteritidis, and listeria monocytogenes growth in chicken, smoked salmon, and meats (Takeuchi

& Yuan, 2002). Despite their benefits, applications of food packaging have been limited.

Allyl-Isothiocyanate

Allyl-isothiocyanate (AITC) is the main pungent, volatile component in an isothiocyanate

family, which can be extracted from various cruciferous plants, such as mustard, horseradish, or

wasabi (Zhang, Jiang, & Li, 2006). Since the 20th century, AITC has been proven to be an

effective food agent and fortifier (Shin, Harte, Ryser, & Selke, 2010). According to (Isshik,

Tokuoka, Rori, & Chiba, 1992 as cited in Shin, 2007), AITC is an efficient food preserving

agent, showing a 5 log reduction of aspergillus niger growth in both fresh beef and raw tuna

packaging. When directly applied, it can increase the shelf-life of food (e.g. sandwiches and

pizza) for an additional month (Ford, 1991 as cited in Shin, 2007). Other studies have also

reported the positive effect AITC had against a broad range of microorganisms (Isshiki et al.,

1992; Lin et al., 2000; Mari et al., 2002; Winther &Nielsen 2006 as cited in Shin et al., 2010). If

it is frequently consumed, (Conaway & Munday as cited in Zhang et al., 2006) stated that AITC

can provide essential benefits against deoxyribonucleic acid (DNA) damage and cancer due to its

high biological activities. In addition, ATIC is also known for its high anti-oxidative, superoxide

scavenging potency, and anti-mutagenic activities; this means it has a wide range of possibilities

for food application (Kinae et al. as cited in Zhang et al., 2006). Although its popularity as a

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food preserving agent has increased over the years, commercial or practical applications for food

packaging are still limited.

With its highly concentrated flavor, AITC may be insufficient in many food applications.

According to (Delaquis & Mazza, 1995 as cited in Shin, 2007), the quantity of applied AITC

should be limited because it can change the packaged food’s natural flavor. A related study has

also indicated that a concentration of 2,000µg AITC can easily become inedible for most people

due to its pungent taste (Kim et al., 2002 as cited in Shin, 2007). Based on a study conducted by

(Dunnick et al., 1982 as cited in Shin, 2007), high concentrations of AITC may have a negative

effect. In that study, a high dosage of AITC (25 mg/kg/day) was given to 49 rats, and

approximately 8% of the rats were later tested positive for urinary bladder damage. Limiting or

controlling the concentration of AITC may be a more efficient, safer alternative.

In spite of this shortcoming, AITC has been approved for certain food applications in

Japan (Isshiki et al., 1992 as cited in Shin, 2007). In fact, commercialization has already been

achieved by Mitsubishi-Kagaku Foods Corporation, producing a number of different Wasaouro

products that have been reported to possess antimicrobial activities (Shin, 2007). In 2006, AITC

was approved for food applications by the Food and Drug Administration (FDA) as GRAS

(generally recognized as safe) in the United States.

AITC can be implemented into food packaging either as a gas or liquid substance.

However, studies have indicated that AITC vapor is more efficient at generating inhibiting

activities, producing similar results as its liquid counterpart even at a lower dosage or

concentration (Zhang et al., 2006; Shin, 2007). According to (Sekiyama et al., 1996 as cited in

Shin et al., 2007), AITC vapor is approximately 500 to 1,000 times more effective if similar

dosages are used. Another study has also indicated that the minimum inhibitory concentration of

AITC vapor required in preventing pathogenic microorganism growth is significantly lower (250

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times) when compared to AITC liquid (Suhr & Nielsen, 2003 as cited in Shin et al., 2010).

However, controlling these components is much simpler in a liquid solution. The reason for this

being that depletion of AITC vapor can easily occur due to its lack of stability and encapsulation

capacity.

AITC can easily become volatile when exposed to food and/or any aqueous solutions

(Han, 2000; Zhang et al., 2006; Shin et al., 2010). The carbon atom, an electrophilic reagent,

shown below in Fig. 1 has been suspected as the main reason for AITC’s volatile nature (Zhang

et al., 2006). Like most electrophilic reagents, AITC is opposed to nucleophilic compounds such

as amino, hydroxyl, thiol, β-dicarbonyl, carboxylic acids, and water, all of which are found in

food (Zhang et al., 2006). In addition to its instability, AITC’s reaction can be temperature

dependent. Also, depletion can easily occur during its migration processes and/or when in

contact with foods. Therefore, its progression as a food preserving agent has been limited.

However, (Depree, Howard, & Savage, 1998 as cited in Zhang et al., 2006) reported that certain

organic compounds (ex. citric acid, sugar ester or salad oil) can stabilize its volatile nature, thus

enhancing AITC’s chemical compatibility.

CH2=CH−CH2−N=C=S

Figure 1. The molecular formula for allyl-isothiocyanate

Cyclodextrin

Cyclodextrin (CD) is a family of natural sugar molecules that are bound together by alpha

(1-4) linkages, forming a torus-shaped ring structure known as cyclic oligosaccharide (Zhang et

al., 2006; Ashnagar, Naseri, & Khanak, 2007). As shown below in Fig. 2, most common CDs

are composed of six, seven or eight glucopyranose units; these are presented as alpha (α), beta

(β), and gamma (γ) respectively (Almenar, Auras, Rubino, & Harte, 2007). Among them, beta-

cyclodextrin (β-CD) has been the ideal option as the control release method in a packaging

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system. CDs are considered as a non-reducing crystalline and water-soluble substance that can

be produced by means of extraction or obtained by enzymatic degradation of starch (Hedges,

1998; Zhang et al., 2006). They have been used in a number of different applications ranging

from food to pharmaceutical and industries related to agricultural and environmental engineering

(Zhang et al., 2006; Ashnagar et al., 2007).

Figure 2. Chemical structure of the three main types of cyclodextrins (Harris et al., 2007)

CD has a complex chemical structure, consisting of large solvent secondary groups and

small primary hydroxyl groups in its external toroid openings, while its glycosidic oxygen and

non-exchangeable hydrogen atoms are oriented within the interior of its cavity (Almenar et al;

2007). This combination gives CDs an excellent polar hydrophilic outer shell and relatively

hydrophobic cavity (Hedges, 1998; Szejtli as cited in Siro et al., 2006). Recently, CDs have

gained interest in various applications due to their aptitude of forming inclusion complexes with

a variety of guest hydrophobic molecules. Furthermore, there is a direct correlation between the

number of glucose molecules and sizes of the cavity. CDs have three different cavity sizes: α:

4.7-5.3 angstrom (Å), β: 6.0-6.5 angstrom (Å) and γ: 7.5-8.3 angstrom (Å) in which, the

differences can enhance their application’s flexibility since bonding with a variety of different

molecular sizes can be difficult (Almenar et al; 2007).

Complexation with CD can enhance the solubility of the guest molecules (food agent),

stabilize against light, temperature, oxidation and hydrolysis damages, reduce volatility, and

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mask unpleasant odors (Hedges, 1998). In a related study, (Ohta, Takatani, & Kawakishi, 1999

as cited in Zhang et al., 2006) reported that CDs, particularly β-CD, can reduce the volatile

nature of AITC and its reaction with nucleophilic compounds. (Siro et al., 2006) had also

reported that encapsulation of α-tocopherol in β-CD can an efficient method for controlling and

sustaining the migration rate for an anti-oxidative packaging system. The total depletion of α-

tocopherol activities is significantly slower with β-CD complex than without, approximately

3,500 hours and 1,000 hours, respectively. According to (Chen, Diao, & Zhang, 2005),

complexation with β-CD has enhanced the solubility of nitrobenzene in water, which means it

can easily dissolve in materials. Complexation with β-CD may reduce defects in food agents,

thus widening their applications.

Low-Density Polyethylene

There has been a variety of different food agents used in packaging (e.g. organic acids,

fungicides, antibiotics, bacteriocins, nisins, nitrobenzenes, and isothiocyanates) which have

shown to sustain inhibiting activities, especially when encapsulated in β-CD (Chen et al., 2005;

Siro et al., 2006; Zhang et al., 2006; Vartiainen, Skytta, Enqvist, & Ahvenainen-Rantala, 2010).

However, the most consistently used component thus far has been synthetic plastics, particularly

low-density polyethylene (LDPE). LDPE is the ideal packaging material due to its toughness,

flexibility, inexpensiveness, relative transparency, and availability (Vartiainen et al., 2010).

LDPE has been reported to have a higher migration rate than other common packaging materials,

such as high density polyethylene (HDPE), polypropylene (PP), and polyethylene terephthalate

(PET) (Han & Floros, 1997 as cited in Vartiainen et al., 2010). Furthermore, LDPE has the

capacity to be incorporated in various packaging systems (Nadarajah, 1994; La Storia, Mauriello,

& Musso, 2008; Vartiainen et al., 2010).

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(Siragusa, Cutter, & Willett, 1999 as cited in Vartiainen et al., 2010) reported that direct

implementation of food preserving agents (e.g. nisins) into LDPE film can generate adequate

inhibiting activities against Lactobacillus helveticus and Brochothrix thermosphacta; however, it

is more efficient with a control agent (e.g. ethylenediaminetetraacetic acid). Impregnation of

potassium sorbate powder into LDPE resins by means of extrusion has shown to be significantly

effective against the spread of microorganisms (Cutter, Willet, & Siragusa, 2001). Mechanical

properties (e.g. tensile strength) were not significantly affected when compared with control

extruded LDPE films; however, its transparency decreased when there was an increase in

concentration of impregnated agent.

Statement of the Problem

Recently, AITC was approved by the FDA for food applications and quickly became one

of the most used food preserving agents due to its high biological activities and health benefits

(Jiang et al., 2006; Shin et al., 2010). However, its application as an effective food agent is

limited because it can easily become volatile when contacted with food. Furthermore, if applied

without an effective control release system, it may affect the integrity or purity of the packaged

foods. Therefore, encapsulation with β-CD may be a more effective method in controlling and

sustaining the release of AITC.

Purposes of the Study

The purposes of this study are to develop efficient antimicrobial packaging films that are

composed of AITC and β-CD compounds, analyze the migration rate of AITC agent, and

compare the mechanical properties, barrier property, and optical property of antimicrobial films

with control LDPE films.

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Assumptions of the Study

AITC is selected for this study because it is believed to be one of the most effective and

safest food preserving agents known thus far. Based on Dr. Han’s studies, there are three types

of characteristic when selecting a food preserving agent: (1) safety assurance, (2) ease and

quality of the maintenance of the compound, and (3) shelf life extension of the product. While

there are many other factors in which to determine the quality of food packaging compounds,

this study has isolated these ideas as the main focus to simplify the designing process of an

efficient antimicrobial packaging system. β-CD will be used as the control release system to

prevent or hinder AITC’s pungent flavor and volatile nature when exposed to food or when in an

aqueous environment. If the preparation procedures are carried out with limited human error, the

findings should be accurate based on related studies. Impregnating AITC-β-CD complex into

LPDE should have positive results.

Definition of Terms

Hydrophilic. Polar side groups compound that have an affinity to water (charge).

Hydrophobic. Compound that repelled water due to its neutral chemical structure (no

charge).

Inclusion complex. Complex chemical structures that consist of a host forming a cavity

in which guest molecules can be encapsulate.

Limitations of the Study

1. Materials used in this study are limited to these components: LDPE (Industrial Arts

Supply), AITC - ≥ 93%, FCC, Food Grade (SAFC), and β-CD (Sigma-Aldrich)

2. Equipment used for analyzing are as follow: gas chromatography-mass spectrometry

(Agilent Technologies 6890N), LRX tensile tester (NEXGEN, Lloyd instruments),

Illinois Oxtran 8001, and Mocon Permatran 3/31

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3. Impregnation of AITC-β-CD complex into LDPE is limited to compression

4. Mechanical, barrier, and optical properties of antimicrobial films will be compared with

control LDPE films

Methodology

The present study will be carried out using the following materials: AITC, β-CD, and

LDPE. Variation of AITC-β-CD complex will be impregnated into LDPE resins by means of

compression to produce antimicrobial films. Release of AITC reagent from both AITC-β-CD

complex and antimicrobial films will be analyzed by gas chromatography-mass spectrometry

(GC-MS). A variety of testing procedures will be conducted to determine the barrier,

mechanical, and optical properties. Finally, evaluation of antimicrobial films will be compared

with control LDPE films to determine if there are significant changes.

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Chapter II: Literature Review

Progress in Food Packaging

In today’s society, people often rely on technologies to simplify their life. Food

packaging has changed people’s perceptions on production, distribution, storage, and

consumption of foods by inhibiting environmental, chemical, and physical damages (Risch,

2009). These protections can be as simple as preventing breakage or as complex as enhancing

the interaction between packaging materials and the surrounding environment to maintain

desirable conditions for the packaged foods. As a result, foods are now expected to have a

longer shelf-life while sustaining their nutritional quality. This has given the consumers an

opportunity to enjoy a wider variety of foods year-round.

Although conventional food packaging can provide adequate protection for basic

containment, innovations in packaging are both essential and necessary for a society that is

becoming increasingly complex and demanding. In addition to quality, consumers also want to

be assured that basic standards are met. These include safety, recyclability, affordability, and

whether or not the packaging system is resealable (La Storia, Mauriello, & Musso, 2008).

Furthermore, strict regulations have limited or prevented the usage of certain food preserving

agents. As a result, packaging techniques have been forced to evolve.

Active Packaging

Active packaging can simply be defined as an intelligent or smart system that can sustain

a safe and desirable condition for foods (Han, 2000). The main purpose of any active packaging

systems is to delay the deterioration of foods from exposure to oxygen, water vapor, and/or light.

In addition, developments have also enhanced the interaction of packaging components with

their internal environments, allowing any package leaks to be displayed, thus preventing the

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spread of contaminations (Ozdemir & Floros, 2004). These attributes are often achieved by

incorporating food agent into the packaging system or using actively functional polymers.

A variety of different factors can influence the atmosphere surrounding a packaging

system. Even after being packaged, most fresh foods will still maintain some of their biological

systems (e.g. produce). During the metabolic processes of produce, oxygen is absorbed while

carbon dioxide is generated. At the same time, water or humidity will also accumulate inside the

headspace, creating an ideal condition for the growth of pathogenic and microorganisms.

Furthermore, the type of packaging materials used can also have an influence because each

material provides a different level of protection (absorption) against gases and moisture. Each

food can have a different reaction towards carbon dioxide or oxygen. As stated above, a highly

concentrated carbon dioxide and a low oxygen packaging atmosphere can easily increase

deterioration in produce; however, this condition may enhance the shelf-life of processed meat.

Active packaging has several systems that can extend the shelf-life and enhance the

quality of foods by providing specific conditions for each product (Han, 2000; Ozdemir &

Floros, 2004). Although these systems vary, they can be classified into three categories: (1)

absorbers, (2) releasers or emitters, and (3) miscellaneous systems. An absorbing system can

isolate or eliminate undesired conditions like oxygen, carbon dioxide, ethylene, water and

volatile odors. In a releasing system, food agent is impregnated into the packaging material to

create a desirable atmosphere. The last system be defined as miscellaneous; this system provides

temperature and surface control. Table 1 shown below lists all the best known commercial and

non-commercial concepts of the active packaging systems. The hope is that these systems can

provide additional benefits that a conventional system cannot.

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Table 1. Schematic of different active packaging systems (Han, 2000; La Storia et al., 2008)

Active Packaging System & Form Working Principle Purpose & Application Examples

Antimicrobial releasing/emitters

Use organic agents, such as silver zeolite, allyl-isothiocyanate, and lysozyme bacteriocins

Prevent the growth of pathogenic and spoilage microorganisms in meat, poultry, fish, bread, cheese, fruit, and vegetables

Carbon dioxide absorbers

Potassium hydroxide calcium, silica gel, calcium hydroxide, and sodium hydroxide

Inhibit carbon dioxide levels. Used for dehydrated poultry products, roasted coffee, and beef jerky

Cholesterol absorbers

Polymer containing cinnamon, ginger, garlic, cayenne pepper and olive oil flavor

Reduce cholesterol level in food products to ensure safety or health

Ethylene Scavenging (sachets and films)

Alumina, vermiculite, or silica gel with potassium permanganate. Zeolite, clay or Japanese oya stone include with films

Increase shelf-life of produce by slowing down their metabolic cycle. Incorporate to fruits (apples, apricots, bananas, and mango) and vegetables (carrots, potatoes, and sprout)

Flavor releasing or emitters (films)

Emit flavor compound into polymers

Enhancing the flavor of a variety of food products

Lactose absorbers Incorporate lactase enzymes into polymer (milk jar)

Enhance the quality or safety of milk and other dairy products by eliminating lactose

Moisture or Humidity absorbers (sachets, films, sheets)

Silica gel (sachets) Moisture or Humidity absorbers (sachets, films, sheets)

Light absorbers (films)

UV-absorbent agent impregnate with polymers, such as polyolefin and polypropylene. Crystallinity in nylon with UV-stabilizer

Induce oxidation growth in light sensitive foods such as ham, pizza and drinks by reducing exposure to UV-light.

Oxygen scavenging or absorbers (sachets, films, and corks)

Ferro-compound, metal salts, ascorbic acid, and oxidation of glucose and alcohol are often incorporate

Prevent the growth of mold, yeast and aerobic bacteria by controlling the oxygen level. Cheese, meat, coffee, tea, nuts and bakery products are some possible applications

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Antimicrobial Packaging

Antimicrobial is one the most promising applications of active packaging systems (Han,

2000). Although conventional packaging can provide adequate protection against basic

contaminant, it cannot maximize the full potential of a food product’s shelf-life, quality, and

safety. Antimicrobial packaging can effectively do these things because it is specifically

designed to prevent and inhibit the growth of pathogenic and spoilage microorganisms (e.g.

bacteria, yeasts, molds). Safety is the most essential feature for an antimicrobial packaging

system even though it has similar objectives as a conventional system (La Storia et al., 2008). Its

popularity has significantly increased because it can be incorporated with any perishable

products susceptible to contamination.

Antimicrobial packaging has been used to prevent deterioration in food worldwide for

over a decade (Vermeiren, Devlieghere, & Debevere, 2002). Studies have shown that

antimicrobial can be an effective and safe application if the right food agent is incorporated

properly. Various food agents have been used for antimicrobial packaging applications, and

each one has a different influence due to their mechanisms and the various physiologies of the

microorganisms (Han, 2000). Categorizing microorganisms based on their ideal conditions for

growth can be an effective method in selecting an efficient preserving agent. For example, the

presence of oxygen slows the growth of aerobes and anaerobes bacteria; hence selecting an

antimicrobial agent that can produce excessive oxygen may hinder these bacteria. Other

examples of categorization are thermophilic, mesophilic and psychrotropic; these bacteria thrive

at relatively high temperatures, therefore selecting a temperature or humidity control agent is the

key component.

Antimicrobial packaging is different in many ways when compared with conventional

methods; antimicrobial is the more efficient and reliable system (Han, 2000; Vermeiren et al.,

23

2002). However, both of these packaging systems share a similar hurdling concept, shown

below in Fig. 3. In general, conventional packaging can sustain either one or two layers of

protection depending on its composite structure. In their natural composition (single layer),

LDPE and EVOH (ethylene vinyl alcohol) have proven to be efficient barriers against humidity

and gases, respectively (Dogan, Koral, & Inan, 2009). Although a complex conventional system

like LDPE-EVOH, shown below can sustain two layers of barrier properties against both

humidity and gas accumulation, it is insufficient when compared to antimicrobial packaging. In

addition to being humidity and gas resistant, antimicrobial possesses an extra barrier against

microorganisms. Each barrier provides a specific resistant component, inhibiting harmful

accumulations within the packaged food.

Humidity barrier Food

Gases barrier Food

Humidity Oxygen barrier Food

Single Package (LDPE) Single Package (EVOH) Dual-layer Package (LDPE-EVOH)

(A) Conventional packaing system

Humidity barrier

Gases barrier

Microbial barrier

Food

Multi-layer Package System

(B) Antimicrobial packaging system

Figure 3. Hurdle technology: Conventional vs. Antimicrobial (Han, 2000)

Antimicrobial activities can be achieved by impregnating food agent into the packaging

material or food (Appendini & Hotchkiss, 2002). In most cases, direct impregnation of agent

24

into food may not be an ideal application. This method can alter the packaged food’s

composition or flavor due to the high-level of concentrated food agent being absorbed. The best

and most commonly used application is impregnating food agent into the packaging material

which generally consists of three different modes: (1) release, (2) absorption, and (3)

immobilization (Han, 2000).

Release mode is used predominately either in a solute or a gas form in which the

antimicrobial agent can migrate into food or headspace to inhibit the growth of microorganisms.

Both of these forms have their advantages and disadvantages. A solute form lacks the migrating

capabilities of a gas substance; however, it is easier to control. Absorption mode prevents or

inhibits the growth of microorganisms by removing the essential or optimal living conditions for

microorganisms; these conditions may include excessive oxygen, carbon dioxide, and humidity.

Immobilization mode shares similar mechanisms as a release mode system. However, it can

only restrain microorganisms when food is in direct contact with the packaging material. The

antimicrobial agent is chemically bound to the packaging material, thus it cannot migrate into

food or the headspace inside a package system (Han, 2000). Hence, it is more effective when

incorporated into liquid foods or a non-headspace packaging system.

Like most active packaging systems, antimicrobial can be developed either in single or

multi-layer films, consisting of an outer, barrier, matrix, and control layer (Ozdemir & Floros,

2004). Fig. 4 shown below illustrates a typical antimicrobial composition and its migration

profiles. The purpose of an outer and barrier layer is to prevent physical damages like humidity

accumulation and excessive gases (e.g. oxygen or carbon dioxide). Antimicrobial agent is

generally incorporated into the matrix layer by means of impregnation or immobilization. The

control layer is implemented to sustain a specific releasing rate of agent, from the matrix layer

into the packaged food or headspace (Han, 2000).

25

Outer Layer

Inhibit humidity or use for printing and/or labeling

Barrier Layer

Inhibit excess gases such as oxygen, carbon dioxide…etc.

Matrix Layer Impregnated or immobilization layers

Control Layer

Control releasing system

Food

Figure 4. Complex layers of antimicrobial packaging (Ozdemir & Floros, 2004)

As shown below in Fig. 5, antimicrobial systems can be categorized into two distinct

groups: with or without a headspace feature. A non-headspace package system can be defined as

a direct contact application of food and packaging material. Various food products or forms can

be incorporated within this system such as solid, viscosity or liquid (La Storia et al., 2008). The

migration process of antimicrobial agent from package into food is due to diffusion or interaction

between the packaging material and food. A headspace package system is defined as an air gap

between food and packaging material. Most headspace systems are designed to sustain a

viscosity or liquid form in which the food can be packed in either flexible package or carton.

The evaporation or equilibrated distribution of the applied agent within the headspace, packaging

material, and/or food is an essential component to the success of this packaging system.

Oftentimes, volatile an antimicrobial agent is used because it can easily migrate through the

headspace or air gaps between the package material and the food.

Package

Diffusion

Immobilization

Food

Package

Diffusion

Headspace

Evaporation

Food

Equilibrium

Sorption

Figure 5. Schematic of different antimicrobial features (Han, 2000)

26

Critical Factors to Consider

Several conditions can significantly influence the efficiency of any antimicrobial system

(Cooksey, 2005). The incorporation methods, permeation, and evaporation of antimicrobial

agent may be some of the most influential factors. However, other factors should be considered

prior to designing (Han, 2000). These additional factors include the following: specific

inhibition activity, controlled release mechanisms, chemical compatibility between agents and

packaging materials, solubility concentration levels, and regulatory status of the antimicrobial

agent.

Selecting the correct antimicrobial agent is a critical factor. There is not a specific agent

that can effectively inhibit all spoilage and pathogenic microorganisms. Each agent has a

different essence and/or inhibition activity which can vary against different microorganisms (La

Storia et al., 2008). Nevertheless, selecting an effective agent can be a straightforward process

because it is based on the targeted microorganisms. Most spoilage and pathogenic

microorganisms’ growths can be anticipated when analyzing the pH, water activity, composition,

and storage temperature of the packaged food (Han, 2000). The purpose of any antimicrobial

agent is to eliminate or hinder any desirable environmental conditions that spoilage and

pathogenic microorganisms might thrive in.

Most antimicrobial packaging systems generally require a controlled release system. The

probability for contamination is high if the migration rate of release agent is not at a specific

ratio with its targeted microorganisms (Cooksey, 2005). Depletion in antimicrobial activities

may occur if the migration rate surpasses its targeted microorganisms; a packaging system can

only sustain a certain volume of released antimicrobial agent. As a result, packaged foods may

easily become contaminated due to an insufficient concentration level of antimicrobial activities.

27

However, when the released rate of antimicrobial agent is slower than the growth rate of its

targeted microorganism, deterioration in food can also occur (Han, 2000).

The chemical compatibility between antimicrobial agent and packaging material

(polymer) is also a critical factor (Han, 2000). All antimicrobial agents can be classified either

as soluble or non-soluble in water. When a water-soluble agent is impregnated into polymer to

produce antimicrobial films, quality is a concern due to the hydrophilic and hydrophobic nature

of the agent and plastic. As a result, hole and powder blooming may form within the packaging

film, thus significantly affecting its mechanical and/or transparency properties (Cooksey,

Gremmer, & Grower, 2000 as cited in Han, 2000). One possible solution is to reduce the

concentration of applied antimicrobial agent. Extensive polymer morphological studies can be

conducted to obtain a precise ratio of agent-to-polymer mixtures.

The solubility between antimicrobial agent and food is another influential component. In

most situations, the degree of solubility between agent and food can change the package’s

activities. For instance, a low soluble agent in food will have a monolithic migration rate;

however, at a higher solubility level, the migration rate will have an unconstrained free diffusion

movement (Han, 2000). The concentration of applied agent is higher during a diffusion

migration; however, depletion can occur during the migration process or when in contact with

food. In a monolithic system the concentration is much lower; however, the antimicrobial

activity maintains at a constant rate until completely depleted within the packaging system

and/or food. Each migration profile has its pros and cons, but understanding their attributes and

how this may help to develop a control system may enhance the effectiveness of any

antimicrobial system.

Although the preceding conditions are critical, regulation is considered the most

significant factor in creating any packaging systems (Cooksey, 2005). Most, if not all food

28

preserving agents have to be approved by various regulatory agencies (e.g. Food and Drug

Administration) before any commercial applications can be applied. A highly concentrated and

volatile agent may change the packaged food’s integrities, thus stricter legislations have been

passed (Miltz, Wesslinget & Onyeneho as citied in Siro 2006).

Gas Chromatography Mass Spectrometry

Gas chromatography-mass spectrometry (GC-MS) is an analytical technique that

combines aspects of gas chromatography (GC) and mass spectrometry (MS). According to

(Santos and Galceran, 2002 as cited in as cited in Kin, 2006) GC-MS has shown be to a leading

confirmation method for identification and quantitation in chemical analysis. It was one of the

first known techniques that can identify and separate chemical compounds in a sample. A

typical GC-MS system, as shown below in Fig. 6, is composed of a carrier gas, flow controller,

injector port, column oven, detector, and a chromatogram. MS is one of the most commonly

used detectors in a GC system (Raymond, 1995 as cited in Kin, 2006).

Figure 6. Schematic of a GC-MS system

In GC analysis, a known volume of a chemical substance can be injected either

automatically or manually into an injector port. The chemical substance is then swept along a

29

column by a carrier gas (e.g. helium). Inside the column, the injected substance’s compounds

will be separated from each other based on their strengths of absorption (Kin, 2006); hence,

some compounds will take longer to than others to exit. The chemical characteristics of the

substance have a direct relationship to its progression along the column. The concept of a race

around a track can be used as an analogy to explain this process; everybody starts at the same

time and location and become separated from one another at the end with a different retention

time. As chemical compounds exit the column, a detector (e.g. mass spectrometry) is used to

monitor its fragmentation by ionization. Retention time and/or fragmentation pattern can be used

to identify the injected chemical’s unique characteristic.

As stated above, direct coupling of GC to MS has shown to be one of the most conclusive

techniques for identifying chemical characteristics. However, during its developmental phase,

the MS detector was shown to be incompatible with a GC system that consisted of a packed

column which was commonly used at the time (Kin, 2006). Packed columns required a higher

carrier gas flow to be efficient, thus inhibiting the application for MS. In the early 1980s, direct

coupling of GC to MS became more efficient due to new developments such as jet separation,

capillary columns, and quadrupole mass analyzer (Verzera et al., 2004; Gonzalez et al., 2005 as

cited in Kin, 2006).

Solid Phase Microextraction

According to (Kataoka et al., 2000 as cited in Kin, 2006), the application and efficiency

of GC-MS analysis has significantly increased since the early 1990s due to the introduction of

solid-phase microextraction (SPME). SPME is a simple and inexpensive preparation technique

that uses a fused-silica fiber to directly extract reagents from a sample. This technique has been

shown to be more applicable in reducing preparation time, limiting solvent usage, and enhancing

the detection limits (Pawliszyn, 1997 as cited in Kin, 2006). It has shown to be a feasible

30

extraction technique for both GC and High Performance Liquid Chromatography (HPLC)

systems. SPME can be implemented to various chemical compounds, especially in the

extraction of volatile organic compounds (Eisert and Levsen, 1996; Pawliszyn, 1997; Prosen and

Zupancic-Kralj, 1999 as cited in Kin 2006).

SPME has the appearance of a modified syringe consisting of a fiber holder and a fiber

assembly as shown below in Fig. 7. The fiber assembly contains a 1-2 cm retractable fused-

silica optical fiber that is coated with a thin polymer film. As stated above, SPME can be used in

two applications: GC, sampling gases (headspace), and HPLC, sampling solution (direct

immersion). In the case of GC analysis, the SPME needle (protective casing) is inserted through

a septum and into the headspace where it exposes the fused-silica optical fiber to an environment

covered with chemical reagent by means of retraction (Kin, 2006). The polymer coated fiber

absorbs the reagent vapor similar to a sponge absorbing water. Upon completion of this

absorption process, the fiber is retracted back into its protected casing (needle) and then

transferred into the GC instrument.

Figure 7. Design and enlarged view of commercial SPME device

31

Chapter III: Methodology

Defined Molar Ratio

AITC has been shown to be an effective antimicrobial agent; however, its efficiency can

be limited due to its natural volatile component. Encapsulating AITC into β-CD may be an

effective solution. In order to begin this process, the molar ratio between AITC and β-CD

compounds must be determined. Molar ratio is defined as the coefficients in a balanced

chemical equation that can be used to determine the relative number of molecules, formula units,

or moles of any compounds involved in a chemical reaction.

In order to obtain the ideal complexation of AITC and β-CD compounds, several molar

ratios of AITC-β-CD complex were prepared at (0.5:1.0, 1.0:1.0, 2.0:1.0, 3.0:1.0, and 1.0:2.0),

respectively. The following equation was used to determine the best molar ratio of a

concentrated AITC-β-CD complex. Based on Eq. 1 shown below, 0.431 mL of AITC is the

correct 1:1 molar ratio for 5g of β-CD compound. Known variables: 5g of β-CD will be used

which has a molecular weight of 1135.12 g/mole, various quantity of AITC will be used with a

molecular weight of 99.15 g/mole, and its density is 1.013 g/cm3. Unknown variables: X

represents number of moles and D, M, and V represent density, mass, and volume, respectively.

0.431 mL of AITC = 5g of β-CD

Equation 1. Define molar ratios between AITC and β-CD

32

Encapsulation of AITC in β-CD

AITC-β-CD complex were produced by encapsulating AITC into β-CD compound inside

a fume hood chamber as shown below in Fig. 8A. Four different molar ratios of AITC-β-CD

complex were prepared at (0.5:1.0, 1.0:1.0, 2.0:1.0, and 3.0:1.0), respectively. Each of the four

molar ratios was prepared three different times in an effort to obtain reproducible results.

Figure 8. Experimental setup for encapsulation of AITC in β-CD

For the initial process, inside an Erlenmeyer flask, β-CD compound was suspended in

100 mL distilled water by stirring at an ambient temperature for ten seconds. AITC compound

was then added to the aqueous β-CD sample while continuous stirring. A plunger containing a

thermometer was introduced, and continuous stirring was carried on for three hours at 65 °C as

shown above in Fig. 8B. Temperature was examined every 30 minutes to ensure a consistent

encapsulation process for all AITC-β-CD complexes.

Encapsulated AITC-β-CD complex was then extracted and transferred into a centrifuge.

It was spun for five minutes at 2500 rpm to remove excess water. The complex sample was then

placed in a seal tight beaker and stored in a vacuum refrigerator (<25 mmHg pressure) for

approximately 24 hours. To obtain the effect of a seal tight beaker, aluminum foil and masking

tape was used, limiting the possibility of escaping vapor (AITC reagent). The AITC-β-CD

A B

33

complex was extracted and placed in a convection oven at 60°C the next day. This process

ensured the removal of any residual AITC that may have been absorbed on the β-CD surface.

SPME Extraction of AITC

Extraction of AITC reagent from β-CD compound was conducted using a solid phase

microextraction (SPME). 25mL of distilled water was placed in a 425mL Mason jar (Buffalo,

NY, USA). A tinfoil baking cup, containing 1g of finely ground AITC-β-CD complex, was

suspended on a tripod fixture inside the jar as shown below in Fig. 9A. The jar was sealed, and

testing was halted for 15 minutes to ensure a full chemical reaction. A Sigma-Aldrich 65 micron

DVB/CAR/PDMS Supellco-SPME fiber (St. Louis, MO, USA) was inserted into the jar’s

headspace for five minutes as shown in Fig. 9B. The SPME used for this procedure consists of a

1cm length fused-silica fiber bonded with a stainless steel plunger and a holder. The fused-silica

fiber was then exposed into the headspace to absorb the release of AITC reagent from β-CD.

This process was repeated three times for each of the four prepared molar ratio AITC-β-CD

complexes.

Figure 9. Schematic of sample preparation with SPME

External Calibration with GC-MS

External calibration was constructed using samples peak from four different

concentrations of AITC-β-CD complex. The complex solutions (input) over the concentration

A B

34

range of unknown (output) were prepared by serial dilution of AITC-β-CD complex using

ethanol to obtain liquid standard solution. The detector response linearity was obtained in

triplicates by GC-MS at each concentration level.

GC-MS analysis of Released AITC from β-CD

The GC-MS process for analyzing the release of AITC reagent from β-CD was conducted

using an Agilent Technologies system 6890N gas chromatograph (Palo Alto, CA, USA) which

was equipped by a XL inert (5973B) mass spectrometer as shown below in Fig. 10. Data was

collected and processed by a MSD computer software program (Chem-Station). SPME fiber

containing an unknown quantity of AITC reagent was inserted into the injector port; it was held

there for two minutes. The AITC reagent was separated using a splitless injection mode on a

HP-5 capillary column (12 cm in length, 23 cm inner diameter, and 2.3 µm film thicknesses). In

this analysis, helium was used as the carrier gas and was held at a constant flow rate of 20psi and

2.5mL/min. The initial oven temperature was 40°C; it then increased at a rate of 10°C/min to a

final temperature of 150°C. The oven temperature was maintained at150°C for eight minutes

until completion. This process was repeated three times for each of the four prepared molar ratio

AITC-β-CD complexes.

Figure 10. Example of a GC-MS Instrument

35

Impregnation of AITC-β-CD with LDPE

Impregnation of 2:1 AITC-β-CD complex (ideal ratio) in LDPE was conducted by means

of compression. A 40 ton TMP compression molding machine (Menomonie, WI, USA)

equipped with a stainless steel platen (14in x 14in) was used. Seven different concentrations of

antimicrobial mixtures were achieved using 0%, 1%, 2%, 4%, 6%, 8%, and 10% AITC-β-CD

complex to 1g of Industrial Arts Supplier LDPE powder resin (Minneapolis, MN, USA). This

mixture was blended in a glass grinder for one minute. Antimicrobial films were achieved by

placing the complex mixtures onto a Teflon sheet and pressed at 250°F for three minutes with a

purging time of one minute as shown in Fig. 11. Each concentration was recreated three times

and used to achieve antimicrobial films.

Figure 11. Schematic of an antimicrobial film by mean of compression

GC-MS Analysis of Released AITC from Films

Analysis of released AITC reagent from antimicrobial film was carried out using similar

procedures as described in the previous sections SPME Extraction of AITC and GC-MS analysis

of Released AITC from β-CD. Antimicrobial films at various concentrations were cut into 3cm x

3cm segments at a thickness of 0.5mm. Each segment was submerged in 25mL distilled water

inside a sealed Mason jar for 15 minutes prior to testing. The release of AITC reagent from

antimicrobial film was prepared by using a SPME as the main extracting technique. As stated

36

above, conditions and procedures for SPME extraction remained the same as described in section

SPME Extraction of AITC. The release of AITC reagent from various concentrated antimicrobial

films was analyzed by GC-MS under the conditions and procedures as described in section GC-

MS analysis of Released AITC from β-CD.

Determination of AITC in the Microcapsules

Microcapsules of AITC were conducted by introducing 1mL of distilled water and 50mL

of hexane into various samples of AITC-β-CD complex (30-50mg). AITC was extracted by

hexane from the microcapsule for ten minutes, twice in the ultrasonic condition. The supernatant

containing AITC was obtained by centrifugation at 2500 rpm for ten minutes. The content of

AITC in hexane was determined using ultraviolet spectrophotometry at 249 nm (its maximum

absorbance).

Optical Property

The evaluation of optical property was carried out using a Nikon Coolpix L810 16.1

megapixel digital camera with 26X optical zoom. Antimicrobial films at various concentrations

were placed on a black surface to ensured adequate lighting. Each image (0.8in x 0.8in) was

capture three times at a distance of five inches.

Mechanical Properties

Tensile strength (TS), elongation at break (EB), and toughness were measured using a

LRX tensile tester (NEXGEN, Lloyd instruments) according to ASTM D 882 as shown below in

Fig. 12. Films were cut into strips (1in width) using a Precision Sample Cutter (Thawing Albert

Instrument Co.,Philadelphia, PA) and conditioned according to ASTM D 618at 23 2 °C/50% 5%

RH. The thickness of each film was determined at three locations with a TMI 549M Micromiter

(Testing Mechanes Inc., Amityville, New York) according to ASTM D 374-99.

37

Figure 12. Sample Film Preparation for Mechanical Properties

Measurement of OTR and WVTR

The oxygen transmission rate (OTR) analysis was conducted using an Illinois Oxtran

8001 unit (Illinois instrument Inc., IL) as shown below in Fig.13A according to ASTM D 3985,

at 0% RH and 23°C. Water vapor transmission rate (WVTR) was determined using a Mocon

Permatran 3/31 (MOCON, Minneapolis, MN) as shown below in Fig. 13B, according to ASTM

F 1249, at 100% RH and 38°C. For both OTR and WVTR analysis, various concentrated

antimicrobial films were cut into segment with a precise area of 50 cm2. Testing of antimicrobial

films was continued until their steady state was reached. Each concentration of films was

analyzed three different times.

Figure 13. Instruments used to analysis OTR and WVTR in antimicrobial film

A B

38

Chapter IV: Results

This chapter examines the process of encapsulating AITC in β-CD and impregnation of

AITC-β-CD complex into LDPE by means of compression molding technique. The release of

AITC from β-CD at various concentrated levels of antimicrobial film was analyzed. In addition,

the mechanical and optical properties was analyzed and compared with control film.

Effect of Encapsulated of AITC in β-CD

Prior to the creation of antimicrobial films by means of compression, encapsulation of

AITC in β-CD must be achieved. GC-MS analysis technique was used to determine the best

complex ratio. Encapsulation was needed to control the volatile tendency of AITC. As

discussed in Chapter I section Allyl-Isothiocyanate, AITC has a highly concentrated flavor and

may have a negative impact on food if not properly controlled. Furthermore, depletion can

easily occur during its migration process and/or when in contact with foods. β-CD is the

preferred method to control AITC’s volatile nature due to β-CD’s ability to form an inclusive

complex with AITC, controlling the release rate of AITC reagent. Fig. 14 illustrates the

encapsulation process of AITC in β-CD, forming a complex AITC-β-CD compound. Pure AITC

sample was prepared using similar procedures as described in section SPME Extraction of AITC

and GC-MS analysis of Released AITC from β-CD to determine its degradation rate if not

encapsulated in β-CD. Initially, the AITC concentration in the jar was 11µL/L as shown below

in Fig. 15. However, gradual decline in concentration occurred over time. On day 12, the AITC

concentration was at 2µL/L; additional details of the degradation in pure AITC are presented in

Appendix A.

39

Figure 14. Schematic of encapsulated AITC in β-CD

Figure 15. Degradation of AITC without β-CD encapsulation

Molar ratio was used to define the volumes at which AITC and β-CD reached

equilibrium. It was concluded that the desired volume to balance 5g β-CD was 0.431mL AITC.

Four different molar ratios were prepared and analyzed for optimal desired effect; these molar

ratios were 0.5:1.0, 1.0:1.0, 2.0:1.0, 3.0:1.0, and 1.0:2.0. According to Siro's method (2006), the

encapsulation process was conducted in distilled water at 65°C for a minimum of one hour. For

research purposes, this process was conducted for three hours due to different components and

also to ensure full encapsulation. Efficiency percentage of the four prepared AITC-β-CD

0

2

4

6

8

10

12

-2 0 2 4 6 8 10 12 14

AIT

C C

once

ntra

tion

(µL

/L) i

n gl

ass j

ar

Storage Time (Day)

β-CD AITC AITC-β-CD

40

complexes was determined using GC-MS analysis and a regression line. Collected data from the

GC-MS analysis for released concentrations of AITC was inserted in the regression line, and the

efficient rate was determined based on the external calibration graph as shown below in Fig. 16.

Based on results shown in Table 2, the molar ratio of AITC-β-CD complex of 2:1 was the most

efficient, sustaining 91.10% of AITC.

Figure 16. External calibration graph of AITC-β-CD complex

Table 2. Effect of molar ratio of AITC- β-CD on encapsulation efficiency % *Encapsulation efficiency of β-CD (%) = (Recovered AITC/Initially input AITC) (100)

Molar Ratio of AITC/β-CD Efficiency (%)*

0.5 63.84

1.0 76.16

2.0 91.10

3.0 92.59

y = 2E+08x + 1E+07 R² = 0.9988

0

500

1,000

1,500

2,000

2,500

0.00 2.50 5.00 7.50 10.00

Are

a o

f R

esp

on

se

Mill

ion

s

AITC Conentration (µL/L) Glass Jar

41

Development of AITC-β-CD Complex Impregnated Film

Prior to impregnation of 2:1 molar ratio of AITC-β-CD complex in LDPE, seven

antimicrobial film mixtures at different concentrations (0%, 1%, 2%, 4%, 6%, 8%, and 10%)

were prepared. These concentrations were grinded with 1g LDPE to ensure even distribution of

AITC-β-CD complex in LDPE prior to compression. 250°F was the ideal temperature for

compression because it met the melting temperature for LDPE without significantly damaging

AITC-β-CD complex. Upon completion of compression, the antimicrobial films at different

concentrations were analyzed for optical and mechanical properties and their oxygen and water

vapor transmission rate.

Mechanical Properties of Compressed Film

Antimicrobial films at different concentrations were analyzed for their strength and

durability by measuring their tensile and elongation properties. AITC-β-CD complex’s effect on

LDPE’s tensile and elongation properties were studied by measuring and comparing the peak

load of films containing AITC-β-CD complex versus the peak load of the control film (0%); all

of which were conducted under the same conditions. Table 3 shown below indicates that the

addition of AITC-β-CD complex did not significantly influence the tensile strength of the film

until concentration level was at least 4%; however, elongation was affected as low as 1%. At 2%

there was not a significant change in tensile strength when compared to the control (0%); hence,

this may be the ideal concentration level of AITC-β-CD complex to LDPE.

42

Table 3. Mechanical strength of the antimicrobial film with different AITC-β-CD concentration ⃰ Means with different letter in each column indicate significantly different (p<0.05)

% of AITC-β-CD Complex TS (Mpa) Elongation at break (in) 0 7.6219a 13.2567a 1 7.6234a 2.5654b 2 7.5654a 2.0641b 4 6.6223b 1.4039c 6 6.5640b 1.1343d 8 6.4538c 0.6931e 10 5.2010d 0.3880f

Optical Property of Compressed Film

Optical property of films is critical for marketability purposes. Consumers need to be

able to see the product through the packaging. Hence, a clean, clear packaging film is desirable.

To evaluate the optical property of the compressed antimicrobial film and control film, images

were taken against a black setting at a set distance and observations were made. Fig. 17 shown

below indicates that at 4% of AITC-β-CD, significant change in optical property occurred. As

described in Chapter II section Critical Factors to Consider, compatibility between antimicrobial

agent, packaging material and food is a concern when a high concentration of agent is applied.

For example, at concentration levels of 4% and higher, AITC-β-CD complex was unevenly

distributed throughout the LDPE, thus giving a grainy appearance (holes and powders). When

comparing the control film to 2% and lower concentrated antimicrobial films, there was not a

significant change in the optical property; hence 2% may be the ideal concentration level to

achieve the desirable optical property.

@ 6% of AITC-β-CD @ 8% of AITC-β-CD

43

Figure 17. Distribution of AITC-β-CD complex in film (0-10%) * Capture image is (0.8in x 0.8in)

Control Film (0% of AITC-β-CD) @ 1% of AITC-β-CD

@ 2% of AITC-β-CD @ 4% of AITC-β-CD

@ 6% of AITC-β-CD @ 8% of AITC-β-CD

@ 10% of AITC-β-CD

44

Measurement of Oxygen and Water Vapor Transmission Rate

The measurements of oxygen and water vapor rates were conducted to determine the

amount of oxygen and water vapor permissible through both antimicrobial and control films. As

stated in Chapter II section Active Packaging, this is critical because a high amount of oxygen

and water vapor within the packaging system permits for an environment where spoilage

microorganisms can grow and thrive. Hence, it is ideal to achieve the lowest oxygen and water

vapor transmission rate possible. Table 4 illustrates that there was a significant change in OTR

and WVTR when comparing antimicrobial film to control film. In control film, there was an

oxygen transmission rate of 863.2±42.99cc/m2 per day. However, at 2% concentration,

antimicrobial film showed an OTR of 1330.0±52.46cc/m2 per day; this is a substantial difference

of approximately 466.8±9.47cc/m2 per day. In addition, there was also a significant change

when comparing the WVTR of the control film to the antimicrobial film at 2% concentration.

The OTR is reflective of the thickness of the film and the compatibility of AITC-β-CD complex

to LDPE. The results in Table 4 conclude that the antimicrobial films may be too thin and

AITC-β-CD complex is not sufficiently compatible enough with LDPE to form a concrete barrier

to efficiently prevent the permeation of oxygen and water vapors. With regards to OTR and

WVTR, these antimicrobial films are ineffective and new components must be explored.

Table 4. Barrier properties of the various concentrated compressed films *Means with different letter in each column indicate significantly different (p<0.05)

% of AITC-β-CD OTR (cc/m2 per day) WVTR (g/m2 per day)

0 863.2±42.99a 2.02±0.21a

2 1330.3±52.46b 2.68±0.41b

4 1007.8±11.89c 4.43±1.67c

6 1269.5±28.12d 4.68±0.002d

8 2869.3±53.49e 7.11±13.36e

45

Release of AITC reagent from Antimicrobial Films

Various techniques were carried out to analyze the release of AITC agents from

antimicrobial films. The release rates of the six different concentrated antimicrobial films were

compared in Fig. 18; additional details are presented in Appendix B and C. It should be noted

that the control film (0%) was not analyzed because it does not contain any AITC agent. As

shown, the difference of migration rate between film 2% and film 1% was significantly less than

the difference in migration rate between film 10% and film 1%. All concentrated antimicrobial

films reached their equilibrium migration rates from day 2 to day 8. Depletion of AITC agent

occurred from day 10 to day 18. It should be noted that the depletion rate in film 1% was

significantly higher when compared to other antimicrobial films. From day 18 onward, all

concentrated antimicrobial films once again reached their equilibrium migration rates, however

at a significantly lower concentration. These results are significant because they mirror the

results of similar studies by (Heirlings et al. 2004 as cited in Siro et al., 2006). The AITC

concentration release at 2% was sustainable and its depletion rate was tolerable, hence 2% may

be the ideal concentration level to create an efficient antimicrobial film, hindering the growth of

microorganisms.

46

Figure 18-AITC release from AITC-β-CD impregnated LDPE film for 30 days (@23°C)

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

-5 0 5 10 15 20 25 30 35

AIT

C c

once

ntra

tion

(µL

\L) i

n gl

ass j

ar

Storage time (day)

Film 1%Film 2%Film 4%Film 6%Film 8%Film 10%

47

Chapter V: Conclusions

Conclusions

Complexation of AITC by β-CD was determined to be a sufficient technique in

controlling and sustaining the release of AITC reagent. This is evident in the comparison of Fig.

15 and Fig. 18. Without encapsulating in β-CD, AITC depleted at a high rate, reaching full

depletion after 12 days. However, with complexation, the depletion rate of AITC was greatly

mitigated, and full depletion did not occur till well after 30 days. The results from Fig. 15 and

Fig. 18 parallel the experimental results of (Siro et al., 2006). Since the complexation of AITC

by β-CD mitigated its depletion rate, sustaining itself for a long duration of time, encapsulating

AITC into β-CD may be a feasible antimicrobial application.

Antimicrobial film had some significant changes with regards to its mechanical (tensile

strength and elongation) and optical properties when compared to the control film. Change in

tensile strength was not significant until AITC-β-CD concentration levels reached 4%. However,

significant change in elongation was observed even at low concentration levels. With regards to

optical properties, significant change did not occur till after 2%. With these results, it can be

concluded that antimicrobial film with a concentration level of 2% AITC-β-CD provides the

most desirable mechanical and optical properties when compared to films of other concentration

levels.

Impregnation of AITC-β-CD complex in LDPE led to significant changes in the barrier

properties of the antimicrobial film when compared to the control film. This is critical because a

poor barrier that allows the permeation of oxygen and water vapor will permit an environment

where spoilage microorganism can grow and thrive regardless of the amount of AITC reagents

released into that environment. As stated above in the results and literature review, this may be

due to two things (1) the thinness of the film and (2) the lack of sufficient compatibility between

48

the AITC and β-CD. This information has led to the conclusion that an alternative component or

method must be explored.

Recommendations

An area of recommendation for improvement is the method of film development. The

current method, usage of the compression machine where distribution of AITC-β-CD complex

within LDPE was accomplished by finely grinding and mixing the AITC-β-CD complex and

LDPE by hand, is inadequate and does not evenly distribute the AITC-β-CD complex throughout

the LDPE. A solution to this is the usage of a single-screw extruder with a normal gravity feed

hopper. This commonly used machine will efficiently blend the AITC-β-CD complex in LDPE.

Another critical area that needs to be addressed is the compatibility between AITC-β-CD

with LDPE. Studies on a different control agent must be explored. The reasoning is that as a

control agent, β-CD has the most significant influence on the structural composition of the

antimicrobial film. This report shows that for the purposes of antimicrobial food packaging, β-

CD is an insufficient control agent due to its weak barrier property and weak elongation

property. DM-β-CD, TA-β-CD, and DMA-β-CD should be explored to see if they’re more

compatible with LDPE.

Future Studies

One area of interest to be addressed is the effectiveness of AITC-β-CD antimicrobial film

as a food preserver. The goal of this study will be to see whether this application will be

successful in extending the shelf of food products and if so, for how long. In addition to shelf

life, two other areas should be addressed: how much nutritional value is lost and when such an

amount is lost that insufficient nutritional value remains.

49

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52

Appendix A: Collected Data for Degradation of AITC

Day Jar # Quantity RT (min) Corrected Areas (in2) AITC Concentration (µL/L)

1

1 AITC (5.0ul) 4.033, 4.390 1,187,539,196 11.00 1 AITC (5.0ul) 4.034, 4.391 1,097,172,658 10.16 1 AITC (5.0ul) 4.033, 4.392 1,040,186,557 9.63 2 AITC (5.0ul) 4.032, 4.081, 4.439 1,119,027,382 10.36 2 AITC (5.0ul) 4.032, 4.448, 4.509 1,279,042,764 11.85 2 AITC (5.0ul) 4.031, 4.2058, 4.458 1,270,341,964 11.77 3 AITC (5.0ul) 4.031, 4.483 1,276,141,445 11.82 3 AITC (5.0ul) 4.035, 4.481 1,121,685,032 10.39 3 AITC (5.0ul) 4.032, 4.482 1,205,577,094 11.17 4 AITC (5.0ul) 4.032, 4.418 1,113,426,607 10.31 4 AITC (5.0ul) 4.031, 4.425 1,009,027,367 9.34 4 AITC (5.0ul) 4.032, 4.390 1,140,423,294 10.56

2

1 AITC (5.0ul) 4.038, 4.357 937,583,686 8.68 1 AITC (5.0ul) 4.035, 4.376 915,391,634 8.47 1 AITC (5.0ul) 4.035, 4.353 919,563,526 8.51 2 AITC (5.0ul) 4.033, 4.440 1,076,627,379 9.97 2 AITC (5.0ul) 4.033, 4.440 1,012,552,520 9.37 2 AITC (5.0ul) 4.032, 4.111, 4.440 1,057,935,100 9.79 3 AITC (5.0ul) 4.033, 4.425 1,001,284,412 9.27 3 AITC (5.0ul) 4.032, 4.398 957,608,098 8.86 3 AITC (5.0ul) 4.031, 4.304, 4.398 943,605,419 8.73 4 AITC (5.0ul) 4.036, 4.376 916,758,262 8.48 4 AITC (5.0ul) 4.037, 4.356 930,997,898 8.62 4 AITC (5.0ul) 4.034, 4.378 1,032,313,849 9.56

4

1 AITC (5.0ul) 4.033, 4.348 716,475,218 6.62 1 AITC (5.0ul) 4.036, 4.349 716,524,693 6.62 1 AITC (5.0ul) 4.035, 4.345 729,883,546 6.75 2 AITC (5.0ul) 4.032, 4.402 755,695,970 6.99 2 AITC (5.0ul) 4.033, 4.408 794,826,408 7.35 2 AITC (5.0ul) 4.034, 4.403 790,945,172 7.31 3 AITC (5.0ul) 4.038, 4.375 782,967,403 7.24 3 AITC (5.0ul) 4.308, 4.376 758,008,701 7.01 3 AITC (5.0ul) 4.034, 4.378 793,224,115 7.34 4 AITC (5.0ul) 4.034, 4.348 798,776,812 7.39 4 AITC (5.0ul) 4.033, 4.100, 4.349 767,948,453 7.10 4 AITC (5.0ul) 4.034, 4.105, 4.345 777,552,568 7.19

53

Appendix A: Continued

Day Jar # Quantity RT (min) Corrected Areas (in2) AITC Concentration (µL/l)

6

1 AITC (5.0ul) 4.030, 4.348 595,170,123 5.50 1 AITC (5.0ul) 4.032, 4.188, 4.341 629,263,188 5.81 1 AITC (5.0ul) 4.030, 4.205, 4.358 534,069,185 4.93 2 AITC (5.0ul) 4.036, 4.391 603,583,462 5.57 2 AITC (5.0ul) 4.031, 4.335 591,505,214 5.46 2 AITC (5.0ul) 4.034, 4.387 587,358,792 5.42 3 AITC (5.0ul) 4.033, 4.196, 4.342 515,679,289 4.76 3 AITC (5.0ul) 4.032, 4.349 550,102,016 5.08 3 AITC (5.0ul) 4.035, 4.194, 4.345 558,969,873 5.16 4 AITC (5.0ul) 4.032, 4.190, 4.341 591,233,732 5.46 4 AITC (5.0ul) 4.033, 4.188, 4.341 616,776,984 5.70 4 AITC (5.0ul) 4.034, 4.194, 4.345 588,098,983 5.43

8

1 AITC (5.0ul) 4.035, 4.114, 4.304 470,789,300 4.34 1 AITC (5.0ul) 4.032, 4.148, 4.322 436,465,513 4.02 1 AITC (5.0ul) 4.035, 4.118, 4.303 461,362,368 4.25 2 AITC (5.0ul) 4.033, 4.349 456,324,036 4.21 2 AITC (5.0ul) 4.032, 4.356 535,600,033 4.94 2 AITC (5.0ul) 4.035, 4.353 535,564,090 4.94 3 AITC (5.0ul) 4.032, 4.167, 4.334 434,593,298 4.00 3 AITC (5.0ul) 4.033, 4.161, 4.327 499,608,146 4.61 3 AITC (5.0ul) 4.034, 4.169, 4.336 425,959,283 3.92 4 AITC (5.0ul) 4.032, 4.126, 4.307 489,731,775 4.52 4 AITC (5.0ul) 4.033, 4.140, 4.315 424,231,711 3.91 4 AITC (5.0ul) 4.035, 4.127, 4.311 461,653,364 4.26

10

1 AITC (5.0ul) 4.030, 4.116, 4.308 350,387,692 3.22 1 AITC (5.0ul) 4.032, 4.126, 4.302 339,031,794 3.12 1 AITC (5.0ul) 4.033, 4.130, 4.320 355,718,407 3.27 2 AITC (5.0ul) 4.032, 4.153, 4.325 391,383,643 3.60 2 AITC (5.0ul) 4.032, 4.159, 4.324 360,293,909 3.31 2 AITC (5.0ul) 4.035, 4.152, 4.328 366,019,181 3.37 3 AITC (5.0ul) 4.032, 4.126, 4.307 374,981,106 3.45 3 AITC (5.0ul) 4.034, 4.160, 4.328 344,220,641 3.17 3 AITC (5.0ul) 4.035, 4.127, 4.311 393,907,752 3.63 4 AITC (5.0ul) 4.033, 4.128, 4.309 350,604,718 3.22 4 AITC (5.0ul) 4.032, 4.128, 4.305 342,822,351 3.15 4 AITC (5.0ul) 4.033, 4.126, 4.313 357,687,988 3.29

54

Appendix A: Continued

Day Jar # Quantity RT (min) Corrected Areas (in2) AITC Concentration (µL/L)

12

1 AITC (5.0ul) 4.071, 4.278 227,332,496 2.08 1 AITC (5.0ul) 4.068, 4.277 229,470,731 2.10 1 AITC (5.0ul) 4.076, 4.286 226,673,680 2.07 2 AITC (5.0ul) 4.077, 4.285 228,876,853 2.09 2 AITC (5.0ul) 4.049, 4.266 220,405,080 2.02 2 AITC (5.0ul) 4.051, 4.261 238,150,698 2.18 3 AITC (5.0ul) 4.048, 4.265 219,508,811 2.01 3 AITC (5.0ul) 4.051, 4.269 211,118,784 1.93 3 AITC (5.0ul) 4.050, 4.266 228,113,585 2.09 4 AITC (5.0ul) 4.068, 4.277 202,072,806 1.84 4 AITC (5.0ul) 4.066, 4.277 236,282,973 2.16 4 AITC (5.0ul) 4.065, 4.282 211,547,343 1.93

55

Appendix B: Collected Data for Antimicrobial Films: Encapsulation of AITC in β-CD

Day Jar # Thickness (in.) Weight (g) AITC+CD Corrected Areas (in2) Concentration (µL/L)

2

1 0.0070 4.698 1% 168,279,442 1.536 1 0.0070 4.698 1% 165,273,067 1.507 1 0.0070 4.698 1% 163,495,353 1.489 2 0.0075 5.025 1% 167,271,867 1.526 2 0.0075 5.025 1% 164,391,219 1.498 2 0.0075 5.025 1% 168,238,625 1.536 3 0.0085 4.819 1% 166,175,059 1.516 3 0.0085 4.819 1% 162,079,709 1.476 3 0.0085 4.819 1% 166,578,047 1.520 1 0.0065 4.400 2% 221,858,032 2.059 1 0.0065 4.400 2% 225,433,552 2.094 1 0.0065 4.400 2% 224,936,784 2.089 2 0.0075 4.860 2% 223,681,270 2.077 2 0.0075 4.860 2% 234,166,185 2.179 2 0.0075 4.860 2% 224,965,982 2.090 3 0.0070 4.828 2% 220,798,562 2.049 3 0.0070 4.828 2% 227,740,055 2.117 3 0.0070 4.828 2% 223,408,166 2.074 1 0.0075 5.147 4% 247,036,251 2.305 1 0.0075 5.147 4% 237,957,003 2.217 1 0.0075 5.147 4% 238,662,869 2.223 2 0.0080 5.138 4% 236,258,399 2.200 2 0.0080 5.138 4% 237,234,477 2.209 2 0.0080 5.138 4% 238,210,555 2.219 3 0.0080 5.069 4% 236,905,173 2.206 3 0.0080 5.069 4% 235,594,031 2.193 3 0.0080 5.069 4% 234,819,131 2.186 1 0.0075 5.060 6% 264,003,125 2.471 1 0.0075 5.060 6% 269,946,909 2.529 1 0.0075 5.060 6% 274,618,154 2.574 2 0.0080 5.130 6% 261,951,549 2.451 2 0.0080 5.130 6% 262,962,325 2.461 2 0.0080 5.130 6% 264,273,101 2.473 3 0.0080 5.200 6% 266,708,375 2.497 3 0.0080 5.200 6% 267,850,598 2.508 3 0.0080 5.200 6% 265,638,794 2.487

56

Appendix B: Continued

Day Jar # Thickness (in.) Weight (g) AITC+CD Corrected Areas (in2) Concentration (µL/L)

2

1 0.0075 5.158 8% 280,468,155 2.632 1 0.0075 5.158 8% 278,632,242 2.614 1 0.0075 5.158 8% 280,107,740 2.628 2 0.0075 5.156 8% 287,573,565 2.701 2 0.0075 5.156 8% 270,668,802 2.536 2 0.0075 5.156 8% 279,289,967 2.620 3 0.0075 5.328 8% 280,223,343 2.629 3 0.0075 5.328 8% 276,927,616 2.597 3 0.0075 5.328 8% 286,048,246 2.686 1 0.0075 5.341 10% 301,385,204 2.836 1 0.0075 5.341 10% 308,497,489 2.905 1 0.0075 5.341 10% 303,828,949 2.860 2 0.0065 5.065 10% 300,145,884 2.824 2 0.0065 5.065 10% 292,261,527 2.747 2 0.0065 5.065 10% 298,145,884 2.804 3 0.0080 5.485 10% 303,805,499 2.860 3 0.0080 5.485 10% 303,075,265 2.852 3 0.0080 5.485 10% 297,406,625 2.797

Day Jar # Thickness (in.) Weight (g) AITC+CD Corrected Areas (in2) Concentration (µL/L)

8

1 0.0070 4.698 1% 154,489,458 1.401 1 0.0070 4.698 1% 158,046,191 1.436 1 0.0070 4.698 1% 163,070,760 1.485 2 0.0075 5.025 1% 151,928,584 1.376 2 0.0075 5.025 1% 157,724,364 1.433 2 0.0075 5.025 1% 160,464,124 1.460 3 0.0085 4.819 1% 163,604,183 1.490 3 0.0085 4.819 1% 161,899,087 1.474 3 0.0085 4.819 1% 164,658,305 1.501 1 0.0065 4.400 2% 222,050,132 2.061 1 0.0065 4.400 2% 220,735,887 2.048 1 0.0065 4.400 2% 228,249,504 2.122 2 0.0075 4.860 2% 226,172,636 2.101 2 0.0075 4.860 2% 222,962,183 2.070 2 0.0075 4.860 2% 221,585,608 2.057

57

Appendix B: Continued

Day Jar # Thickness (in.) Weight (g) AITC+CD Corrected Areas (in2) Concentration (µL/L)

8

3 0.0070 4.828 2% 222,173,844 2.062 3 0.0070 4.828 2% 223,937,182 2.080 3 0.0070 4.828 2% 224,137,290 2.082 1 0.0075 5.147 4% 238,895,379 2.226 1 0.0075 5.147 4% 232,631,307 2.165 1 0.0075 5.147 4% 230,591,034 2.145 2 0.0080 5.138 4% 227,057,876 2.110 2 0.0080 5.138 4% 226,134,837 2.101 2 0.0080 5.138 4% 231,299,465 2.152 3 0.0080 5.069 4% 235,161,512 2.189 3 0.0080 5.069 4% 235,669,213 2.194 3 0.0080 5.069 4% 239,367,449 2.230 1 0.0075 5.060 6% 261,284,632 2.444 1 0.0075 5.060 6% 252,168,643 2.355 1 0.0075 5.060 6% 258,007,053 2.412 2 0.0080 5.130 6% 255,276,553 2.386 2 0.0080 5.130 6% 259,845,005 2.430 2 0.0080 5.130 6% 260,577,845 2.437 3 0.0080 5.200 6% 249,983,773 2.334 3 0.0080 5.200 6% 258,598,483 2.418 3 0.0080 5.200 6% 268,285,411 2.513 1 0.0075 5.158 8% 269,390,708 2.523 1 0.0075 5.158 8% 279,551,183 2.623 1 0.0075 5.158 8% 270,986,394 2.539 2 0.0075 5.156 8% 272,850,266 2.557 2 0.0075 5.156 8% 277,371,822 2.601 2 0.0075 5.156 8% 274,073,159 2.569 3 0.0075 5.328 8% 276,355,132 2.591 3 0.0075 5.328 8% 277,041,525 2.598 3 0.0075 5.328 8% 274,153,122 2.570 1 0.0075 5.341 10% 288,049,479 2.706 1 0.0075 5.341 10% 307,197,869 2.893 1 0.0075 5.341 10% 304,251,243 2.864

58

Appendix B: Continued

Day Jar # Thickness (in.) Weight (g) AITC+CD Corrected Areas (in2) Concentration (µL/L)

8

2 0.0065 5.065 10% 301,936,364 2.841 2 0.0065 5.065 10% 306,175,310 2.883 2 0.0065 5.065 10% 294,383,246 2.767 3 0.0080 5.485 10% 301,689,835 2.839 3 0.0080 5.485 10% 298,032,286 2.803 3 0.0080 5.485 10% 301,258,078 2.835

Day Jar # Thickness (in.) Weight (g) AITC+CD Corrected Areas (in2) Concentration (µL/L)

10

1 0.0070 4.698 1% 154,489,458 1.401 1 0.0070 4.698 1% 145,046,191 1.309 1 0.0070 4.698 1% 153,070,760 1.388 2 0.0075 5.025 1% 149,253,685 1.350 2 0.0075 5.025 1% 152,034,128 1.378 2 0.0075 5.025 1% 148,139,222 1.339 3 0.0085 4.819 1% 151,277,706 1.370 3 0.0085 4.819 1% 154,674,310 1.403 3 0.0085 4.819 1% 153,422,645 1.391 1 0.0065 4.400 2% 216,211,990 2.004 1 0.0065 4.400 2% 216,177,441 2.004 1 0.0065 4.400 2% 216,568,641 2.008 2 0.0075 4.860 2% 213,598,774 1.979 2 0.0075 4.860 2% 218,562,723 2.027 2 0.0075 4.860 2% 216,633,407 2.008 3 0.0070 4.828 2% 214,173,844 1.984 3 0.0070 4.828 2% 210,225,702 1.946 3 0.0070 4.828 2% 212,154,888 1.965 1 0.0075 5.147 4% 227,388,893 2.113 1 0.0075 5.147 4% 233,637,099 2.174 1 0.0075 5.147 4% 230,643,658 2.145 2 0.0080 5.138 4% 230,206,562 2.141 2 0.0080 5.138 4% 228,527,662 2.124 2 0.0080 5.138 4% 228,806,257 2.127

59

Appendix B: Continued

Day Jar # Thickness (in.) Weight (g) AITC+CD Corrected Areas (in2) Concentration (µL/L)

10

3 0.0080 5.069 4% 227,785,660 2.117 3 0.0080 5.069 4% 228,905,108 2.128 3 0.0080 5.069 4% 236,472,688 2.202 1 0.0075 5.060 6% 259,487,964 2.427 1 0.0075 5.060 6% 258,622,416 2.418 1 0.0075 5.060 6% 254,125,857 2.374 2 0.0080 5.130 6% 259,334,311 2.425 2 0.0080 5.130 6% 257,774,855 2.410 2 0.0080 5.130 6% 260,658,535 2.438 3 0.0080 5.200 6% 253,078,503 2.364 3 0.0080 5.200 6% 252,162,486 2.355 3 0.0080 5.200 6% 251,419,445 2.348 1 0.0075 5.158 8% 273,898,905 2.567 1 0.0075 5.158 8% 277,274,538 2.600 1 0.0075 5.158 8% 278,033,525 2.608 2 0.0075 5.156 8% 275,121,449 2.579 2 0.0075 5.156 8% 263,968,289 2.471 2 0.0075 5.156 8% 269,768,666 2.527 3 0.0075 5.328 8% 272,978,798 2.558 3 0.0075 5.328 8% 276,603,129 2.594 3 0.0075 5.328 8% 264,039,632 2.471 1 0.0075 5.341 10% 286,016,559 2.686 1 0.0075 5.341 10% 301,059,271 2.833 1 0.0075 5.341 10% 291,251,243 2.737 2 0.0065 5.065 10% 300,352,038 2.826 2 0.0065 5.065 10% 306,186,310 2.883 2 0.0065 5.065 10% 302,289,504 2.845 3 0.0080 5.485 10% 283,490,148 2.661 3 0.0080 5.485 10% 294,691,047 2.771 3 0.0080 5.485 10% 301,258,078 2.835

60

Appendix B: Continued

Day Jar # Thickness (in.) Weight (g) AITC+CD Corrected Areas (in2) Concentration (µL/L)

18

1 0.0070 4.698 1% 116,454,765 1.030 1 0.0070 4.698 1% 116,823,255 1.034 1 0.0070 4.698 1% 120,019,737 1.065 2 0.0075 5.025 1% 114,281,736 1.009 2 0.0075 5.025 1% 119,738,993 1.062 2 0.0075 5.025 1% 118,977,883 1.055 3 0.0085 4.819 1% 109,292,576 0.960 3 0.0085 4.819 1% 118,837,218 1.053 3 0.0085 4.819 1% 116,328,604 1.029 1 0.0065 4.400 2% 208,087,602 1.925 1 0.0065 4.400 2% 200,491,004 1.851 1 0.0065 4.400 2% 199,863,847 1.845 2 0.0075 4.860 2% 207,111,847 1.915 2 0.0075 4.860 2% 211,624,259 1.959 2 0.0075 4.860 2% 201,294,599 1.859 3 0.0070 4.828 2% 191,878,057 1.767 3 0.0070 4.828 2% 200,584,341 1.852 3 0.0070 4.828 2% 198,925,253 1.835 1 0.0075 5.147 4% 222,817,966 2.069 1 0.0075 5.147 4% 225,034,629 2.090 1 0.0075 5.147 4% 220,777,895 2.049 2 0.0080 5.138 4% 220,109,019 2.042 2 0.0080 5.138 4% 222,067,834 2.061 2 0.0080 5.138 4% 223,405,567 2.074 3 0.0080 5.069 4% 213,934,106 1.982 3 0.0080 5.069 4% 206,870,265 1.913 3 0.0080 5.069 4% 197,042,479 1.817 1 0.0075 5.060 6% 252,626,890 2.360 1 0.0075 5.060 6% 244,213,009 2.278 1 0.0075 5.060 6% 251,309,193 2.347 2 0.0080 5.130 6% 239,039,928 2.227 2 0.0080 5.130 6% 243,485,021 2.270 2 0.0080 5.130 6% 242,825,296 2.264 3 0.0080 5.200 6% 249,389,385 2.328 3 0.0080 5.200 6% 249,056,586 2.325 3 0.0080 5.200 6% 247,610,184 2.311

61

Appendix B: Continued

Day Jar # Thickness (in.) Weight (g) AITC+CD Corrected Areas (in2) Concentration (µL/L)

18

1 0.0075 5.158 8% 271,268,431 2.542 1 0.0075 5.158 8% 277,080,708 2.599 1 0.0075 5.158 8% 273,893,312 2.567 2 0.0075 5.156 8% 261,753,130 2.449 2 0.0075 5.156 8% 265,601,400 2.486 2 0.0075 5.156 8% 272,233,690 2.551 3 0.0075 5.328 8% 274,276,582 2.571 3 0.0075 5.328 8% 268,150,997 2.511 3 0.0075 5.328 8% 276,424,088 2.592 1 0.0075 5.341 10% 288,265,847 2.708 1 0.0075 5.341 10% 267,110,026 2.501 1 0.0075 5.341 10% 285,945,704 2.685 2 0.0065 5.065 10% 295,141,445 2.775 2 0.0065 5.065 10% 288,052,919 2.706 2 0.0065 5.065 10% 279,722,797 2.624 3 0.0080 5.485 10% 299,195,267 2.814 3 0.0080 5.485 10% 288,700,561 2.712 3 0.0080 5.485 10% 299,195,267 2.814

Day Jar # Thickness (in.) Weight (g) AITC+CD Corrected Areas (in2) Concentration (µL/L)

30

1 0.0070 4.698 1% 102,292,220 0.892 1 0.0070 4.698 1% 106,047,284 0.928 1 0.0070 4.698 1% 107,985,421 0.947 2 0.0075 5.025 1% 108,975,442 0.957 2 0.0075 5.025 1% 110,853,482 0.975 2 0.0075 5.025 1% 103,678,981 0.905 3 0.0085 4.819 1% 106,471,561 0.933 3 0.0085 4.819 1% 109,948,963 0.967 3 0.0085 4.819 1% 99,165,579 0.861 1 0.0065 4.400 2% 174,124,026 1.593 1 0.0065 4.400 2% 172,634,820 1.579 1 0.0065 4.400 2% 173,540,917 1.588

62

Appendix B: Continued

Day Jar # Thickness (in.) Weight (g) AITC+CD Corrected Areas (in2) Concentration (µL/L)

30

2 0.0075 4.860 2% 176,712,406 1.618 2 0.0075 4.860 2% 172,927,924 1.582 2 0.0075 4.860 2% 176,782,478 1.619 3 0.0070 4.828 2% 178,553,197 1.636 3 0.0070 4.828 2% 178,364,952 1.635 3 0.0070 4.828 2% 185,101,910 1.700 1 0.0075 5.147 4% 207,829,964 1.922 1 0.0075 5.147 4% 203,747,144 1.882 1 0.0075 5.147 4% 199,406,595 1.840 2 0.0080 5.138 4% 213,591,633 1.979 2 0.0080 5.138 4% 220,321,209 2.044 2 0.0080 5.138 4% 218,532,096 2.027 3 0.0080 5.069 4% 196,841,139 1.815 3 0.0080 5.069 4% 194,099,373 1.788 3 0.0080 5.069 4% 200,964,822 1.855 1 0.0075 5.060 6% 235,149,149 2.189 1 0.0075 5.060 6% 236,733,306 2.205 1 0.0075 5.060 6% 235,975,025 2.197 2 0.0080 5.130 6% 241,695,588 2.253 2 0.0080 5.130 6% 244,962,017 2.285 2 0.0080 5.130 6% 235,793,999 2.195 3 0.0080 5.200 6% 231,408,113 2.153 3 0.0080 5.200 6% 243,763,740 2.273 3 0.0080 5.200 6% 245,745,422 2.293 1 0.0075 5.158 8% 264,933,842 2.480 1 0.0075 5.158 8% 263,935,019 2.470 1 0.0075 5.158 8% 261,558,324 2.447 2 0.0075 5.156 8% 256,109,365 2.394 2 0.0075 5.156 8% 251,348,957 2.347 2 0.0075 5.156 8% 260,850,041 2.440 3 0.0075 5.328 8% 251,444,324 2.348 3 0.0075 5.328 8% 258,047,654 2.413 3 0.0075 5.328 8% 268,747,352 2.517 1 0.0075 5.341 10% 278,407,188 2.611 1 0.0075 5.341 10% 290,986,594 2.734 1 0.0075 5.341 10% 285,060,927 2.676 2 0.0065 5.065 10% 288,802,917 2.713 2 0.0065 5.065 10% 286,224,843 2.688 2 0.0065 5.065 10% 281,734,091 2.644 3 0.0080 5.485 10% 287,140,170 2.697 3 0.0080 5.485 10% 274,635,483 2.575 3 0.0080 5.485 10% 281,408,168 2.641

63

Appendix C: Chromatogram of Antimicrobial Films: Encapsulation of AITC in β-CD

AITC

Noise

AITC

Noise

Concentration of 2%

Concentration of 8%