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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
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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
References
Aleemomair, A. (2008, October 27). Cyclodextrins in pharmaceuticals: An overview. Retrieved
from http://www.pharmainfo.net/pharma-student-magazine/cyclodextrins-
pharmaceuticals-overview-0
Appendini, P. (2002). Review of antimicrobial food packaging. Innovative Food Science &
Emerging Technologies, 3(2), 113-126.
Cooksey, K. (2005). Effectiveness of antimicrobial food packaging materials. Food Additives &
Contaminants, 22(10), 980-987. doi: 10.1080/02652030500246164
Dogan, H., Koral, M., & İnan, T. Y. (2009). Ag/zn zeolite containing antibacterial coating for
food-packaging substrates. Journal of Plastic Film & Sheeting, 25(3), 207-220. doi:
10.1177/8756087909354479
Farber, J.M. (1991). Microbiological aspects of modified atmosphere packaging: A review.
Journal of Food Protection, 54(1), 58-70.
Han, J.H. 2000. Antimicrobial food packaging. Food Technology, In R. Ahvenainen (Series
Ed.),. Novel food packaging techniques, (pp. 50-56). Sawston, Cambridge, England:
Woodhead Publishing Limited.
Hedges, A. R. (1998). Industrial applications of cyclodextrins. Chemical Reviews, 98(5), 2035-
2044.
Huff, K. (2008). Active and intelligent packaging: Innovations for the future. Unpublished
manuscript, University of VirginiaTech, Retrieved from
http://www.iopp.org/files/public/VirginiaTechKarleighHuff.pdfIsshiki,
K., Tokuoka, K., Rori, R., & Chiba, S. (1992). Preliminary examination of allyl-isothiocyanate
vapor for food preservation. Bioscience, Biotechnology, and Biochemistry, 56, 1476–
1477.
50
Kin, C. (2008). Development and validation of a solid phase microextraction method for
simultaneous determination of pesticide residues in fruits and vegetables by gas
chromatography. (Doctoral dissertation). Retrieved from
http://dspace.fsktm.um.edu.my/bitstream/1812/506/1/PhDThesis(ChaiMeeKin).pdf
Morris, D. (2000, December 13). Today’s food trends: Are consumers eating to live…or living to
eat?. Smoke Signals, 6, 25-30.
Ozdemir, M., & Floros, J. D. (2004). Active food packaging technologies. Critical Reviews in
Food Science & Nutrition, 44(3), 185-193.
Qing-Feng, Z., Zi-Tao J., & Rong L. (2007). Complexation of allyl isothiocyanate with β-
cyclodextrin and its derivatives and molecular microcapsule of allyl isothiocyanate in β-
cyclodextrin. European Food Research & Technology, 225(3), 407-413.
Rhoades J., & Rastall, B. (2000), Chitosan as an antimicrobial agent, Food Technology
International, Ingredients & Additives, 32-33.
Shin, J., Harte, B., Ryser, E., & Selke, S. (2010). Active packaging of fresh chicken breast, with
allyl isothiocyanate (AITC) in combination with modified atmosphere packaging (MAP)
to control the growth of pathogens. Journal of Food Science, 75(2), M65-M71. doi:
10.1111/j.1750-3841.2009.01465.x
Siró, I., Fenyvesi, É., Szente, L., De Meulenaer, B., Devlieghere, F., Orgoványi, J., Sényi, J., &
Barta, J. (2006). Release of alpha-tocopherol from antioxidative low-density polyethylene
film into fatty food simulant: Influence of complexation in beta-cyclodextrin. Food
Additives & Contaminants, 23(8), 845-853. doi: 10.1080/02652030600699064
Suppakul, P., Miltz, J., Sonneveld, K., Bigger, S.W. (2003). Active packaging technologies with
an emphasis on antimicrobial packaging and its applications. Journal of Food Science
68(2), 408–420.
51
Takeuchi, K., & Yuan, J. (2002). Packaging tackles food safety: A look at antimicrobials,
Industrial case study presentation during IFT annual meeting (June 15–19, 2002,
Anaheim, CA).
Vartiainen, J., Skyttä, E., Enqvist, J., & Ehvenainen-Rantala, R. (2003). Antimicrobial and
barrier properties of LDPE films and containing imazalil and EDTA. Journal of Plastic
Film & Sheeting, 19(4), 249-261. doi: 10.1177/8756087903042808
Vermeiren, L., Devlieghere, F., & Debevere, J. (2002). Effectiveness of some recent
antimicrobial packaging concepts. Food Additives and Contaminants, 19 Suppl, 163-171.
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%