Control Release of Allyl Isothiocyanate Vapor from Mustard Seed

Meal Powder Through Encapsulation in Electrospun

Poly(lactic acid) and Poly(ethylene oxide) Fibers

By

Ruyan Dai

A Thesis Presented to The University of Guelph

In partial fulfillment of requirements for the degree of Master of Science in Food Science

Guelph, Ontario, Canada © Ruyan Dai, September 2013

! ABSTRACT

Control Release of Allyl Isothiocyanate Vapor from Mustard Seed Meal

Powder Through Encapsulation in Electrospun Poly(lactic acid) and

Poly(ethylene oxide) Fibers

Ruyan Dai Advisor: University of Guelph, 2013 Dr. Loong-Tak Lim

There is a growing interest in using antimicrobial components from edible plants to prolong the shelf-life of food due to consumer preference for naturally occurring preservatives. In this thesis research, the release of allyl isothiocyanate (AITC), a potent antimicrobial volatile from mustard seed, was investigated. The formation of AITC in mustard seed is mediated by an enzymatic reaction, in which myrosinase catalyzes the hydrolysis of sinigrin - a glucosinolate naturally presents in mustard seed. The effects of relative humidity (85 to 100% RH), temperature (5 to 35°C), and lipid content on the release kinetics of AITC from mustard seed meal powder (MSMP) were evaluated using headspace gas chromatography. The MSMP samples were prepared by a ball milling treatment, resulting in particle diameters ranging from 10 to 500 µm. The kinetic data showed that both the release rate and the maximum amount of AITC released significantly (p< 0.05) increased with elevated RH and temperature. The finer the particle size of the MSMP, the higher the AITC release rate. To manipulate the AITC release kinetics, defatted ground MSMP samples were encapsulated within electrospun fibers.

The defatted ground MSMP was dispersed into 9% poly(lactic acid) (PLA), 3% poly(ethylene oxide) (PEO), or blends of the two polymer solutions at weight ratios of

75:25, 50:50 and 25:75, respectively. The release kinetics of AITC from the composite fibers were monitored using a headspace gas chromatograph. The AITC release data in the headspace air showed that the addition of PEO into PLA polymers significantly increased (p< 0.05) the AITC release rate comparing to that from the 100PLA0PEO

MSMP-containing membrane and indicated that electrospun fibers with different PLA and PEO ratios could be used as carriers to control the release of AITC from MSMP.

In view of the antimicrobial properties of AITC, MSMP and MSMP-containing electrospun fibers could control the release of AITC and may be feasible for use in food active packaging applications to extend food shelf-life.

iii

ACKNOWLEDGEMENTS

I would like to start off with thanking my advisor and mentor, Dr. Loong-Tak Lim.

Without his guidance, dedication and patience, I would never have had the chance to explore the fascinating world of electrospinning and active packaging. I would like to convey my gratitude to my committee members Drs. Massimo Marcone and Robert

Lencki, for their expertise, encouragement, and exceptional suggestions, especially with writing this thesis. I would like to acknowledge the contribution to Dr. Sandy Smith for her professional knowledge and continuous help with SEM analysis.

A special thanks goes to my seniors, group members and friends in the Food

Packaging Research group: Xiuju Wang, Suramya Minhindukulasuria, Solmaz Alborzi,

Ana Cristina Vega-Lugo, Khalid Moomand, Roc Chan, Alex Jensen, Yucheng Fu, Niya

Wang, Gloria Wang, Vania Pinto and Vinicius Deon. Thank you all for your advices, supports, tolerance and friendship. You made my “lab time” a really wonderful experience and I will always treasure the memories. I am also grateful for my previous professors in Soochow University, Dr. Ye Yuantu and Dr. Cai Chunfang. Without their help, I would not have had the opportunity to pursue my degree in Canada.

My utmost appreciation goes to my parents Li Conggan and Dai Wenfu, thank you for your love, unconditional understanding, support and encouragement. I also want to thank my lovely brother, Dai Xincheng, who always counts down the days before I go back home. I would like to express my gratitude to my boyfriend Benny (Cheung Ming)

Shiu for his continuous encouragement, patience, tolerance and support. Many thanks go to my dear friends Jenifer Thi Lu and Sarah Malik for their company during my happy and hard times throughout my studies in Canada and their patience in reading and

! "#! revising my writing. I thank my friends Fan Xu, Xiaoyan Liu, Ying Ji, Yuqing Li, Lijuan

Zheng for their friendship and support. I also thank the Natural Science and Engineering

Research Council of Canada (NSERC) and Biofume Technologies Inc. for their essential financial support.

! #! Table of contents 1. Introduction ...... 1 2. Literature Review ...... 4 2.1 Overview of Food Active Packaging ...... 4 2.2 Active Packaging with an Emphasis on Antimicrobial Packaging ...... 5 2.2.1 Overview of Food Antimicrobial Active Packaging ...... 5 2.2.2 Synthetic Chemical Antimicrobial Agents ...... 5 2.2.3 Naturally Derived Antimicrobial Agents ...... 7 2.2.3.1 Probiotics ...... 8 2.2.3.2 Bacteriocins ...... 8 2.2.3.3 Enzymes ...... 9 2.2.3.4 Plants Extracts and Essential Oils ...... 10 2.3 AITC as an Antimicrobial Compound ...... 11 2.4 Mustard Seed as a Source of AITC ...... 14 2.4.1 Overview of Mustard Seed and its Antimicrobial Efficiency ...... 14 2.4.2 Sinigrin ...... 15 2.4.3 Myrosinase ...... 16 2.5 Control Release using Encapsulation ...... 18 2.6 Electrospun Fibers for Controlled Release of Bioactives ...... 22 2.6.1 Overview of Electrospun Fibers ...... 22 2.6.2 Electrospun Fibers as Encapsulation Carriers ...... 23 2.7 Poly(lactic acid) (PLA) ...... 25 2.7.1 Overview ...... 25 2.7.2 Electrospun PLA Fibers ...... 26 2.8 Poly(ethylene oxide) ...... 26 2.8.1 Overview of PEO ...... 26 2.8.2 PEO as an Aid Polymer in Electrospinning ...... 27 3. Justifications and Objectives ...... 28 3.1 Justifications ...... 28 3.2 Objectives ...... 29 4. Release of Allyl Isothiocyanate from Mustard Seed Meal Powder ...... 30 4.1 Introduction ...... 30 4.2 Materials and Methods ...... 32 4.2.1 Materials ...... 32 4.2.2 Preparation of MSMP ...... 32 4.2.3 Physical and Chemical Characterization of MSMP ...... 33 4.2.4 Scanning Electron Microscopy (SEM) Analysis ...... 34 4.2.5 Headspace Analysis of MSMP ...... 34 4.2.6 Data Analysis ...... 37 4.3 Results and Discussion ...... 37 4.3.1 Characterization of MSMP ...... 37 4.3.2 AITC Release Kinetics as Affected by Temperature and RH ...... 41 4.3.3 Particle Size Effects on AITC Release Kinetics ...... 45 4.3.4 Lipid Content Effects on AITC Release Kinetics ...... 47 4.4 Conclusions ...... 47 5. Control Release of AITC from Defatted Ground MSMP with Blended Electrospun Fibers of PLA and PEO ...... 49

! #"! 5.1 Introduction ...... 49 5.2 Materials and Methods ...... 51 5.2.1 Materials ...... 51 5.2.2 Methods ...... 52 5.2.2.1 MSMP Preparation ...... 52 5.2.2.2 Solution Preparation ...... 52 5.2.2.3 Electrospinning ...... 53 5.2.3 Fiber Morphology and Size Analysis ...... 54 5.2.4 Attenuated Total Reflection–Fourier Transform Infrared (ATR-FTIR) Evaluation ..... 55 5.2.5 Thermal Analysis ...... 56 5.2.6 Headspace Analysis ...... 56 5.2.7 Data Analysis ...... 58 5.3 Results and Discussion ...... 59 5.3.1 Surface Morphology of Electrospun Fibers ...... 59 5.3.2 Fiber Size Analysis ...... 63 5.3.3 Release of AITC from Encapsulated MSMP ...... 65 5.3.4 FTIR analyses on Pristine and MSMP-Containing Electrospun Fiber Membranes ... 71 5.3.5 Thermal Analyses ...... 74 5.4 Conclusions ...... 79 6. Thesis Conclusions and Future Directions ...... 81 6.1 Conclusions ...... 81 6.2 Future Directions ...... 83 7. References ...... 84 8. Appendices ...... 96 8.1 Surface-to-volume ratio distribution of MSMP particle samples as examined in Chapter 4...... 96 8.2 RH building up curves in various experiment conditions as examined in Chapters 4 and 5...... 99

! #""! LIST OF TABLES

TABLE 2.1 - SOME ANTIMICROBIAL AGENTS OF POTENTIAL USE IN THE ACTIVE PACKAGING APPLICATIONS...... 6

TABLE 2.2 - SOME ENCAPSULATED FOOD INGREDIENTS AND THE RESEARCHED ENCAPSULATION METHODS...... 20

TABLE 4.1- PHYSICAL AND CHEMICAL PROPERTIES OF VARIOUS MSMP SAMPLES...... 39

TABLE 4.2- FITTED MODEL PARAMETERS (QMAX, K AND ") OF MODIFIED GOMPERTZ EQUATION FOR VARIOUS MSMP SAMPLES UNDER DIFFERENT TEMPERATURE (5, 20, AND 35°C) AND RH (85 AND 100% RH) CONDITIONS...... 45

TABLE 5.1 - MEAN FIBER DIAMETER AND STANDARD DEVIATIONS OF PRISTINE AND MSMP-CONTAINING ELECTROSPUN FIBER MATS...... 64

TABLE 5.2 - MODEL FITTED PARAMETERS FROM MODIFIED GOMPERTZ EQUATION OF AVERAGED QMAX, K AND LAG TIME CONSTANTS FOR THE AITC RELEASE PROFILES FROM FIVE MSMP-CONTAINING ELECTROSPUN FIBER MATS AT 20°C AND 100% RH CONDITIONS...... 69

! #"""! List of Figures

FIGURE 2.1 - SINIGRIN STRUCTURE. ADAPTED FROM SAIMI 2009...... 16

FIGURE 2.2 - SINIGRIN HYDROLYSIS TO ISOTHIOCYANATES AND GLUCOSE...... 16

FIGURE 2.3- PHYSICAL AND CHEMICAL TYPES OF ENCAPSULATION SYSTEMS. ADAPTED FROM POTHAKAMURY AND BARBOSA-GNOVAS 1995...... 19

FIGURE 2.4 - VARIOUS FORMS OF CAPSULES. ADAPTED FROM GIBBS AND OTHERS 1999...... 21

FIGURE 2.5 - CHEMICAL SCHEMATIC STRUCTURE OF PLA...... 25

FIGURE 2.6 - CHEMICAL SCHEMATIC STRUCTURE OF PEO...... 27

FIGURE 4.1 - HEADSPACE SAMPLING SYSTEM TO STUDY THE AITC RELEASE KINETICS FROM MSMP...... 35

FIGURE 4.2 - PARTICLE SIZE DISTRIBUTION OF MUSTARD SEED SAMPLES: (A) APR-MSMP; (B) GROUND-MSMP; (C) DEFATTED-MSMP; AND (D) GROUND-DEFATTED-MSMP...... 40

FIGURE 4.3 - REPRESENTING SCANNING ELECTRON MICROGRAPHS FOR VARIOUS MSMP SAMPLES...... 40

FIGURE 4.4 - REPRESENTATIVE AITC RELEASE PROFILES FROM VARIOUS MSMP SAMPLES IN 1 L HEADSPACE GLASS JAR UNDER DIFFERENT TEMPERATURE AND HUMIDITY CONDITIONS. SYMBOLS ARE EXPERIMENTAL DATA, WHILE CONTINUOUS LINES ARE FITTED CURVES BASED ON THE MODIFIED ...... 44

FIGURE 5.1 - SCHEMATIC DIAGRAM OF THE EXPERIMENTAL SETUP FOR ELECTROSPINNING OF MSMP-CONTAINING FIBERS...... 54

FIGURE 5.2 - SCANNING ELECTRON MICROGRAPHS OF THE PRISTINE ELECTROSPUN ULTRAFINE FIBERS: (A) 100PLA0PEO; (B) 75PLA25PEO; (C) 50PLA50PEO; (D) 25PLA75PEO; AND (E) 0PLA100PEO...... 61

FIGURE 5.3 - SCANNING ELECTRON MICROGRAPHS OF THE MSMP-CONTAINING ELECTROSPUN ULTRAFINE FIBERS: (A) 100PLA0PEO; (B) 75PLA25PEO; (C) 50PLA50PEO; (D) 25PLA75PEO; AND (E) 0PLA100PEO...... 63

FIGURE 5.4 - FIBER DIAMETER DISTRIBUTIONS OF THE PRISTINE AND MSMP CONTAINING ELECTROSPUN FIBERS WITH VARIOUS RATIOS OF PLA AND PEO. .. 66

FIGURE 5.5 - REPRESENTATIVE CUMULATIVE AITC RELEASE PROFILES FROM VARIOUS MSMP-CONTAINING ELECTROSPUN FIBERS IN 1 L HEADSPACE GLASS JAR UNDER SATURATED RH AT 20°C. SYMBOLS ARE EXPERIMENTAL DATA, WHILE CONTINUOUS LINES ARE FITTED CURVES BASED ON THE MODIFIED GOMPERTZ EQUATION...... 67

! "$! FIGURE 5.6 - COMPARISON OF FTIR SPECTRA OF THE PRISTINE ELECTROSPUN ULTRAFINE FIBERS: (A) 100PLA0PEO; (B) 75PLA25PEO; (C) 50PLA50PEO; (D) 25PLA75PEO; AND (E) 0PLA100PEO...... 72

FIGURE 5.7 - COMPARISON OF FTIR SPECTRA OF MSMP- CONTAINING ELECTROSPUN FIBERS: (A) 100PLA0PEO; (B) 75PLA25PEO; (C) 50PLA50PEO; (D) 25PLA75PEO; AND (E) 0PLA100PEO...... 74

FIGURE 5.8 - DSC THERMOGRAMS OF PRISTINE ELECTROSPUN ULTRAFINE FIBERS: (A) 100PLA0PEO; (B) 75PLA25PEO; (C) 50PLA50PEO; (D) 25PLA75PEO; AND (E) 0PLA100PEO...... 75

FIGURE 5.9 - DSC THERMOGRAMS OF MSMP AND MSMP-CONTAINING ELECTROSPUN ULTRAFINE FIBERS...... 78

FIGURE A.1- PARTICLE SURFACE-TO-VOLUME RATIO DISTRIBUTION OF MUSTARD SEED SAMPLES: (A) APR-MSMP; (B) GROUND-MSMP; (C) DEFATTED-MSMP; AND (D) GROUND-DEFATTED-MSMP...... 98

FIGURE A.2 - BUILDING UP OF RELATIVE HUMIDITY (WITH 6 AND 12 #L DISTILLED WATER RESPECTIVELY) IN THE HEADSPACE OF 1 L HERMETIC SEALED BOTTLE AT 5°C IN CHAPTER 4...... 99

FIGURE A.3 - BUILDING UP OF RELATIVE HUMIDITY (WITH 15 AND 30 #L DISTILLED WATER RESPECTIVELY) IN THE HEADSPACE OF 1 L HERMETIC SEALED BOTTLE AT 20°C IN CHAPTER 4...... 100

FIGURE A.4 - BUILDING UP OF RELATIVE HUMIDITY (35 AND 70 #L DISTILLED WATER RESPECTIVELY) IN THE HEADSPACE OF 1 L HERMETIC SEALED BOTTLE AT 35°C IN CHAPTER 4...... 100

FIGURE A.5 - BUILDING UP OF RELATIVE HUMIDITY (WITH 1000 #L DISTILLED WATER) IN THE HEADSPACE OF 1 L HERMETIC SEALED BOTTLE AT 20°C IN CHAPTER 5...... 101

! $! 1. Introduction

Packaging is an essential element for product development and commercialization (Lopez-Rubio and others 2004). To maintain product quality and adequate shelf-life, much research has been done over the past several decades and many innovative packaging concepts have been developed. Among them, the active packaging approach has received considerable interests from academia and various industries. Active packaging has been defined as “packaging in which subsidiary constituents have been deliberately included in or on either the packaging material or the package headspace to enhance the performance of the package system” (Robertson

2006). One of the most promising sub-categories of active packaging is the antimicrobial food packaging technologies (Suppakul and others 2003). Various antimicrobial agents have been studied extensively in antimicrobial packaging applications (Han 2005;

Suppakul and others 2003).

Allyl isothiocyanate (AITC) is an antimicrobial compound that has gained great interest in food antimicrobial packaging applications (Han 2000; Jung and others 2009;

Nielsen and Rios 2000). Researchers have been exploiting synthetic and/or pure AITC to prolong the shelf-life of food products. AITC has been shown to be effective in eliminating Escherichia coli O157:H7 and Aspergillus glaucus in beef and peanuts, respectively (Chacon and others 2006a; Dhingra and others 2009b). However, direct addition of AITC to food can be problematic due to rapid release of the volatile compounds, causing organoleptic issues (Ko and others, 2012). In order to overcome this shortcoming, some researchers have attempted to deliver AITC through the use of natural products derived from the Cruciferae family, such as mustard seed and ! %! ! horseradish (Jung and others 2009; Kinae and others 2000; Mustakas and others 1961;

Nilson 2011; Shofran and others 1998). These plants are known to produce AITC when their tissue is disrupted due to the myrosinase-mediated hydrolysis of a glucosinolate known as sinigrin. The release kinetics of AITC from these plants are related to extrinsic

(e.g., relative humidity [RH], temperature, pH) and intrinsic (e.g., pH, lipid content, moisture content, particle size) properties (Li and others, 2005, Tsao and others, 2000).

Thus, it can be hypothesized that mustard seed may be used as a natural carrier to control AITC release kinetics by adjusting the extrinsic and intrinsic factors discussed above.

Another avenue to deliver AITC in a controlled manner is to encapsulate AITC in polymeric carriers (Ko and others, 2012; Vega-Lugo and Lim, 2009). One popular polymer carrier that has been examined as a good candidate to control the release of active compounds is electrospun fibers (Vega-Lugo and Lim 2009; Neo and others 2013;

Zhou and Lim 2009). This may be attributed to several advantages of electrospun fibers, including large surface-to-volume ratio (Frenot and Chronakis 2003; Huang and others

2003b) and the facility by which of the physical-chemical properties of the carriers can be adjusted by altering polymer composition (Huang and others 2003b; Kenawy and others

2002; Uhrich and others 1999). Thus, electrospun fibers are versatile carriers not only for preventing the uncontrolled release of bioactive compounds, but also for allowing the development of a triggering mechanism that activates their release (e.g., by exploiting the desirable interaction(s) between the fiber and their surrounding matrices). To this end, electrospun fibers may be applied for controlled release of various volatile compounds according to the product end use and the headspace conditions.

! &! ! Although a number of studies related to the application of pure AITC for food product preservation have been reported over the past decade, investigations related to the use of processed Cruciferae plants, such as mustard seeds, for active packaging applications are limited. Moreover, methodologies to encapsulate processed mustard seed to achieve controlled release of AITC have not been explored. Currently, mustard seeds are mainly used as a condiment. Considering the fact that Canada is one of the leading producers and exporters of mustard seed (AAFC, 2013), the development of a novel use of mustard seed will expand its use, thereby benefiting the Canadian mustard industry.

! '! ! 2. Literature Review

2.1 Overview of Food Active Packaging

Packaging plays an important role in the protection, distribution, and commercialization of foodstuffs. The demand for optimal packaging is increasing due to the development of global commercialization and consumers’ preferences for high quality, minimally processed products, both of which require extended product shelf-life and enhanced food safety. From a product end-use standpoint, as projected by Smithers

Pira (2010), the increasing consumers’ demand for convenient packaged foods will drive sales in the food packaging industry, especially in the areas of moisture scavengers, self-venting films, microwave susceptors, and other innovative packaging systems

(Anonymous 2010). In the highly competitive marketplace, food companies will continue to find ways to reduce production costs and maximize profitability, while being vigilant in reducing their impact to the environment (Ozdemir and Floros 2004).

To produce food products with enhanced quality and extended shelf-life, many innovative packaging approaches have been taken. Among them, active packaging has been reported to have an important effect in achieving these goals. Active packaging is defined as “packaging in which subsidiary constituents have been deliberately included in or on either the packaging material or the package headspace to enhance the performance of the package system” (Robertson 2006). Based on this definition, active packaging includes components of packaging systems that are capable of: scavenging oxygen, absorbing carbon dioxide, moisture, ethylene and/or flavor/odor taints, releasing carbon dioxide, ethanol, antioxidants and/or other preservatives, maintaining

! (! ! temperature control and compensating for temperature changes, and eliminating microorganisms with antimicrobial systems (Ozdemir and Floros 2004).

2.2 Active Packaging with an Emphasis on Antimicrobial Packaging

2.2.1 Overview of Food Antimicrobial Active Packaging

Packaging technologies that focus on antimicrobial purposes are one of the most promising sub-categories of active packaging for food products (Suppakul and others

2003). Antimicrobial packaging is a system capable of killing or inhibiting the growth of microorganisms, thereby extending food shelf-life (Han and others 2000). The efficacy of these antimicrobial systems is generally achieved through the delivery of antimicrobial agents via packaging materials into the package headspace or the packaged food product.

Various antimicrobial agents can be incorporated into conventional food packaging systems to create new antimicrobial packaging systems. Table 2.1 shows some of the potential antimicrobial agents that have been studied. According to the origin of antimicrobial agents, they can be classified into two categories: synthetic chemical antimicrobial agents, and naturally derived agents. Selected examples are discussed below.

2.2.2 Synthetic Chemical Antimicrobial Agents

Synthetic chemical antimicrobial agents are either produced by chemical synthesis or chemical modification of natural materials. They have been widely

! )! ! investigated and applied in various food systems. As shown in Table 2.1, some common antimicrobial chemical agents are organic acids and salts (Melnick and others 1954), alcohol (Shapero and others 1978), fatty acids (Vojdani and Torres 1990), fungicides and phenolic compounds (Rauha and others 2000). Among them, organic

Table 2.1 - Some antimicrobial agents of potential use in the active packaging applications.

Category Types Examples

Organic acid and Acetic acid, citric acid, benzoic acid, propionic salt acid, potassium sorbate

Alcohol Ethanol

Synthetic Chelating agents EDTA chemical Fatty acids Lauric acid, palmitoleic acid

Fungicides Benomyl, sulfur dioxide, imazalil

Phenolic Catechin, cresol, hydroquinone

Enzymes Lysozymes, glucose oxidase

Bacteriocins Nisin, pediocin, subtilin, lacticin

Butylatedhydroxyanisole, Antioxidants butylatedhydroxytoluene, tertiary butylhydroquinone Naturally derived Antibiotics Natamycin

Polysaccharides Chitosan

Plant volatiles AITC, thymol, eugenol, citral

Lactic acid bacteria such as Lactobacilli, Probiotics Lactococci, Entorococci,

From (Brody and others 2001; Galvez and others 2011; Han 2005; Juneja and others 2012; Suppakul and others 2003). acids and fungicides have been widely applied by researchers due to their high antimicrobial efficiency and their relatively low cost (Han 2005). For example, Limjaroen and coworkers (2005) prepared sorbic-acid-containing (0%, 1.5%, and 3.0% w/v) ! *! ! polyvinylidene chloride films and claimed that these films might be useful in enhancing the safety and shelf-life of ready-to-eat delicatessen products by reducing the growth of

Listeria monocytogenes (Limjaroen and others 2005). Moreover, sorbic acid (0.25-0.5%, w/v wrapper) containing wrappers (300-gauge moisture-proof cellophane) adequately protected cheese from surface mold spoilage, and less than 0.1% sorbic acid (w/w, cheese) migrated to the cheese (Melnick and others 1954). Other organic acids such as citric acid (Dole$alová and others 2010), propionic acid (Ouattara and others 1997) and lactic acid (Dole$alová and others 2010; Greer and Dilts 1995) have also been reported to be effective in inhibiting the growth of food-borne microorganisms on various food products. By and large, chemical antimicrobial agents are efficient in retarding the growth of microorganisms and hence are currently being widely applied in food products

(Dole$alová and others 2010; Greer and Dilts 1995; Melnick and others 1954; Ouattara and others 1997).

2.2.3 Naturally Derived Antimicrobial Agents

The increasing interest in natural preservatives in the food industry is primarily driven by the consumer’s perception that naturally-occurring antimicrobial agents possess lower risks than their synthetic counterparts (Nicholson 1998). In response to this trend, many studies have attempted to incorporate or immobilize natural antimicrobial agents into the matrix or onto the surface of packaging materials to increase the shelf stability of processed meats, cheeses, and other products. As listed in

Table 2.1, the most common natural-occurring agents are bacteriocins, enzymes, and

! +! ! essential oils derived from herbs and spices.

2.2.3.1 Probiotics

Probiotics are defined by the Food and Agriculture Organization (FAO) as ‘‘live microorganisms (bacteria or yeasts), which when ingested or locally applied in sufficient numbers confer one or more specified demonstrated health benefits for the host’’

(Anonymous 2011). Many traditional fermented food products contain antimicrobial probiotics and there has been some research and developments regarding the function of antimicrobial probiotics for the preservation of fermented foods (Anal and Singh 2007;

Han 2005). The most commonly used antimicrobial probiotics are lactic acid bacteria.

The antimicrobial activity of lactic acid bacteria involves producing bacteriocins and non-peptide growth-inhibiting chemicals. Studies showed that probiotics are effective in retarding the growth of the E. coli O157:H7 in refrigerated ground beef

(Muthukumarasamy and others 2003) and dry fermented sausages (Muthukumarasamy and Holley 2007). Probiotics also efficiently inhibited the growth of E. coli O157:H7 and

Listeria monocytogenes in dry sausages (Tyopponen and others 2003). These results suggest the potential use of probiotics in antimicrobial packaging applications.

2.2.3.2 Bacteriocins

Bacteriocins are proteins produced by bacteria in probiotic systems. These peptides are effective against the growth of some microorganisms (Brody and others

2001; Galvez and others 2011). For instance, nisin is produced by the lactic acid bacteria,

! ,! ! Lactococcuslactis, and effectively eliminates gram-positive organisms, notably the

Clostridia species (Schillinger and others 1996). Nisin has been accepted by regulatory authorities for food use in some countries, such as Japan (Brody and others 2001).

Bacteriocins could be used as preservatives in various foods, such as in raw meat, egg products, seafood, milk and dairy products (Brody and others 2001; Cha and Chinnan

2004; Galvez and others 2011; Nicholson 1998). However, since bacteriocins are proteins and/or peptides, their lack of resistance to thermal treatments and pH has limited their application (Galvez and others 2011).

2.2.3.3 Enzymes

Antimicrobial enzymes are another group of naturally derived agents that are of great interest in the development of active food packaging. Enzymes may be bound to the inner surface of food contact films, catalyzing the production of antimicrobial agents.

Natural enzymes that have been studied extensively are: lysozymes (Brody and Budny

1995; Cha and Chinnan 2004; Cha and others 2002), glucose oxidase (GOX) (Field and others 1986; Fuglsang and others 1995; Vartiainen and others 2005), and other enzymes (Brody and Budny 1995; Han 2000). Lysozyme lyses the %-1, 4-glycosidic linkages between N-acetylmuramic acid and N-acetylglucosamine found in peptidoglycan in cell membrane of gram positive bacteria (Cha and Chinnan 2004). For instance, zein-casting films which incorporated partially purified hen egg lysozyme were effective in eliminating Bacillus subtilis and Lactobacillus plantarum culture (Padgett and others 1998). On the other hand, GOX can retard the growth of bacteria by forming a potent antimicrobial, hydrogen peroxide (Brody 2001). A study that applied 0.067% GOX

! -! ! aqueous solution (v/v) to refrigerated, fresh, whole winter flounder and winter flounder filets improved the shelf-life of fish from 15 days to 21 days (Field and others 1986). In another study, GOX that was immobilized on amino- and carboxyl-plasma-activated biorientated polypropylene films also exhibited antimicrobial activity against the growth of

E. coli and B. subtilis (Vartiainen and others 2005). Other antimicrobial enzymes such as

%-glucanases and chitinases also have been applied in many food systems and have demonstrated antimicrobial efficacy against many foodborne microorganisms (Fuglsang and others 1995; Suppakul and others 2003). Similar to bacteriocins, antimicrobial enzymes are sensitive to intrinsic (substrate) and extrinsic factors (temperature and pH)

(Brody and Budny 1995; Han 2005), which can limit their practical application.

2.2.3.4 Plants Extracts and Essential Oils

Historically, natural extracts from herbs and spices have been used to extend the shelf life of food and improve their sensory quality (Han 2005; Nadajarah and others

2005b). Extracts and essential oils from herbs and spices (e.g., cinnamon, allspice, clove, oregano, thyme, rosemary, mustard, and oregano) have been shown to possess a wide spectrum of antimicrobial properties (Cha and Chinnan 2004; Nielsen and Rios 2000;

Winther and Nielsen 2006). Some extracts also offer other advantages such as antioxidative (Padhye and others 2009) and nutraceutical bioactivities (Kapadia and Rao

2011).

The antimicrobial efficiency of plant extracts and essential oils has been studied extensively in food systems. Nielsen and Rios (2000) investigated the inhibition of bread spoilage fungi (Penicillium commune, Penicillium roqueforti, Aspergillus flavus, and

! %.! ! Endomyces fibuliger) by volatile components from spices and herbs. It was found that cinnamon, garlic, and clove had high antimicrobial activity, whereas oregano oleoresin weakly inhibited microbial growth. In their study, mustard essential oil, which primarily consists of AITC, had the strongest effect in inhibiting all the tested fungi. Other studies have also demonstrated that AITC effectively inhibits a variety of pathogenic microorganisms (Isshiki and others 1992; Nadajarah and others 2005a; Suhr and

Nielsen 2003). Therefore, extracts from herbs and spices are promising natural antimicrobial agents for food preservation, increasing shelf-life as well as enhancing product safety.

2.3 AITC as an Antimicrobial Compound

When plant tissue from the Cruciferae family (e.g., mustard, horseradish,

Japanese wasabi, brussels sprouts, broccoli, kale and turnip) are disrupted, AITC is produced as a result of enzymatic hydrolysis of a glucosinolate (Jung and others 2009;

Kinae and others 2000; Nadajarah and others 2005a; Winther and Nielsen 2006). The unique pungent odor produced is in part due to the formation of AITC. The antimicrobial properties of AITC have been well demonstrated in the literature (Delaquis and Sholberg

1997; Suhr and Nielsen 2003; Winther and Nielsen 2006). AITC is potent against both

Gram-negative and -positive bacteria, as well as molds and yeasts in both the liquid and vapor phases. In the liquid phase, its minimum inhibition concentration (MIC) is 50-1000 ppm for bacteria, 1-4 ppm for nonxerotolerant yeasts, and 50 ppm for xerotolerant yeasts.

In the vapor phase, the MIC of AITC is 34-110, 16-37, and 16-62 ng/mL, for bacteria, yeasts, and molds, respectively (Delaquis and Mazza 1995; Isshiki and others 1992;

! %%! ! Shofran and others 1998). The bactericidal effect of AITC is mainly achieved by disrupting microbial cell membrane integrity, resulting in the leakage of the cellular metabolites. Moreover, it has been shown that the effectiveness of AITC against bacteria is sustained throughout the various bacterial growth stages (Lin and others 2000b).

Researchers have exploited the antimicrobial efficiency of AITC for food preservation. For example, Lin and others (2000) reported the bactericidal effects of

AITC against E. coli O157:H7 and Salmonella montevideo in fresh produce (Lin and others 2000a). Han (2000) showed that AITC inhibited the growth of Salmonella in beef

(Han 2000). Besides the direct addition to food, AITC has been used in conjunction with modified atmosphere packaging (MAP) to extend the shelf-life of food products. This method leverages the volatility of AITC to modify the composition of the internal headspace atmosphere of a package to achieve longer product shelf-life (Floros 1990).

To this end, beef chops, cured pork, sliced raw tuna, cheese, rye bread, and pears have been effectively preserved with MAP containing AITC vapor (Chacon and Buffo 2006;

Isshiki and others 1992; Mari and others 2002; Nielsen and Rios 2000; Winther and

Nielsen 2006).

AITC can be synthesized from chemical precursors or naturally extracted from

Cruciferae plants, and their use in food systems is widely accepted in many countries.

For example, in Japan, AITC is permitted for use as a preservative and AITC-containing antimicrobial films are commercially available (Lee 2005). The U.S. Food and Drug

Administration considers AITC as being generally recognized as safe (GRAS) in meat, fish, shellfish, and poultry products and in baked pies (Anonymous 2005). In Europe,

AITC is accepted as an additive for food or packaging purposes (Anonymous 1995) with

! %&! ! an allowable daily intake (DI) of 0.06 mg/kg of body weight (Winther and Nielsen 2006).

The MIC of gaseous AITC to microorganisms is 0.016 to 0.11 ppm (Delaquis and Mazza

1995), which is below the European AITC DI level, thereby further supporting the feasibility of applying the AITC in food packaging to extend their shelf-life.

AITC is volatile and has a pungent odor. The inherently pungent odor of AITC might be unacceptable when it is applied at high concentrations in food (Ko and others

2012b; Winther and Nielsen 2006). To reduce the volatility of AITC and limit its pungent odor, Chacon and Buffo (2006) have developed a method of encapsulating AITC in gum

Acacia to deactivate E. coli O157:H7 and reduced the strong AITC odor. Their encapsulation method involved freeze-drying an AITC-in-corn-oil/gum Acacia emulsion.

The dried AITC-containing gum Acacia mix was then chopped in a food processor to obtain microencapsulated AITC-gum Acacia powders with an AITC loading of 3.7–54.8 mg AITC/g. The microcapsules at 5% or 10% (w/w) were then added to finely chopped beef that was packed under nitrogen, and stored at 4°C for 18 days. The researchers found that microencapsulated AITC-gum Acacia microencapsules that produced greater than 1000 ppm of AITC in the beef samples were effective against E. coli O157:H7 as compared to those control treatments. In contrast, the AITC-gum Acacia microcapsules has milder odor than their unencapsulated counterpart (Chacon and Buffo 2006). A similar finding was reported by Ko and others (2012). These researchers spray dried an emulsion of AITC (purity> 95%), gum Arabic aqueous solution (25%, w/w), chitosan

(2.5%, w/v lactic acid solution) and Tween 20 (0.5%, w/w) to obtain gum Arabic-AITC microparticles with the loading of gum Arabic to AITC ratio of 3:1 (w/w). Gum

Arabic-AITC microparticles at loadings less than 0.1% (w/w, kimchi) were proven

! %'! ! effective in bacteria elimination without reducing product sensory quality after 15 days of kimchi storage (at both 4 and 10°C) (Ko and others 2012b). In another study, Vega-Lugo and Lim (2009) developed a method of producing AITC-%-cyclodextrin complex (0.4:2.4 mole ratio), followed by encapsulating the complex in electrospun poly(lactic acid) (PLA) and soy protein isolate (SPI) ultrafine fibers. They demonstrated that the kinetics of AITC release were RH-dependent and suggested that moisture could be used for activating the release of AITC from the PLA and SPI fiber carriers (Vega-Lugo and Lim 2009).

2.4 Mustard Seed as a Source of AITC

2.4.1 Overview of Mustard Seed and its Antimicrobial Efficiency

As discussed above, one natural source of AITC is mustard seed. Canada is among the largest exporters of mustard, and accounts for 35% of the world’s production and 50% of global exports (AAFC, 2013). The majority of mustard seed is used as a condiment in the form of either seed or oil by the food industry (AAFC 2013).

Recently, there is an increased interest in utilizing mustard seed and its derivatives for shelf-life extension of food, in light of the presence of AITC in the mustard seed. Nadajarah and others (2005) have applied mustard seed flour (5% to 20%, w/w) to ground beef that was packaged in nitrogen-flushed packages. They reported that at greater than 10% concentration, the mustard seed flour effectively inhibited the growth of

E. coli O157:H7 on meat during refrigerated storage (Nadajarah and others 2005b). In another study, Nilson (2011) applied deodorized yellow mustard powder at 4-6% (w/w) on dry cured Westphalian ham, and reported that the mustard powder eliminated E. coli

! %(! ! O157:H7 at a faster rate than the control during the curing process (Nilson 2011). Similar findings were reported by Graumann and Holley (2009) when the mustard seed flour

(60g/kg of ham, w/w) was applied to the surface of dry cured Westphalian ham. This study suggested that mustard flour can reduce about 40–50% the time that was required for the E. coli concentration to decrease to the target level (5 log10 CFU g&1) as compared to the untreated control group (Graumann and Holley 2009). These studies show that mustard seed flour/meal has the potential for retarding the growth of foodborne pathogens.

2.4.2 Sinigrin

Glucosinolates (thioglucosides) (Figure 2.1) are a group of nitrogen and sulphur containing compounds present in Cruciferae plants (Delaquis and Sholberg 1997). The characteristic flavor and aroma of Cruciferous vegetables are due, in part, to the presence of volatile compounds released from the enzymatic hydrolysis of glucosinolates (Fenwick and others 1983). Sinigrin is one of the major glucosinolates that presents in mustard seeds. Sinigrin itself has little antibacterial efficacy in its natural state in Cruciferous plants (Shofran and others 1998). But upon injury or mechanical disruptions of plant tissues, sinigrin undergoes a hydrolysis reaction to produce a variety of products depending on processing methods, including AITC, allyl thiocyanate (ATC), allyl cyanide, and 1-cyano-2,3-epithiopropane (Bones and Rossiter 1996; Fenwick and others 1983; Olsen and Sorensen 1981). Among these, AITC is the one that exhibits the strongest antimicrobial properties (Figure 2.2) (Shofran and others, 1998). It is reported that ATC may also exhibit antimicrobial properties, but its efficacy is unclear as it can be

! %)! ! converted into its AITC isomer via a quasi 6-membered ring transition state (Kyung and

Fleming 1994). However, AITC along with free glucose and sulphate produced from the hydrolysis reaction of sinigrin only could be produced under neutral pH condition in the presence of myrosinase (MacGibbon and Allison 1970 ; Shofran and others 1998).

Figure 2.1 - Sinigrin structure. Adapted from Saimi 2009.

!"#$%&'(%)* +&'&,#&'* -./0$%)* 1%$23&$0"('(2)%*

Figure 2.2 - Sinigrin hydrolysis to isothiocyanates and glucose.

2.4.3 Myrosinase

Myrosinase (!-thioglucoside glucohydrolase; EC 3.2.3.147) presents in many plants that contain glucosinolates (Li and Kushad 2005; MacGibbon and Allison 1970 ;

Yen and Wei 1993). In Cruciferae family plants, myrosinase usually presents as a group of isoenzymes, which are enzymes that differ in amino acid sequence but catalyze the same chemical reaction (Bones1990; Bones and Rossiter 1996). In Brassica napus and

Sinapis alba seeds, myrosinases are dimeric proteins with apparent molecular masses

! %*! ! of 135-150 kDa (Fenwick and others 1983; Lönnerdal and Janson 1973). Myrosinase and glucosinolates are separated into different cell compartments in plants, and can be brought together during cell disruption to form hydrolysis products. The activity of myrosinase is affected by several intrinsic and extrinsic factors, some of which are discussed below.

Myrosinase activity could be affected by several factors including pH, temperature, pressure of the headspace, and the presence of some chemical compounds (Bjorkman and Lonnerdal 1973; Li and Kushad 2005; MacGibbon and Allison 1970 ). Li and Kushad

(2005) showed that purified myrosinase from horseradish (Armoracia rusticana) roots exhibits high activity at 37-45°C when the pH was in the range of 5 to 8. Under optimal conditions, using sinigrin as a substrate, the Km and Vmax values were 0.128 mM and

0.624 µmol min–1, respectively (Li and others, 2005). They also found that the purified enzyme remained stable at 4°C for more than one year. Studies conducted by Van Eylen and coworkers (2008) observed that in Bis–Tris buffer solution (2 mmol/l, pH 6.5), the optimal temperature for yellow mustard seed myrosinase activity at atmospheric pressure was 60°C. As pressure increased, the reaction rate increased up to 200 MPa

(gauge pressure) and the optimal temperature shifted to 40°C (Van Eylen and others

2008). In their study, myrosinase from yellow mustard seed is stable at 60°C, but its activity was reduced by 94% after heating at 72.5°C for 10 min (Van Eylen and others

2008). With regards to the pressure, the enzyme activity began to decrease above 300

MPa, and at 600 MPa, no myrosinase activity was detected regardless of the temperature. On the other hand, the myrosinase activity could also be significantly (p<

0.05) increased in the presence of ascorbic acid (Fenwick and others 1983; Li and

! %+! ! Kushad 2005; Van Eylen and others 2008) and MgCl2 (Van Eylen and others 2008). In buffer solution (Bis–Tris, pH 6.5, 2 mmol/L), ascorbic acid drastically enhanced the myrosinase activity even at low concentrations (0.5 mmol/L), and a similar effect was found with MgCl2, although to a lesser extent.

Particle size of the mustard seed flour/meal powder is another parameter that affects AITC release kinetics. A previous study showed that particle size reduction in mustard seed flour/meal powder can increase the total amount of AITC released as compared to samples with larger particle sizes (Mazzola and Zhao 2010). Furthermore, since AITC is aliphatic, the presence of lipid in the meal can also affect its release behavior. Lim and Tung (1999) determined the vapor pressure of pure AITC and demonstrated that blending AITC with canola oil is effective in depressing the vapor pressure of AITC.

By manipulating the factors discussed above, mustard seed flour/meal powder can act as a promising natural carrier of AITC. To deliver AITC into packaging headspace, mustard seed meal powders could be packaged/immobilized in various carriers, such as sachets, films or fibers. Release of volatile compounds from different carriers will be discussed in the following section.

2.5 Control Release using Encapsulation

Controlled release is defined as “a method by which one or more active agents or ingredients are made available at a desired site and time at a specific rate” (Desai and

Park 2005). Many active compounds (antimicrobials, vitamins, nutraceuticals, flavors, etc.) are reactive, sensitive to ambient factors and/or volatile. Therefore, they need to be

! %,! ! protected. With appropriate controlled release technology, these materials can be used more conveniently and effectively in the food, cosmetic and pharmaceutical industries to reduce nutritional loss, mask or preserve flavors and aromas, and transform liquids into easily handled solid ingredients, while simultaneously prolonging shelf-life (Desai and

Park 2005). In addition, it could also be used to incorporate otherwise sensitive and volatile ingredients into a formulation to produce a time-release effect. Another advantage of encapsulation is its flexibility of formats when combined with foods (Figure

2.3). In light of the advantages of encapsulation, selected methods to encapsulate food compounds are discussed below.

Figure 2.3 - Physical and chemical types of encapsulation systems. Adapted from Pothakamury and Barbosa-Gnovas 1995.

One widely adopted method to control the release of bioactives is encapsulation, ! %-! ! which involves the coating or entrapment of the active compounds in single or a mixture of materials (Desai and Park 2005; Gibbs and others 1999). The coated or entrapped materials are usually identified as the core material, actives, fill, internal phase or payload (Gibbs and others 1999). Numerous ingredients (Table 2.2) including minute particles of ingredients (e.g., acidulants, fats, and flavors) and whole ingredients (e.g., raisins, nuts, and confectionary products) could be encapsulated in various formats

(Figure 2.4) (Desai and Park 2005). On the other hand, the coating material which is usually known as wall material, carriers or capsule, could be produced from a wide variety of sources (Gibbs and others 1999). For instance, the carrier can be made from gums (Ko and others 2012a), proteins (Fernandez and others 2009; Neo and others

2013), natural or modified polysaccharides (Poverenov and others 2013), synthetic polymers (Kayaci and Uyar 2012), and so on. The selection of microencapsulation

Table 2.2 - Some encapsulated food ingredients and the researched encapsulation methods.

Category Examples Researched method examples

Acidulants Lactic acid, acetic acid Fluidized bed techniques, extrusion

Sweeteners Sugars, aspartame Spray drying, fluidized bed techniques

Enzymes and Lipase, Penicilliumroqueforti Liposome entrapment microorganisms

Antioxidants Vitamin E, vitamin C Spray drying, liposome entrapment

Lipids Fish oil, rice bran oil Electrospinning, spray drying

Flavoring agents Citrus oil, mint oils Spray drying, extrusion

Preservatives AITC, gallic acid, thymol Electrospinning, spray drying

Colorants %-carotene, turmeric Extrusion, electrospun fiber

Source from (Gibbs and others 1999; Pothakamury and Barbosa-Gnovas 1995). ! &.! ! method and coating material is interdependent, and is governed by the coating-core materials’ properties (physical and chemical) and the intended applications

(Brannon-Peppas 1993; Desai and Park 2005).

Figure 2.4 - Various forms of capsules. Adapted from Gibbs and others 1999.

Generally, in an encapsulated system, the wall material protects the core material from undesirable environment factors while allowing small molecules or volatiles to pass in or out of the capsule carrier. Controlled release of the encapsulated core material can then be achieved by the influence of a specific stimulus present in the surrounding environment at a specified stage (Brannon-Peppas 1993; Desai and Park 2005;

Pothakamury and Barbosa-Gnovas 1995), such as pH change (enteric and anti-enteric coating), mechanical stress, temperature change, enzymatic activity, time, osmotic pressure and so on (Desai and Park 2005).

! &%! ! Various techniques have been developed for the encapsulation of bioactive compounds, such as spray drying, extrusion coating, liposome entrapment and electrospinning (Gibbs and others 1999; Pothakamury and Barbosa-Gnovas 1995;

Vega-Lugo and Lim 2009). Among these methods, electrospun fibers are one of the most promising carriers for controlled release applications (Fernandez and others 2009;

Kayaci and Uyar 2012; Neo and others 2013) and will be further discussed in the following section.

2.6 Electrospun Fibers for Controlled Release of Bioactives

2.6.1 Overview of Electrospun Fibers

Electrospinning is a process by which polymeric continuous ultrafine fibers, with diameters ranging from hundreds of nanometers to a few micrometers, can be produced by drawing a polymer solution (or polymer melt) using electrostatic force (Frenot and

Chronakis 2003). Although the process of electrospinning has been known for almost 80 years and the first patent was issued to Formhals in 1934 (US Patent, 1975,504), electrospinning and electrospun fibers still draw a great deal of interest (Reneker and

Chun1996). As studied by Frenot and Chronakis, nanofibers with a diameter of 100 nm have a geometrical surface area to mass ratio of approximately 100 m2/g (Frenot and

Chronakis 2003). Apart from the advantage of a high surface-to-volume ratio, three-dimensional yet seamless ultrafine fiber network assemblies (ultrafine fibrous membranes) can be formed onto the target substrate in a single step. This fiber spinning technique also can be used to lace together a variety of polymers, fibers, and particles to

! &&! ! produce ultrathin layers. The broad spectrum of applicable molecules, such as synthetic and biological polymers and polymerless sol-gel systems (Matsumoto and Taniola 2011), have further expanded the use of electrospun fibers for many applications. Their high surface-to-volume ratio and small pore size of electrospun ultrafine fibers make them interesting candidates for a wide variety of applications such as medical tissue scaffolds, wound dressing carriers for drugs, protective fabrics, high performance filter media, filler for nanocomposite materials, etc. (Matsumoto and Taniola 2011).

2.6.2 Electrospun Fibers as Encapsulation Carriers

More recently, researchers have found ways of dissolving or dispersing active compounds and small insoluble particles into the polymer solution, which upon electrospinning and evaporation of the solvent, results in functional ultrafine fibers.

Furthermore, since electrospun fibers can be applied for a wide range of active compounds simply by proper selection of an electrospinnable polymer, one can encapsulate drugs or other active compounds with different physical-chemical properties

(Huang and others 2003b; Potinenia and others 2003; Sill and von Recum 2008). In electrospun fibers, the active compound is dispersed throughout the matrix, and thus preventing the ‘‘burst release’’ in the applied environment (Potinenia and others 2003).

As a result, research on the use of electrospun fibers as carriers to facilitate the control release of active compounds has drawn increasing attention (Agrawal and others 2006).

For instance, Fernandez and coworkers (2009) have encapsulated %-carotene into electrospun zein (maize protein) nanofibers, demonstrating the potential of nanotechnology in food and nutraceutical formulation and coatings, bioactive food

! &'! ! packaging, and food processing industries (Fernandez and others 2009). Neo and others

(2013) have successfully encapsulated gallic acid into zein ultra-fine fibers at different loadings (5%, 10% and 20%) in order to control the burst release of the core material.

The encapsulated gallic acid was shown to retain its antioxidant activity after incorporation in zein electrospun fibers (Neo and others 2013). Vega-Lugo and Lim

(2009) used PLA and SPI to encapsulate AITC and the electrospun fibers they created were able to control the AITC release behavior as a function of RH. Overall, electrospinning techniques have shown promising results as an efficient and effective method for the preparation of many functional ingredients. Therefore, it is feasible to apply electrospun fiber methods to encapsulate mustard seed, and better control the release behavior of AITC by manipulating the polymer characteristics.

Because AITC is evolved during the hydrolysis reaction of glucosinolate in which water is a substrate, electrospun fiber carriers having moderate hydrophilicity may be preferred to control AITC release behavior. On the other hand, the hydrolysis of glucosinolate during the electrospinning polymer preparation processes must be inhibited. This requirement mandates that the encapsulating polymers should dissolve in relatively hydrophobic solvents to form electrospinnable fiber-forming solutions, so that when mustard seed powders are incorporated, premature release of AITC is minimized.

The moisture sensitivity of the carrier fiber could be further manipulated by blending of polymers with different hydrophilic properties. A literature survey indicated that several polymers possess these two criteria (e.g., PLA and poly(ethylene oxide) [PEO]). The material properties of PLA and PEO polymers are discussed in the following sections.

! &(! ! 2.7 Poly(lactic acid)

2.7.1 Overview

PLA (Figure 2.5) is a versatile biodegradable aliphatic polyester that has excellent mechanical properties, good biocompatibility and low toxicity. It has been used extensively in fields ranging from the pharmaceutical, biomedical, and automobile industries. It is also been used as a biodegradable plastic for disposable consumer products and food packaging (Ahmed and Varshney 2011; Albertsson and others 2011;

Lim and others 2008). The polymer can be processed into different forms using conventional processing methods including: extrusion, injection molding, film and sheet casting, stretch blow molding, extrusion blown film, thermoforming, and electrospinning

(Lim and others 2010). PLA is generally considered as a green polymer since its raw material (lactic acid) is derived from renewable sources, such as corn and sugarcane.

Furthermore, PLA is compostable through hydrolysis and enzymatic processes, during which it is converted to carbon dioxide and water (Ahmed and Varshney 2011; Jasim

Ahmed 2011; Saltzman 2001). PLA based packaging materials are GRAS, which puts it in a unique position for food applications (Ahmed and Varshney 2011).

Figure 2.5 - Chemical schematic structure of PLA. ! !

! &)! ! 2.7.2 Electrospun PLA Fibers

Electrospun PLA fibers are being used primarily in the tissue engineering and biomedical fields as tissue scaffolds and carriers for bioactive agents (Kenawy and others 2002), antibacterial silver nanoparticles (Xu and others 2006) and enzymes (Zhou and Lim 2009). However, with the vast amount of evidence supporting the efficient controlled release capacity of electrospun PLA fibers for various active compounds, the use of electrospun PLA fibers as carriers for active antimicrobial compounds in food systems is very promising. When electrospun PLA fibers are used to encapsulate bactericidal active compounds, for instance, and are then inserted into the food headspace, the polymer matrices is capable of extending the food shelf-life. Previously,

Vega Lugo and Lim (2009) and Zhou and Lim (2009) applied electrospun PLA fiber to encapsulate AITC and GOX, respectively. Both studies demonstrated that electrospun

PLA fibers are promising for active packaging applications.

2.8 Poly(ethylene oxide)

2.8.1 Overview of PEO

PEO, or polyethylene glycol (PEG) when the molecular weight is less than about

20 kDa, is biocompatible, nontoxic and has been recognized as an effective polymer for numerous applications (Griffith 2000; Honarbakhsh and Pourdeyhimi 2011). PEO is an amphiphilic uncharged polymer with the molecular formula (-CH2CH2O-)n (Figure 2.6).

The amphiphilicity is due to the ether oxygen that interacts with water, while the ethylene part participates in hydrophobic interactions (Bekiranov and others 1997; Maxfield and

! &*! ! Sheperd 1975). These properties enable PEO to be solubilized in various solvents, and it is compatible with many polymers (Agrawal and others 2006).

2.8.2 PEO as an Aid Polymer in Electrospinning

PEO can be electrospun alone to produce continuous PEO fibers (Doshi and

Reneker 1995). This polymer is also used as an electrospinning aid agent to promote the electrospinning of other polymers that otherwise cannot be electrospun (Honarbakhsh and Pourdeyhimi 2011; Vega-Lugo 2012). For instance, PEO has been added to SPI polymer as a spinning aid agent, as SPI itself is not electrospinnable (Vega-Lugo 2012).

In the study conducted by Honarbakhsh and Pourdeyhimi (2011), PEO was blended with

PLA in chloroform and electrospun into fibers. They reported that the incorporation of

PEO into PLA resulted in composite fibers that are more hydrophilic than those made of pure PLA (Honarbakhsh and Pourdeyhimi 2011). In another study, PEO was blended with chitosan to produce stable yet antimicrobial membrane structures that are showing promise for pharmaceutical and antimicrobial packaging applications (Zivanovic and others 2007).

Figure 2.6 - Chemical schematic structure of PEO.

! &+! ! 3. Justifications and Objectives

3.1 Justifications

The literature review in Section 2 shows that mustard seeds are a natural source of AITC, and thus possess strong antimicrobial properties (Nadajarah and others 2005b;

Nilson 2011; Rhee and others 2002). The use of mustard seed products, such as mustard seed meal powder (MSMP), for antimicrobial active packaging applications could potentially be promising. However, in order to facilitate its deployment in packaging systems, MSMP will need to be encapsulated or contained within a carrier so that the release rate of AITC can be optimized for the target applications.

To this end, electrospun fibers have high surface-to-volume ratio, which could be beneficial as an encapsulant for MSMP to facilitate the release of AITC. Moreover, the production of electrospun fiber membranes is considerably simple. From a material properties standpoint, the barrier properties of the fiber, which governs the release profile of the encapsulated AITC, can be manipulated readily by changing the formulation of the fiber-forming solution.

The efficacy of the envisaged MSMP-based active packaging system is hypothesized to depend on the release rate and maximum total amount of AITC released from the MSMP. Since AITC is generated by the enzymatic hydrolysis reaction of sinigrin, temperature and RH (which determines water activity) are expected to play important roles. If MSMP are encapsulated in electrospun fibers, the hydrophilicity of the electrospun carrier membranes, which is dependent on the polymer used, will also affect the release rate profile of AITC.

! &,! ! 3.2 Objectives

On the basis of the above considerations, the overall objective of this project is to utilize MSMP in conjunction with electrospinning technology to develop active membrane structures suitable for controlled release of AITC from MSMP to inhibit the growth of spoilage microorganisms in food products.

Specific objectives are:

• Investigation of AITC release kinetics from MSMP by studying the effect of

temperature and water activity on the enzymatic reaction between sinigrin and

myrosinase;

• Modification of MSMP physical properties to manipulate the AITC release kinetics

from sinigrin;

• Development and optimization of encapsulation methodology of MSMP in

electrospun PLA, PEO and PLA-PEO blend fibers;

• Investigation of AITC release kinetics from MSMP encapsulated in PLA, PEO and

PLA-PEO fibers;

• Characterization of surface morphology, chemical composition, and thermal

properties of the MSMP-contained electrospun membranes.

! &-! ! 4. Release of Allyl Isothiocyanate from Mustard Seed Meal Powder

4.1 Introduction

Sinigrin is a glucosinolate naturally present in mustard seeds and other edible plants from the Cruciferae family (Delaquis and Mazza 1995; Delaquis and Sholberg

1997; Lin and others 2000a; Lin and others 2000b; Nadajarah and others 2005a; Nielsen and Rios 2000; Park and others 2000). When tissues of these plants are disrupted, the cell-bound myrosinase comes into contact with sinigrin, causing the hydrolysis of the glucosinolate. Due to this reaction, the volatile AITC is released, resulting in the characteristic pungent flavor of crushed mustard seeds (Luciano and Holley 2009;

Pechácek and others 2000; Tsao and others 2000).

The broad-spectrum antimicrobial properties of AITC have been well documented.

In the liquid phase, the minimum inhibitory concentrations (MIC) of AITC are 5-1000, 1-4, and 50 ppm for bacteria, nonxerotolerant yeasts, and xerotolerant yeasts, respectively

(Kanemaru and Miyamoto 1990; Kinae and others 2000; Kyung and Fleming 1996). In vapor phase, AITC is more potent against microorganisms; the MIC against bacteria, yeasts, and molds are 34-110, 13-37, and 16-62 g/L, respectively. The antimicrobial properties of AITC have been leveraged for extending the shelf life of various food products, including beef, chicken breast, dried sausage, bread, peanuts and alfalfa seeds (Chacon and Buffo 2006; Chacon and others 2006b; Dhingra and others 2009a;

Nadajarah and others 2005a; Netramai and others 2011; Nielsen and Rios 2000; Park and others 2000; Seo and others 2012; Shin and others 2010; Suhr and Nielsen 2003;

Ward and others 1998). AITC has also been used in conjunction with packaging

! '.! ! structures to extend the shelf-life of foods (Lim and Tung 1997; Lim and others 1998).

Due to its volatility, AITC needs to be temporarily protected within a carrier matrix to prevent its uncontrolled release, which may impact the sensory attributes of the food products. Researchers have encapsulated AITC in various polymer carriers and investigated the release behavior of this volatile antimicrobial agent. For instance, Ko and others (2012) encapsulated AITC in gum Arabic and chitosan microparticles to significantly reduce the odor of AITC in a fermented cabbage product, kimchi, while stil inhibiting the growth of bacteria (Ko and others 2012b). They reported that the loading of

AITC-containing microparticles was limited to less than 0.1% (w/w) of kimchi, which prevented the detection of AITC off-flavor in the product. Vega-Lugo and Lim (2009) encapsulated AITC in electrospun SPI and PLA fibers to control the release of AITC. In their study, they exploited the moisture-sensitivity of SPI and PLA to activate the release of AITC from the fiber carriers. Another mean of reducing the volatility of AITC is by mixing it with a compatible diluent. To this end, canola oil has been shown to be effective for depressing the vapor pressure of AITC (Lim and Tung 1997). Shin and others (2010) investigated the release of AITC from an AITC-canola oil blend sealed in high-density polyethylene film to inhibit the growth of L. monocytogenes and S. typhimurium in raw chicken packaged in MAP.

In the present study, rather than using synthetic or regenerated polymers, a mustard seed matrix was used as a natural carrier to control the release of AITC. Since the liberation of AITC from sinigrin requires the presence of water (Parkin 2008), dry milling of mustard seed into powder does not trigger the release of AITC. However, when the powder is rehydrated with moisture, the hydrolysis reaction will be activated, thereby

! '%! ! causing the release of AITC. By controlling the hydrolysis reaction of sinigrin, the release behavior of AITC from mustard seeds can be manipulated.

Although the use of mustard seed as a natural antimicrobial is promising, to date, a systematic study on AITC release kinetic, as affected by temperature and RH, has yet to be conducted. The objective of this study is to investigate the effects of RH, temperature, particle size, and lipid content on the release kinetics of AITC from mustard seed meal powder (MSMP). Mustard seed meal is a byproduct from mustard oil extraction, and is mainly used as low value animal feeds (Hendrix and others 2012). The development of alternative uses of mustard seed meal for various applications will increase the value of the mustard crop.

4.2 Materials and Methods

4.2.1 Materials

MSMP was donated by Biofume Technologies Inc. (Saskatoon, SK, Canada).

AITC (> 99%, GC grade) and hexane (> 95%) were purchased from Sigma Aldrich

(Oakville, ON, Canada).

4.2.2 Preparation of MSMP

Four varieties of MSMP were prepared: (1) As-received MSMP (APR-MSMP);

(2) Defatted-MSMP obtained by defatting APR-MSMP with hexane using the Soxhlet oil extraction method (Nielson 2003); (3) Ground-MSMP obtained by grinding APR-MSMP with a ball mill (Model PM 100, Retsch GmbH, Haan, Germany) at 450 rpm for one hour;

! '&! ! and (4) Ground-defatted-MSMP obtained by grinding the defatted-MSMP with the ball mill at 450 rpm for one hour. All samples were prepared at 24 ± 2°C and 60 ± 5% RH.

Samples were freshly prepared and stored in airtight glass jars with no more than two weeks before AITC release experiments.

4.2.3 Physical and Chemical Characterization of MSMP

The lipid content of MSMP samples was determined by hexane extraction in a

Soxhlet apparatus for 6 hours at 55 ± 5°C, according to AOAC method (Arif and others

2012; Mustakas and others 1961; Nielson 2003). The crude protein content of MSMP was determined by the Dumas method using a protein/nitrogen analyzer (model FP-528,

LECO, U.S.A.) according to AOAC 992.23 protocol for oil seed products. A nitrogen-to-protein conversion factor of 6.25 was applied for the protein analysis (Arif and others 2012; Ezeagu and others 2002; Siemens 2010; Singh and Sinhal 2011). The moisture content of 5 ± 0.5 g of sample was determined using a microprocessor-based

IR moisture/solid content analyzer (model IR-50, Denver Inc., USA). The ash content was measured by incinerating 5 ± 0.5 g samples at 550°C to a constant weight (Nielson

2003). All of the characterizations procedures were undertaken in triplicate at 24 ± 2°C,

60 ± 5%RH and calculated on a wet basis. Maximum and minimum diameters of MSMP particles were measured using a light microscope (BX60, Olympus America Inc., NY,

U.S.A.). To disperse the MSMP particles, samples were mixed with canola oil before being examined under the microscope. Particle size distribution was analyzed using

Image-Pro Plus 6.0 software package (Media Cybernetics Inc., Betheda, Md., U.S.A.).

The color measurement of each sample was measured using a portable Minolta

! ''! ! spectrophotometer (model CR-300, Minolta, Osaka, Japan). MSMP specimens were spread evenly onto petri dishes before being placed on the Minolta calibration plate

(CR-300). L*, a*, b* values were measured.

4.2.4 Scanning Electron Microscopy (SEM) Analysis

For SEM analysis, a thin layer of MSMP sample was immobilized on carbon tape and then attached to a metal stub. The samples were then coated with gold and palladium (20 nm) using a sputter coater (Model K550, Emitech, Ashford, Kent, UK).

MSMP specimen morphology was examined using a scanning electron microscope

(model S-570, Hitachi High Technologies Corp., Tokyo, Japan) at an accelerating voltage of 10 kV. Micrographs for each MSMP sample were taken from at least five locations. Micrograph analysis was conducted using image-processing software (Image

Pro-Plus 6.0, Media Cybernetics Inc., Bethesda, MD, U.S.A.).

4.2.5 Headspace Analysis of MSMP

AITC release kinetics from MSMP samples were determined using an automatic headspace analysis system consists of an environmental chamber (Model MLR-350H,

Sanyo Corp., Japan), a gas chromatograph (GC) equipped with flame ionization detector

(FID) (GC 6890, Agilent Technologies Inc., Santa Clara, CA, U.S.A.), a controller (SRI

Instruments Inc., Las Vegas, NV, U.S.A.), and stream selection valves (Model

EMTCA-CE, VICI Valco Instruments Co. Inc., Houston, TX, U.S.A.) (Figure 4.1).

Stainless steel tubing with an OD of 1/16 inch was used throughout the system.

! '(! !

Figure 4.1 - Headspace sampling system to study the AITC release kinetics from MSMP.

To study the release kinetic of AITC, 20 ± 0.5 mg of MSMP was spread evenly on the bottom of a beaker and then placed in a sealed glass jar (1 L) equipped with a septum (Figure 4.1). For the first set of samples, an aliquot of 6,15 or 35 µL of distilled water was injected onto a filter paper to provide an initial 85% RH at 5, 20 and 35°C, respectively. In the second set of samples, the amounts of water were increased by a factor of two (12, 30 and 70 µL) to saturate the headspace (i.e., 100% RH). Test jars were kept in environmental chambers maintained at the target temperatures selected to represent typical refrigerated, ambient, and abusive conditions.

! ')! ! Sampling jars and beakers were first purged with dry air for two minutes before each experiment. Three replicates were carried out for each test condition. The AITC concentration in the headspace of the jar was analyzed using a procedure similar to that described by Vega-Lugo and Lim (2009). During headspace analysis, an aliquot of 15 mL jar headspace gas was extracted through the septum and transferred to the GC. The same volume of air was injected back into the glass jar to replace the extracted headspace air. Calibration of the FID was performed by injecting the AITC standard into the jars to provide headspace concentration ranging from 0.1 to 0.5 µL/L. Flow rates for nitrogen (carrier gas), hydrogen and air were 30, 30 and 200 mL/min, respectively. Oven and detector temperatures were set at 90 and 200oC, respectively. Retention time and peak area were analyzed using chromatographic software (Peak Simple 393-32 bit, SRI

Instruments, Torrance, CA, U.S.A.).

The mass of AITC in the extracted headspace gas was calculated by summing the recorded mass (Mr) and the accumulated mass loss (Ml) up to the present sampling point (Eqs. 1-3):

Mr = Cr Vb (1)

Ml = (Cr-1x Ve) + Ml-1 (2)

Mc = Mr + Ml (3) where Cr is the recorded AITC concentration calculated based on calibration constants;

Cr-1 is the recorded concentration of the previous sampling point; Vb is the volume of the glass jar; Ve is the volume of headspace gas extracted; Ml-1 is the sum of mass loss of all the previous sampling points before Ml; and Mc is the corrected AITC mass at any given sampling point.

! '*! ! 4.2.6 Data Analysis

Statistical analysis was conducted using SPSS 20 software (IBM® SPSS Inc.,

Chicago, IL, U.S.A.). One-way Analysis of Variance (ANOVA) was carried out to determine the significance differences between treatments at 95% confidence interval.

AITC release kinetics from various MSMP samples were modeled empirically using the modified Gompertz equation (Lay and others 1999; Mu and others 2006), which is a suitable model for describing the cumulative release kinetics of AITC:

,* $k " exp(1) ',. Qt = Qmax exp+!exp& (# ! t) +1)/ (4) -, % Qmax (0, where Qt is the amount of the AITC released at time t; Qmax is the cumulative amount of

AITC released; k is the rate constant, and l is the lag time. Non-linear regression analyses were conducted to determine the three parameters using the Solver function in the Microsoft® Excel spreadsheet package (Microsoft Office 2011, WA, U.S.A.). The

GRG Nonlinear algorithm option was selected to minimize the residual sum of squares by changing the three parameters, without imposing any constraints.

4.3 Results and Discussion

4.3.1 Characterization of MSMP

The Lipid content of the APR-MSMP and Ground-MSMP was 16.6 and 15.6%, respectively, which is lower than the typical lipid contents (30 to 47%) for yellow mustard seed (AAFC 2013), indicating that the APR-MSMP samples had been partially defatted by the supplier. By contrast, no lipid was detected in the defatted samples (Table 4.1). Sample

! '+! ! moisture content at the time of testing ranged between 7 and 7.5 % (wet basis). Compared with the un-defatted samples, both protein and ash contents were significantly higher in the defatted MSMP due to the increased proportion of these constituents in the test specimens after the lipid component had been removed. Similar findings were reported by Sharma and others (2012).

The color of the four MSMP samples was significantly different (p< 0.05), as reflected by their L*, a*, b* values (Table 4.1). The b* value of APR-MSMP was highest

(13.76), showing that the yellowness of APR-MSMP was most intense when compared to the other three samples. On the other hand, the yellowness of defatted MSMP samples was significantly lower than the un-defatted counterparts, due to the removal of carotenoids and other pigments during the Soxhlet extraction process (Appelovist 1970;

Lindgren and others 2003). The APR-MSMP exhibited a significantly lower L* value, indicating that it had the darkest color as compared to the other three samples.

Changes in a* value were minimal, although the APR-MSMP was significantly more red in color than the others.

The particle size distribution of the APR-MSMP was heterogeneous with greater than 20% of particles exceeding 50 µm, while more than 50% were smaller than 25 µm

(Figure 4.2A). The unground-defatted samples exhibited a similar trend (Figure 4.2C). As expected, the ground MSMP samples (Figures 4.2B, 4.2D) had smaller overall particle sizes than the unground APR-MSMP and defatted-MSMP samples (Figures 4.2A and

4.2C). With both ground samples, approximately 30% of the particles had diameters around 10 µm, with a total of greater than 80% of the particles smaller than 25 µm. About

3% of the ground MSMP samples were greater than 50 µm, as compared with more than

! ',! ! 20% for the unground specimens. These results indicated that substantial size reduction had occurred during the ball milling. The defatting treatment did not affect the grinding of the MSMP, as illustrated by the similar particle size distribution profiles of the

Ground-MSMP and Ground-defatted-MSMP (Figures 4.2B and 4.2D).

Table 4.1- Physical and chemical properties of various MSMP samples.

Ground-defatted-M Properties APR-MSMP Ground-MSMP Defatted-MSMP SMP

Lipid, % 16.6 ± 0.1a 15.6 ± 0.2a 0.0b 0.0b

Moisture, % 7.5 ± 1.0ab 6.9 ± 0.4ab 7.5 ± 0.0b 7.1 ± 0.1a

Protein, % 33.6 ± 0.4a 33.8 ± 0.0a 39.7 ± 0.1b 40.5 ± 0.6b

Ash, % 5.5 ± 0.0a 5.4 ± 0.1a 6.5 ± 0.1a 6.4 ± 0.0a

L* value 45.25 ± 1.01a 51.94 ± 0.26b 53.22 ± 0.55c 52.03 ± 0.35b

a* value 2.34 ± 0.01a 0.0033 ± 0.05b 0.05 ± 0.05b -0.14 ± 0.02c

b* value 13.76 ± 1.00a 11.63 ± 0.15b 7.39 ± 0.14c 7.21 ± 0.07c

Values are mean ± standard deviation (n= 3). Means within a row with different superscript letters are significantly different (p< 0.05).

The microstructures of the MSMP samples are presented in Figure 4.3. The

APR-MSMP contained chunky particles with rough surface textures, which under high magnification, revealed the presence of wavy surfaces (Figure 4.3A). The defatted-MSMP counterpart had similar microstructure except that the particulates observed under high magnification were more jagged in appearance (Figure 4.3C). By contrast, large chunky structures were not observed in the Ground-MSMP and Ground-defatted-MSMP samples

(Figures 4.3B and 4.3D). The fine particulate observed under high magnification could be the contributor to the increased AITC release rate.

! '-! !

Figure 4.2 - Particle size distribution of mustard seed samples: (A) APR-MSMP; (B) Ground-MSMP; (C) Defatted-MSMP; and (D) Ground-defatted-MSMP. !

Figure 4.3 - Representing scanning electron micrographs of various MSMP samples.

! (.! ! 4.3.2 AITC Release Kinetics as Affected by Temperature and RH

AITC release profiles for the various MSMPs are summarized in Figure 4.4, which all show initial lag phases, followed by increasing AITC concentrations as time progressed. Plateaus were detected for samples tested under elevated RH and temperature, while continuously increasing trends were observed for samples tested under relatively lower temperature and RH conditions. The presence of the lag phase is mainly due to the time needed for water to evaporate into the headspace, to the MSMP matrix, to react with sinigrin, as well as for the desorption of AITC from MSMP particles into the headspace. The calculated parameters for the modified Gompertz equation are summarized in Table 4.2. As shown, the predicted maximum amounts of AITC released were significantly lower for samples exposed to 85% RH than those exposed to 100%

RH (p< 0.05). For example, at 20°C, when RH was increased from 85% to the saturation level, the maximum amount of AITC released increased from 4.76 to 10.33, 4.52 to

13.16, 3.33 to 14.41, and 3.04 to 14.75 mg/g for APR-MSMP, Ground-MSMP,

Defatted-MSMP, and Ground-defatted-MSMP, respectively (Table 4.2). This is expected since water is a substrate in sinigrin hydrolysis (Castelo-Branco and Pinto 2010; Korsrud and Bell 1966; Shofran and others 1998). Also, a fraction of the moisture from the headspace could be absorbed by cellulose in the MSMP matrix, which represents approximately 15% of mustard seeds by weight (Ohlson and Oljefabriker 1972). Some of the water molecules that are tightly bound to cellulose might not be available for the sinigrin hydrolysis reaction.

At 35oC and 100% RH condition, assuming that the majority of glucosinolate was hydrolyzed in the presence of an excess amount of water, the predicted total AITC

! (%! ! released ranged from 13.36 to 17.46 mg/g MSMP. These values are within the same order of magnitude as the results reported by Bell and others (1971) for solvent-extracted mustard seed meals (11.8 mg/g). The different maximum amounts of

AITC observed from four MSMP samples may be caused by the different initial sinigrin contents of various MSMP samples due to the different treatments applied. Potentially, a significant amount of sinigrin might have been hydrolyzed due to its exposure to ambient moisture.

AITC release rate increased significantly as temperature increased, as reflected by the increasing k values as a function of temperature (Table 4.2). This is consistent with results reported by other researchers (Bjorkman and Lonnerdal 1973; Eylen and others 2005). Another reason for the increasing release rate of AITC with temperature is related to the enhanced diffusion of water into MSMP and desorption rate of AITC from the MSMP matrices. On the other hand, no correlations between lag time duration and temperature or RH were observed (Table 4.2). The lack of correlation can be attributed to the fact that water vapor buildup inside the sampling bottles during the onset of the experiments was not a strong function of these two variables.

! (&! !

! ('! !

Figure 4.4 - Representative AITC release profiles from various MSMP samples in 1 L headspace glass jar under different temperature and humidity conditions. Symbols are experimental data, while continuous lines are fitted curves based on the modified

! ((! ! 4.3.3 Particle Size Effects on AITC Release Kinetics

Comparing the AITC release profiles of ground and unground MSMP samples, it is evident that the release of AITC was significantly (p< 0.05) faster in the former at all temperature and RH conditions tested (Figure 4.4). For example, at high RH, the rate constants for un-defatted MSMP were 4.11, 12.10, 40.99 min-1 (APR-MSMP) and 6.30,

17.67, 59.78 min-1 (Ground-MSMP) at 5, 20 and 35°C, respectively (Table 4.2). The increased AITC release rates for the ground samples could be attributed to the larger surface-to-volume ratios as compared to the unground counterparts, which enhanced the mass transports of both water and AITC. Similar results were reported by Sharma and others (2012), who observed that the reduction in particle size of mustard meal particles induced significantly faster glucosinolate transformation in soil (Sharma and others 2012).

For the defatted MSMP, size reduction did not significantly change the average rate constants between the ground samples and un-ground samples (p> 0.05), although the average rate constant values were smaller in ground samples. This result indicates that the hexane Soxhlet extraction and/or size reduction procedure considerably affected the myrosinase-catalyzed reaction. The reason for this observation is unknown, but was likely due to partial inactivation of the enzyme and/or hydrolysis/leaching of sinigrin during the lipid extraction and the material handling processes.

! ()! ! Table 4.2 - Fitted model parameters (Qmax, k and !) of modified Gompertz equation for various MSMP samples under different temperature (5, 20, and 35°C) and RH (85 and 100% RH) conditions.

Temp Qmax Rate constant, k Lag time, ! -1 1 Samples eratur (mg/g MSMP) (min ) (x 10 min) e (°C) 85% RH 100% RH 85% RH 100% RH 85% RH 100% RH 5 2.48±0.41Aa 6.25±0.34Ba 2.34±0.29Aa 4.11±0.04Ba 6.81±3.48Aa 16.56±3.66Ba APR-MSMP 20 4.76±0.53Ab 10.33±1.17Bb 11.56±0.45Ab 12.10±2.39Ab 10.24±2.88Aa 1.96±1.75Bb 35 7.07±0.72Ac 13.36±0.30Bc 27.23±3.70Ac 40.99±5.83Bc 7.99±1.33Aa 5.87±1.08Ab 5 5.77±0.66Aa 12.65±1.07Ba 4.19±0.42Aa 6.30±0.85Ba 5.97±5.58Aa 10.40±3.24Aa Ground-MSMP 20 4.52±0.41Ab 13.16±1.03Ba 9.84±0.84Ab 17.67±0.70Bb 5.49±1.55Aa 13.60±2.62Ba 35 4.84±0.48Aab 14.04±0.26Ba 21.08±1.55Ac 59.78±9.35Bc 6.03±0.69Aa 11.98±0.95Ba 5 6.02±0.11Aa 8.57±1.28Aa 5.24±0.89Aa 6.53±0.72Aa 9.56±2.12Aa 10.76±3.41Aa Defatted-MSMP 20 3.33±0.34Ab 14.41±1.62Bb 10.85±1.92Ab 20.67±4.64Bb 8.87±0.85Aa 11.86±1.99Ba 35 5.61±0.52Aa 14.55±0.72Bb 27.36±3.66Ac 41.37±5.11Bc 5.72±0.99Ab 8.17±0.24Ba 5 4.71±1.41Aab 6.72±1.10Aa 2.75±0.79Aa 5.68±0.74Ba 21.41±0.35Aa 10.58±2.98Ba Ground-defatted 20 3.04±2.95Aa 14.75±1.64Bb 6.57±0.34Ab 14.70±1.93Ba 7.62±1.37Ab 25.12±2.84Bb -MSMP 35 5.64±1.01Ab 17.46±0.59Bc 18.22±2.08Ac 35.46±7.84Bb 5.77±0.70Ab 13.12±1.55Ba

Qmax is the predicted maximal amount of AITC release from one gram of MSMP, k is the release rate constant, and ! is the lag time before the release of AITC was detected. Values are means of three replicated measurements ± standard deviation (n=3).

For each of the parameters (Qmax, k or !), mean values with different upper case superscript letters within a row for specific MSMP sample at a given temperature are significantly different due to the RH effect (p< 0.05). Mean values in a column with different lowercase superscript letters are significantly different d ue to the temperature effect (p< 0.05). Data analysis is carried out using One-Way ANOVA with Tukey’s test at "=0.05.

! "#! ! 4.3.4 Lipid Content Effects on AITC Release Kinetics

In the presence of excess moisture (100% RH), the release rate constant values for APR-MSMP were lower than with the Defatted-MSMP specimens (Table 4.2). This observation is expected due to the partitioning of AITC into the lipid phase in the

APR-MSMP, and the solubility of AITC in oil (Lim and Tung 1997). However, an opposite trend was observed for the ground samples: at 100% RH, the AITC release rates at 5, 20 and 35oC, were 6.3, 17.67 and 59.78 min-1 for Ground-MSMP, and 5.68, 14.70 and

35.46 min-1 for Ground-defatted MSMP, respectively. These results indicate that grinding of mustard meal powder might have partially inactivated the myrosinase due to the frictional heat generated.

4.4 Conclusions

This study showed that the release of AITC from mustard seed meal powder can be activated using headspace moisture. The rate and the maximum amount of AITC released are dependent on ambient RH and temperature, as well as particle size and/or oil content of the mustard meal powder. The amount of AITC released from the various

MSMP samples ranged from 2 to 17 mg/g MSMP within 24 h under the experimental conditions tested. With the reported MIC for microorganism in the vapor phase that ranges from 10 to 100 mg/L (Isshiki and others 1992; Shofran and others 1998), the quantities of AITC in the mustard seed meal powders are within the design window for deployment in active packaging, especially in modified atmosphere packaging applications, to inhibit the growth of spoilage microorganisms prevalent in perishable and

! (+! ! minimally processed food products. To this end, further investigations to optimize the

AITC release profile (e.g., burst versus sustained release) by manipulating the release parameters are needed for specific food applications. Due to its strong odor at elevated concentration and propensity to induce discoloration in fresh meats (Shin and others

2010), it is prudent to optimize the delivery system from both shelf-life and sensory perspectives, targeting specific products.

! (,! ! 5. Control Release of AITC from Defatted Ground MSMP with Blended

Electrospun Fibers of PLA and PEO

5.1 Introduction

AITC is a natural compound that has been studied extensively due to its strong ability to eliminate food-borne microorganisms such as molds, yeast and other food-borne pathogens (Delaquis and Mazza 1995; Kinae and others 2000; Mari and others 2002; Nadajarah and others 2005a; Shofran and others 1998). Besides direct application to food products, AITC has also been used in conjunction with MAP to extend the shelf-life of food products, including fresh beef, cured pork, sliced raw tuna, cheese, egg sandwich, noodles, and pasta (Jung and others 2009; Parashar and others 2011;

Serrano and others 2008; Shin and others 2010; Suppakul and others 2003).

The antimicrobial efficacy of synthetic AITC and some of its limitations, such as volatility and strong odor, are discussed in Chapter 3. To facilitate the delivery of AITC to food, encapsulation methods have been developed to encapsulate AITC in various polymer matrices (Ko and others 2012a; Vega-Lugo and Lim 2009). Another method to deliver AITC to food is to utilize natural plant components that contain AITC, such as those from the Cruciferae family. As one of the important oilseeds, mustard seed is usually pressed and ground for the extraction of oil leaving a byproduct mustard seed meal powder (MSMP) that is used for low value applications (Marcone and others 1997).

Since MSMP is rich in the AITC precursor – sinigrin. This byproduct may be beneficial for

MAP applications in food preservation. Because water is a required substrate for the enzymatic hydrolysis of sinigrin (Bjorkman and Lonnerdal 1973), the release rate of AITC

! (-! ! from the mustard seed could be manipulated by controlling its accessibility to water. This behavior is elucidated by studies presented in Chapter 4, in which the effects of RH and temperature were investigated on the rate of release of AITC from various processed

MSMP.

The focus of this chapter is to encapsulate MSMP in electrospun fibers. These fibers have high surface-to-volume ratios, which is beneficial for facilitating the release of the encapsulated active compounds (Pham and others 2006; Schiffman and Schauer

2008; Sill and von Recum 2008). Moreover, the physical-chemical properties of fiber mats can be manipulated readily by altering polymer compositions (Huang and others

2003). For example, by blending different types of polymers, one could develop encapsulants with differing physical-chemical properties such as hydrophilicity, fiber diameter, density, glass transition temperature, etc. (Huang and others 2003a; Potinenia and others 2003; Sill and von Recum 2008). By encapsulating MSMP in electrospun fibers, a triggering mechanism that activates the release of AITC is envisioned that exploits the desirable interaction(s) between the fiber and the moisture in the air.

For the purpose of encapsulating MSMP, the polymer should have moderate hydrophilicity while still being soluble in non-aqueous solvents to prevent sinigrin hydrolysis during solution preparation. PLA is an ideal polymer for this application since it is a weak moisture barrier. It can also be dissolved in chloroform/dimethylformamide

(DMF) blend and the resulting polymer solution can be electrospun readily into fibers

(Huang and others 2003a; Vega-Lugo and Lim 2009). Moreover, the release rate of

AITC from PLA could be modified by blending the polymer with another compatible polymer, such as PEO. PEO is an interesting polymer since it is amphiphilic. It is also

! ).! ! used as a versatile electrospinning aid to promote the formation of fiber (Agrawal and others 2006; Bekiranov and others 1997; Honarbakhsh and Pourdeyhimi 2011; Maxfield and Sheperd 1975; Zivanovic and others 2007). PEO and PLA share similar solubility parameter value ()) in the range of 9.5-9.8. Accordingly, the two polymers are expected to form homogeneous blends (Younes and Cohn 1988).

To the best of our knowledge, no studies have focused on controlling the release of AITC from MSMP by encapsulating it in the nonwoven electrospun membranes.

Therefore, the objective of this study is to investigate the release behavior of AITC from

MSMP encapsulated within nonwoven electrospun PLA-PEO membranes when exposed to saturated humidity. Morphological, thermal, and spectral analyses of the resulting electrospun matrices were also conducted.

5.2 Materials and Methods

5.2.1 Materials

PLA (6201 D) was donated by NatureWorks LLC (Minnetonka, MN, U.S.A.). PEO

(Mw 300 kDa), hexane, chloroform (HPLC grade), and DMF were purchased from Sigma

Aldrich (Oakville, ON, Canada). MSMP was donated by Biofumes Technologies Inc.

(Saskatoon, SK, Canada). An AITC standard (>98% GC purity) was purchased from

Fluka Chemical Co. (Steinheim, Germany).

! )%! ! 5.2.2 Methods

5.2.2.1 MSMP Preparation

MSMP sample was defatted and ground as discussed in Chapter 4. Briefly, the

Soxhlet oil extraction with hexane was used to remove the lipid, while a ball mill (Model

PM 100, Retsch GmbH, Haan, Germany) was used to decrease the particle size. The ball mill was operated at 450 rpm for one hour using eight stainless steel balls (diameter

20 mm) in a 500 mL stainless steel grinding bowl. Sample size distribution and the sample chemical composition of the resulting MSMPs were investigated and discussed in previous chapter. Sample preparation was conducted at 24 ± 2°C and 60 ± 5% RH.

Samples were freshly prepared and stored in airtight glass jars for no more than two weeks ahead of the encapsulation experiments.

5.2.2.2 Solution Preparation

The concentrations of all solution components and solutions were expressed on a w/w basis. In the current study, two batch fiber-forming solutions, i.e., 9% PLA solution and 3% PEO solution, were prepared by dissolving PLA and PEO in a mixture of chloroform and DMF (9:1, w/w), respectively. Polymer solutions were then mixed using magnetic stirrers for 12 h at 24 ± 2oC.

To study the effect of PLA and PEO composition on the fiber characteristics and

AITC release kinetics, 9% PLA and 3% PEO batch solutions were blended at different ratios of 100:0, 75:25, 50:50, 25:75, 0:100 (w/w), and the resulted fiber membranes are designated as 100PLA0PEO, 75PLA25PEO, 50PLA50PEO, 25PLA75PEO,

! )&! ! 0PLA100PEO, respectively, according to their weight ratio of PLA and PEO stock solutions. The same amount of the defatted and ground MSMP was dispersed into the five polymer solutions at a weight ratio of 9% of the polymer solution by mixing using a magnetic stirrer for three hours. All of the experiments were completed in triplicate.

5.2.2.3 Electrospinning

Polymer solutions were electrospun using a horizontal setup (Figure 5.1) to avoid the solution dripping onto the collector. Polymer solutions were drawn into a 3 mL plastic syringe (KD Scientific, Holliston, U.S.A.) and pumped at 3 mL/h via a piston assembled using a syringe pump (Orion M361, Thermo Scientific, Oakville, ON, Canada). A

20-gauge blunt tip stainless steel needle was attached to the 3 mL syringe, which served as a spinneret. The spinneret was connected to the positive electrode of a direct current power supplier (DC model ES50P-50W/DAM, Gamma High Voltage, Ormond Beach, FL,

U.S.A.), operating at a constant voltage of 11 ± 1 kV. A circular stainless steel plate covered by aluminum foil was grounded to collect the electrospun fibers. The distance between the tip of the spinneret and the collector was kept at 15 ± 1 cm. Electrospinning experiments were conducted in an environmental chamber (Model MLR-350H, Sanyo

Corp., Japan) maintained at 25 ± 0.5oC and 20 ± 1% RH. Upon collection, electrospun fibers were transferred into a vacuum oven for 48 h at 40oC to evaporate residual chloroform and DMF. Samples were then stored in a desiccator (Fisher Scientific,

Oakville, ON, Canada) before further characterization.

! )'! !

Figure 5.1 - Schematic diagram of the experimental setup for electrospinning of MSMP-containing fibers.

5.2.3 Fiber Morphology and Size Analysis

Fiber microstructure and morphology was evaluated using a scanning electron microscope (SEM) (Model S-570, Hitachi High Technologies Corp., Tokyo, Japan).

Electrospun fiber samples were mounted on metal stubs using double-adhesive carbon tape, and then coated with an ultrathin (20 nm) layer of gold and palladium using a sputter coater (Model K550, Emitech, Ashford, Kent, UK). An accelerating voltage of 10 kV was applied during the analysis. Samples were firstly detected under the lowest magnification to evaluate the overall uniformity of the fiber samples. Samples were then examined under higher magnifications to evaluate fiber morphology and diameter. ! )(! ! Samples were randomly selected from three different locations on the electrospun fiber membranes. Micrographs that were selected for quantitative analyses had representative morphologies. The tests were undertaken at 22 ± 2oC and 60 ± 5% RH.

To determine the average diameters of the fiber samples, the micrographs obtained were analyzed with image processing software (Image Pro-Plus 6.0, Media

Cybernetics Inc., Bethesda, MD, U.S.A.). For pristine fiber samples, the diameters of the fibers were recorded, while for MSMP-containing samples, only the fibrous portions were measured. The average fiber diameter and size distribution were determined from approximately 200 random measurements from representative micrographs.

5.2.4 Attenuated Total Reflection–Fourier Transform Infrared (ATR-FTIR)

Evaluation

FTIR spectra of pristine and MSMP-containing electrospun fiber samples, and

MSMP were obtained using a FTIR spectrometer (IRPrestige21, Shimadzu Corp., Kyoto,

Japan), equipped with an ATR (Pike Technologies, Madison, Wis., U.S.A.). The test was undertaken by performing an average of 40 scans for each sample at 4 cm-1 resolution.

All of the fiber mats and MSMP samples were directly mounted on the ATR crystal, compressed with a press, and scanned between 700 to 3900 cm-1 at room temperature conditions (22 ± 2°C, 60 ± 5 % RH). Wavenumber values for the peaks of interest were determined with the spectrum analysis software IRsolution (Shimadzu Corp., Kyoto,

Japan). Three sampling spots were randomly selected from the fiber samples and each spot was analyzed three times.

! ))! ! 5.2.5 Thermal Analysis

Differential scanning calorimetry (DSC) analysis was carried out using a DSC

(Q2000, TA Inc. New Castle, DE, U.S.A.) under nitrogen (18 mL/min) purged atmosphere. In all tests, 3 to 5 mg of sample was cut into small pieces from the middle region of the electrospun membranes; the selected fiber membranes were then stacked closely and sealed in alod-aluminum pans. A similar amount of MSMP powder was also analyzed to characterize its thermal properties. The pristine fiber samples were heated from 0 to 220°C, while MSMP and MSMP-containing fiber samples were heated from 0 to 260°C; all samples were heated at 10°C/min. Thermal data were analyzed using TA

Universal Analysis Software (TA Inc. New Castle, DE, U.S.A.). From the thermograms, glass transition temperature (Tg) and the melting temperature (Tm) were determined for

MSMP, pristine and MSMP-containing electrospun fibers. Each Tg and Tm value was expressed as the average of three determinations.

5.2.6 Headspace Analysis

AITC release kinetics from MSMP samples was determined using an automatic headspace analysis system as described in Chapter 4 (Figure 4.1), which consisted of an environmental chamber (Model MLR-350H, Sanyo Corp., Japan), a GC equipped with FID (GC 6890, Agilent Technologies Inc., Santa Clara, CA, U.S.A.), a controller (SRI

Instruments Inc., Las Vegas, NV, U.S.A.), and stream selection valves (Model

EMTCA-CE, VICI Valco. Instruments, Houston, TX, U.S.A.). Stainless steel tubing with an outside diameter of 1/16 inch was used throughout the system.

! )*! ! AITC release kinetics from MSMP-containing electrospun fiber mats at 20oC in saturated RH condition were the focus of the current study. To acquire the relevant information, all MSMP-containing fiber membranes were placed in hermetically sealed jars. Specifically, a 40 cm2 (5 cm x 8 cm) electrospun membrane sample on aluminum foil and a 5 mL beaker containing a glass filter paper that was saturated with one gram

(excessive amount) of distilled water, were placed and sealed in a 1 L glass jar. The sampling jars were kept at 20 ± 0.5oC in the environmental chamber during the experiment. A custom-built septum was attached to the lid of each jar, allowing the sampling needle to pierce through the lid to extract the headspace gas, and to replace the same amount of air after each extraction.

The sampling process has been described previously in the Chapter 4. Briefly, about 15 mL of headspace gas was extracted from the sample system headspace by piercing the septum with a needle. The gas extracted was then injected into the sample inlet loop of the GC module and was conveyed by nitrogen, the carrier gas of the GC, along the capillary column and then passed through the FID, which located was at the temperature-regulated oven. The parameters used for GC detection were: (1) a nitrogen, purge and carrier gas, flow rate of 80 mL/min and 30 mL/min, respectively; (2) a hydrogen flow rate of 30 mL/min; and (3) an airflow rate of 200 mL/min. The oven temperature was kept at 90oC and the FID temperature was set at 200oC.

AITC release kinetics in each sample was determined by analyzing the headspace recorded AITC mass (Mr) that was recorded by the software (Peak Simple

393-32 bit, SRI Instruments, CA, U.S.A.). The mass of AITC was obtained by summing the Mr and the accumulated mass loss (Ml) up to the present sampling point (Eqs. 1-3):

! )+! ! Mr = Cr Vb (1)

Ml = (Cr-1x Ve) + Ml-1 (2)

Mc = Mr + Ml (3)

where Cr is the recorded AITC concentration calculated based on calibration constants;

Cr-1 is the recorded concentration of the previous sampling point; Vb is the volume of the glass jar; Ve is the volume of headspace gas extracted; Ml-1 is the sum of mass loss of all the previous sampling points before Ml; and Mc is the corrected AITC mass at any given sampling point. The AITC was considered not being released when Mc < Mc-1.

5.2.7 Data Analysis

Statistical analysis was conducted using SPSS 20 software (IBM® SPSS Inc.,

Chicago, IL, U.S.A.). One-way ANOVA was carried out to detect the differences of various factors and treatments at a 95% confidence interval with Tukey’s post hoc test.

AITC release kinetics were modeled empirically using the Modified Gompertz equation

(Lay and others 1999; Mu and others 2006):

,* $k " exp(1) ',. Qt = Qmax exp+!exp& (# ! t) +1)/ (4) -, % Qmax (0, where Qt is the amount of the AITC released at time t; Qmax is the cumulative amount of

AITC released; k is the rate constant, and ' is the lag time. Non-linear regression analyses were conducted to determine values for the three parameters using the Solver function in the Microsoft® Excel spreadsheet package (Microsoft Office 2011, WA,

! ),! ! U.S.A.). The GRG Nonlinear algorithm option was selected to minimize the residual sum of squares by changing the three parameters, without imposing any constraints.

5.3 Results and Discussion

5.3.1 Surface Morphology of Electrospun Fibers

Micrographs for pristine fiber and MSMP-containing fibers are shown in Figures

5.2 and 5.3, respectively. Comparing Figures 5.2 and 5.3, there are moderate differences in the fiber membrane morphologies between pristine and MSMP-containing fiber membranes. Fibers that were generated from pristine polymers were homogeneous and smooth (Figure 5.2). With MSMP added into the polymers, the fiber morphologies were different from those of pristine electrospun fibers. Also, the MSMP encapsulated fibers had beaded sections along with the fibers. The observed beads were due to

MSMP particle aggregates that are larger in diameter than the electrospun fibers

(Chapter 4; Figure 4.2). These distinct morphologies were not observed with the pristine electrospun fibers.

As shown in the micrographs with higher magnification, the MSMP was effectively encapsulated within the polymer matrices as evidenced by the absence of jagged surface morphologies of MSMP, which are typical of the unencapsulated MSMP powders. Bulk phase separation of MSMP was not detected, as evidenced by an even distribution of particles throughout the electrospun fiber membranes (Figure 5.3; lower magnification).

Unlike earlier study, where pure and blended PLA and PEO were dissolved in

! )-! ! dichloromethane, resulting in porous electrospun fiber mats, all of the fibers in our study were nonporous. The nonporous fiber morphology was observed are possibly due to the lower vapor pressure of chloroform versus dichloromethane, as pointed out by

Honarbakhsh and Pourdeyhimi (2011).

(a) Pristine100PLA0PEO fiber membranes

1.00 mm 100 µm 10 µm

(b) Pristine75PLA25PEO fiber membranes

1.00 mm 100 µm 10 µm

! *.! ! (c) Pristine50PLA50PEO fiber membranes

1.00 mm 100 µm 10 µm

(d) Pristine25PLA75PEO fiber membranes

1.00 mm 100 µm 10 µm

(e) Pristine0PLA100PEO fiber membranes

Figure 5.2 - Scanning electron micrographs of pristine electrospun ultrafine fibers: (a) 100PLA0PEO; (b) 75PLA25PEO; (c) 50PLA50PEO; (d) 25PLA75PEO; and (e) 0PLA100PEO.

! *%! !

(a) MSMP-containing 100PLA0PEO fiber membranes

1.00 mm 100 µm 10 µm

(b) MSMP-containing 75PLA25PEO fiber membranes

1.00 mm 100 µm 10 µm

(c) MSMP-containing 50PLA50PEO fiber membranes

1.00 mm 100 µm 10 µm

! *&! ! (d) MSMP-containing 25PLA75PEO fiber membranes

1.00 mm 100 µm 10 µm

(e) MSMP-containing 0PLA100PEO fiber membranes

1.00 mm 100 µm 10 µm

Figure 5.3 - Scanning electron micrographs of MSMP-containing electrospun ultrafine fibers: (a) 100PLA0PEO; (b) 75PLA25PEO; (c) 50PLA50PEO; (d) 25PLA75PEO; and (e) 0PLA100PEO.

5.3.2 Fiber Size Analysis

Regardless of the presence of MSMP, fiber diameters from all blended PLA and

PEO polymers resulted in significantly larger (p< 0.05) diameters than those of fibers electrospun from one polymer. The average diameters of the pristine PLA and PEO fibers were about 1.09 ± 0.31 and 1.31 ± 0.34 µm, respectively (Table 5.1). Similar findings were observed for the MSMP-containing fibers (Table 5.1). This trend is in consistent with a study reported by Oliveira and others (2013) with blown-spun ultrafine

! *'! ! fibers made from the same polymers in chloroform at various blend ratios (Oliveira and others 2013). The fiber diameter variance for the polymer blend fibers could possibly be attributed to the limited compatibility of the polymer mixtures (Oliveira and others 2013).

This will be discussed further in Section 5.3.5.

Table 5.1 - Mean fiber diameter and standard deviations of pristine and MSMP-containing electrospun fiber mats.

Fiber membranes Without MSMP, µm With MSMP, µm

100PLA0PEO 1.09 ± 0.31a 0.81 ± 0.40a

75PLA25PEO 1.68 ± 0.85b 1.46 ± 0.58b

50PLA50PEO 2.02 ± 0.44c 1.50 ± 0.46b

25PLA75PEO 1.75 ± 0.27b 1.48 ± 0.39b

0PLA100PEO 1.31 ± 0.34d 0.77 ± 0.37a

Values are mean ± standard deviation (n=200). Numbers within a column with different superscript letters indicate statistical significant difference (p< 0.05). Statistic analysis was conducted using one-way ANOVA with Tukey’s post-hoc test.

For pristine electrospun membranes (no MSMP added), fiber diameters ranged from 1.09 ± 0.31 to 2.02 ± 0.44 µm, while for those dispersed with MSMP, the diameters ranged from 0.77 ± 0.37 to 1.50 ± 0.46 µm. The smaller fibers observed with the MSMP composite fibers were probably caused by the different electrospinning phenomena that occurred in the pristine polymers and MSMP-containing polymers. Typically, during the electrospinning process, the polymer jet is drawn by the applied electrostatic force.

When charged, the polymer solutions at the spinneret tip are first elongated, then whipped and wiggled before attaching to the target collector. With the addition of MSMP to all the polymer solutions, the momentum of the polymer jets was higher due to the extra mass resulting from the embedded MSMP particles as compared to those without

! *(! ! MSMP. This phenomenon would induce a larger stretching force on the fibers as they take flight to the collector, thereby reducing the fiber thickness as compared to polymer jets wherein MSMP particles were absent.

5.3.3 Release of AITC from Encapsulated MSMP

The release profiles of AITC from various encapsulated MSMP samples are illustrated in Figure 5.5. To better understand these AITC release profiles, all of the curves were fitted to the modified Gompertz model and were characterized using different parameters including the cumulated quantity (Qmax), release rate (k), and lag time ('). These values are summarized in Table 5.2.

As shown in Figure 5.5, the AITC release profiles were characterized by an initial lag phase, followed by a quick releasing phase, and finally a plateau phase towards the later part of the experiment. The delay in AITC release during the lag phase was mostly due to the time needed for moisture from the beaker to saturate the sampling headspace, permeate into the PLA-PEO fiber matrices and the MSMP matrix, and initiate sinigrin hydrolysis. Also, the time needed for AITC to diffuse out of the fiber matrix could also contribute to the lag time. Since PEO is a more hydrophilic polymer than PLA, it was expected that the lag phase would be shorter in fibers containing the more hydrophilic

PEO component. However, no such correlation was observed in this study (Table 5.2). It is possibly because the rates of water permeating into the various fiber mats examined in the current study were similar. It may also be a result of the sample variance, such as the differences in contents of water binding constituents in MSMP which may influence the moisture content that is available for the sinigrin hydrolysis reaction. Furthermore,

! *)! ! variation in the initial RH inside of the sampling bottles before experiments may have contributed to this finding. Nevertheless, the lag time (about 5 h, Figure A.5) in current study was considered as small when comparing to the total experiment duration (180 h).

Pristine fiber membranes MSMP-containing fiber membranes

Figure 5.4 - Fiber diameter distributions of the pristine and MSMP containing electrospun! fibers with various ratios of PLA and PEO. ! **! !

Figure 5.5 - Representative cumulative AITC release profiles from various MSMP-containing electrospun fibers in 1 L headspace glass jar under saturated RH at 20°C. Symbols are experimental data, while continuous lines are fitted curves based on the modified Gompertz equation.

Beyond the initial lag phase, the release rate and the amount of AITC released from various MSMP-containing electrospun fibers increased as time progressed.

Evidently, AITC released k and Qmax values varied for different polymer matrices (Table

5.2). In general, the release rates of AITC from fiber mats containing PEO were significantly higher (p< 0.05) than those from pure PLA fiber mats. For instance, the

AITC release rate from 75PLA25PEO was 0.3 h-1, which was more than three times faster than that from the 100PLA0PEO polymers. These results could be attributed to the increased hydrophilic properties of the fiber carriers when PEO was added. The effect of

PEO on enhancing fiber hydrophilicity when blended with PLA polymers has been previously observed by Honarbakhsh and Pourdeyhimi (2011). In their study, PLA and

! "#! ! PEO polymers were blended at various ratios and were electrospun into fiber mats. To determine the hydrophilicity of fiber mats made with different PLA-PEO ratios, they measured the water contact angles of electrospun fiber mats and found that the water contact angle of PEO-PLA (10:90, w%) blended electrospun fibers was at about 47.4°, which was significantly smaller than those of neat PLA fibers(at about 58.4°). On the basis of the reported findings from the literature, it is reasonable to conclude that the addition of PEO to PLA resulted in increased hydrophilicity of the fiber membranes.

The increased hydrophilicity of the blended fiber mats may also have enhanced the total amount of the AITC released (i.e., Qmax). Table 5.2 shows that Qmax was significantly higher (p< 0.05) for the blended fiber membranes as compared to those from the pure PLA fiber membranes. For instance, the Qmax of AITC from blended PEO and PLA polymers reached about 7 to 9 ppm while it was only about 5 ppm from

100PLA0PEO fiber membranes. The Qmax and k values were the highest with the

50PLA50PEO fiber membrane, and reached about 9 ppm from per gram MSMP and

0.38 h-1, respectively. Therefore it was expected that fiber mats with higher PEO concentrations would lead to higher AITC release rates and Qmax, with the highest Qmax and k attained from MSMP embedded in pure PEO polymers. The assumption is based on the fact that pure PEO fiber membranes would be the most hydrophilic among all the fiber membranes examined in the current study. However, only about 6 ppm of AITC was released from the MSMP-containing 0PLA100PEO fiber membranes, which was significantly lower (p< 0.05) than the amount observed from MSMP-containing PEO-PLA blended polymer membranes (Table 5.2). Meanwhile, the AITC release rate from the

MSMP-containing 0PLA100PEO fiber membranes was significantly slower (p< 0.05)

! *,! ! than those from the 75PLA25PEO and the 50PLA50PEO fiber membranes.

Table 5.2 - Model fitted parameters from modified Gompertz equation-Qmax, k and lag time constants calculated for the AITC release profiles from five MSMP-containing electrospun fiber mats at 20°C and 100% RH conditions.

MSMP-containing Qmax, Rate, k, Lag time, ', samples µL/L.g MSMP h-1 h

100PLA0PEO 4.59 ± 0.33a 0.09 ± 0.00a 5.20 ± 0.33a

75PLA25PEO 7.29 ± 0.16b 0.30 ± 0.02b 4.86 ± 0.63a

50PLA50PEO 8.88 ± 0.05c 0.38 ± 0.01c 6.24 ± 1.33a

25PLA75PEO 8.37 ± 0.30c 0.25 ± 0.03d 9.50 ± 0.90a

0PLA100PEO 7.40 ± 1.48b 0.25 ± 0.01d 8.41 ± 2.41a

Values are mean ± standard deviation (n=3). Means within a column with different superscript letters indicate statistical significant difference (p< 0.05). Statistic analysis was conducted using one-way ANOVA with Tukey’s post-hoc test.

This anomaly can be explained by inspecting the appearance of the test specimens at the end of the kinetic studies. At the end of the experimental period, the

MSMP-containing 0PLA100PEO polymer membranes shrunk considerately while other fiber membranes retained their structural integrity. The shrinking process of the PEO fiber membrane during the AITC release experiment reduced the surface area of the electrospun membrane, causing the MSMP particles to clump together, thereby slowing down the moisture sorption and AITC release into the headspace. This effect can be seen in the AITC release profile (Figure 5.5), where the slope for the PEO membrane release curve is comparable to those of PLA and PEO blended membranes during the first 20 hours, but the slope noticeably decreases as time progressed. This reduction in the release rate is probably due to a collapsing fibrous matrix, the result of PEO moisture

! *-! ! uptake. Since PEO is an amphiphilic polymer, moisture could penetrate the cross-linked region inside of the polymer matrix, and form hydrogen bonds with the oxygen on the

PEO backbone (Agrawal and others 2006; Bekiranov and others 1997; Honarbakhsh and Pourdeyhimi 2011; Maxfield and Sheperd 1975; Zivanovic and others 2007).

Hydrogen bonding is one of the main causes of PEO solubility in water, and hence it is possible that some PEO fibers disintegrated in the moisture from the headspace

(Bekiranov and others 1997). By contrast, for electrospun membranes prepared from

PLA and PLA-PEO blends, the relatively hydrophobic PLA provided a mechanical framework that could retain membrane integrity.

When evaluating the AITC release rates from the MSMP-containing PLA and

PEO blended fiber membranes under saturated RH conditions, it was found that the

AITC release rate from 25PLA75PEO was significantly (p< 0.05) slower than that from the 50PLA50PEO. This could be explained based on the fact that PLA is less hydrophilic than PEO, and thus the amount of water available for the hydrolysis of sinigrin in PLA encapsulated MSMP is greater than that available in PEO. On the other hand, the greater k value observed for the 50PLA50PEO membrane versus 75PLA25PEO membrane is likely caused by the higher hydrophilicity and/or increased material plasticization in the former, which outweighed the ability of PEO to retain water.

Findings from the studies in this section show that AITC release kinetics could be controlled by manipulating the ratios of PLA, PEO and MSMP in electrospun fiber membranes, which is desirable when developing AITC release carriers for various MAP applications, especially when optimizing the antimicrobial efficacy for a specific food product. In order to better understand the interaction of PLA, PEO and MSMP, FTIR

! +.! ! analysis was conducted on all the pristine and composite fibers in the following section.

5.3.4 FTIR analyses on Pristine and MSMP-Containing Electrospun Fiber

Membranes

FTIR analysis in the mid IR region (700–3900 cm-1) was performed to elucidate the nature of interactions between different components within the fibers. The spectra of pristine and MSMP-containing fiber samples were plotted together in Figures 5.6 and 5.7 to facilitate comparison. Polymers that are compatible and exhibit strong interaction with each other typically display considerable frequency shifts, band widening, and/or band intensity variations, whereas incompatible polymers will display minimal changes in the individual spectral components (Coleman and others 1992; Kang and others 2001; Kister and others 1998; Oliveira and others 2013; Younes and Cohn 1988).

Figure 5.6 shows the FTIR spectra of the electrospun pristine PLA, pristine PEO and PLA-PEO blended fibers at different ratios. As an aliphatic polyester, PLA contains flexible ester bonds in the backbone structure. PLA fibers demonstrated the characteristic carbonyl (C=O) absorption bands with a peak at 1751 cm&1 (which represented the backbone ester group), whereas the C-O-C stretching bands of PLA

&1 appeared at 1082 cm . Absorption bands that relate to the amorphous and crystalline structure of PLA appeared at 862 and 755 cm&1, respectively. These absorbance peaks are in good agreement with the PLA stretching bands previously reported in the literature

(Auras and others 2010; Kang and others 2001; Kister and others 1998; Xu and others

2009; Younes and Cohn 1988).

By contrast, PEO is a polymer with the molecular formula (-CH2CH2O-)n. The

! +%! ! characteristic C-O-C absorbance band is located at 1097 cm&1 (Pielichowski and

Flejtuch 2005; Wen and others 1996; Zhou and others 2011). The bands detected at 962

&1 and 843 cm have been attributed to the rocking and twisting of C-H2 (Pielichowski and

Flejtuch 2005). Both of these peaks have been associated with the crystalline structure of PEO (Oliveira and others 2013; Younes and Cohn 1988). In addition, the identical spectra observed for PEO electrospun fibers and pure PEO powder indicates that the electrospinning process did not alter the molecular conformation of the polymer (Figure

5.6).

C-O-C PEO 1097 cm-1

60

C-H stretch 2880 cm-1 C=O PLA 1751 cm-1
50

Pure PEO powder

40

0PLA100PEO

30

25PLA75PEO Absorbance

20 50PLA50PEO

75PLA25PEO 10

C-O-C PLA 1082 cm-1

100PLA0PEO 0 3700 3500 3300 3100 2900 2700 2500 2300 2100 1900 1700 1500 1300 1100 900 700 -1 Wavenumber, cm

Figure 5.6 - Comparison of FTIR spectra of the pristine electrospun ultrafine fibers: (a) 100PLA0PEO; (b) 75PLA25PEO; (c) 50PLA50PEO; (d) 25PLA75PEO; and (e) 0PLA100PEO.

! +&! ! In the polymer blend, the C-O-C stretching band of PEO at 1097 cm&1 overlapped and merged with the C-O-C from PLA at 1082 and 1084 cm&1 bands in 75PLA25PEO and 50PLA50PEO, respectively. Two peaks were detected at 1082 and 1097 cm&1 for

25PLA75PEO fibers. No significant shift was observed for these two peaks as the polymer blend proportion changed. Similarly, the C-H stretch at 2880 cm&1 from the PEO backbone remained unchanged at all polymer blend ratios. In two previous studies that involved extruded PLA-PEG films, similar phenomenon was reported (Uhrich and others

1999; Younes and Cohn 1988). The low concentration of the end groups -OH and the absence of other moieties capable of developing the intermolecular interactions were cited as the contributing reasons for the observed absence of frequency shift in the IR spectra.

FTIR spectra of MSMP-containing fibers were evaluated and are shown in Figure

5.7. The absorbance band at 1649 cm-1 can be attributed to the amide I stretching from the protein in MSMP. There is about 40.51% (w %) protein in the tested MSMP as reported in Section 4.3.1. The amide I band was found in all MSMP-containing electrospun fibers regardless of their composition. Comparing Figures 5.6 and 5.7, the characteristic absorption bands of C-O-C (1097 cm-1) and C=O (1751 cm-1) of PEO and

PLA, respectively, remained stationary after the incorporation of MSMP. In summary, the

FTIR analyses did not reveal any specific interaction between MSMP and the polymers for the IR active groups investigated.

! +'! ! 50 Amide - 1649 cm-1
MSMP 45 C-O-C PEO 1097cm-1
C-H stretch 2880 cm-1
40

C=O PLA -1
35 0PLA100PEO 1751cm

30 25PLA75PEO

25

Absorbance 20 50PLA50PEO

15

10 75PLA25PEO

C-O-C 5 PLA 1082 cm-1
100PLA0PEO 0 3700 3500 3300 3100 2900 2700 2500 2300 2100 1900 1700 1500 1300 1100 900 700 -1 Wavenumber, cm

Figure 5.7 - Comparison of FTIR spectra of MSMP- containing electrospun fibers: (a) 100PLA0PEO; (b) 75PLA25PEO; (c) 50PLA50PEO; (d) 25PLA75PEO; and (e) 0PLA100PEO.

5.3.5 Thermal Analyses

DSC tests were performed to evaluate the thermal properties and to investigate the miscibility of PEO and PLA polymers. The thermograms obtained are presented in

Figures 5.8 and 5.9, from which Tg and Tm were determined. The Tg was taken as the temperature at the midpoint of the inflection and the Tm was considered as the peak temperature of the melting endotherm.

PLA and PEO are semi-crystalline polymers made up of amorphous and

! +(! ! crystalline domains (Nijenhuis and others 1996; Oliveira and others 2013; Younes and

Cohn 1988). The crystallinity of polymers can be determined by evaluating their melting enthalpy. Figure 5.8 presents the thermograms for pristine PLA, PEO and PLA-PEO blend fibers. The endothermic melting peaks of 0PLA100PEO and 100PLA0PEO electrospun fibers appeared at 72ºC and 168ºC, respectively, and these were comparable to values previously reported in the literature (Coleman and others 1992;

Nijenhuis and others 1996).





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         ([R8S 7HPSHUDWXUH ƒ& 8QLYHUVDO9$7$ Figure 5.8 - DSC thermograms of the pristine electrospun ultrafine fibers: (a) 100PLA0PEO; (b) 75PLA25PEO; (c) 50PLA50PEO; (d) 25PLA75PEO; and (e) 0PLA100PEO.

The miscibility of PLA and PEO after blending was also investigated by evaluating

! +)! ! the Tg values of the blend polymers. The blend is miscible if it exhibits a single compositional-dependent Tg, while it is considered as immiscible if it presents two different Tg (Kadla and Kubo 2003; Paul and Bucknall 2000; Sheth and others 1997;

Younes and Cohn 1988). Unfortunately, this method could not be applied for all of the fibers in the current study, since the glass transition of PLA and the melting of PEO endotherms overlapped in the range of 60-72°C in most of the PLA and PEO blended polymers, and especially in the 75PLA25PEO polymer blend. Similar difficulties were reported in the literature for thermal analyses of PEG and PLA polymer blends (Nijenhuis and others 1996; Sheth and others 1997; Younes and Cohn 1988). Nevertheless, there is an indication that the added PEO may have also acted as a plasticizer to PLA, as evident from the decreased Tg of PLA polymers as the PEO content increased (Figure

5.8). The plasticizing effect of PEG (< 50%, w%) on PLA in an extrusion polymer, as reported by Sheth and others (1997), led to a higher crystallization exotherm for PLA.

These findings suggested that PLA and PEO are partially miscible depending on the composition.

Younes and Cohn (1988) argued that when the blended polymers are not miscible, phase separation occurs provided the polymers are properly aged. However, the phase separation of PLA and PEO was not observed in the current study, since PLA and PEO polymers were dissolved in chloroform and DMF and spun into fibers shortly after the polymer preparation. Similar finding have been reported by Oliveira and others (2013) when using a solvent blow-spun method to prepare PLA-PEO blend fibers. Moreover, fast solvent evaporation and polymer solidification that is typical of the electrospinning process may also have inhibited phase separation.

! +*! ! The melting peak temperature of pure PEO shifted from 72°C to lower temperatures when the weight ratio of PLA polymer solution increased from 0 to 50%.

This shift is possibly due to the disruption of PEO chain packing by the PLA polymer, producing imperfect crystalline structures, leading to a lower melting temperature for

PEO. On the other hand, the Tm of PLA remained unchanged for all the blend polymers regardless of the PEO ratio, which shows that the crystalline phase of PLA was not affected by the introduction of PEO (Figure 5.8). Similar phenomena were reported by

Younes and Cohn (1988) where PLA and PEG were blended at various ratios for film casting (Younes and Cohn 1988). After adding MSMP into the fibers, the melting temperatures of 100PLA0PEO and 0PLA100PEO shifted from 168 to 162°C, and from

72 to 62°C, respectively (Figure 5.9). This result indicates that the presence of MSMP hindered the organization of PLA and PEO molecular chains into ordered structures during the blending and/or electrospinning processes, resulting in a decreased Tm in of both polymers. When the PLA content in the fiber membranes was decreased from 100% to 75% and 50%, the Tm of PLA shifted from 168°C to about 160°C, indicating a decreased crystalline phase in the PLA polymers. This PLA Tm lowering effect may be due to presence of some constituents in MSMP, such as starch and cellulose. There is about 5% starch (Munshi and others 1990) in the MSMP seed and 15% cellulose

(Ohlson and Oljefabriker 1972) in the dry weight of MSMP. Starch is reportedly capable of altering the melting temperature of PEO when blended with PEO polymers at various ratios (Pereira and others 2009), with the interaction between starch and PEO being due to the hydrogen bonding generated among the hydroxyl groups of starch and the oxygen atoms from repeat units of PEO. In addition, cellulose could decrease the melting

! ++! ! temperature of PLA electrospun fibers by forming smaller and less dense crystalline structures (Shi and others 2012).



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Figure 5.9 - DSC thermograms of MSMP and MSMP-containing electrospun ultrafine fibers.

MSMP alone presented a weak melting endotherm peak at about 73°C and a relatively strong endotherm peak at about 193°C (Figure 5.9). This indicated the presence of some crystalline and/or semi-crystalline polymers/structures in the MSMP, such as proteins and polysaccharides. There is about 40.5% protein in the defatted ground MSMP (Table 4.1) and about 15% of cellulose in the MSMP (Ohlson and

Oljefabriker 1972). These components likely contributed to the changes in thermal properties observed. When MSMP was encapsulated by PLA and PEO fibers, the Tm of

MSMP increased from 193 to 198°C regardless of the fiber mat composition (Figure 5.9). ! +,! ! As shown in Figure 5.3, MSMP was covered with a thin layer of PLA and/or PEO, and the increased Tm of MSMP is likely due to the interactions of these polymers with components in MSMP that may have had been leached out by the solvents during polymer preparation.

5.4 Conclusions

Electrospinning is an efficient method of generating homogeneous and nonporous fiber membranes from PLA, PEO and their polymer blends. Furthermore, these polymers are versatile for encapsulating the defatted ground MSMP into electrospun membranes with homogeneous MSMP distribution. The diameters of the blended PLA and PEO fibers are significantly larger (p< 0.05) than those of pure polymer fiber regardless of the presence of MSMP. For the pristine fiber membranes, the diameters of the blended fiber membranes were in the range of 1.68 ± 0.85 to 2.02 ± 0.44 µm comparing to 1.09 ± 0.31 to 1.31 ± 0.34 µm for the pure PLA or PEO membranes, respectively. In addition, for the

MSMP-containing fiber membranes, the diameters of blended fiber membranes were in the range of 1.46 ± 0.58 to 1.50 ± 0.46 µm comparing to 0.77 ± 0.37 to 0.81 ± 0.40 µm for the polymer membranes electrospun from a single polymer.

The GC analysis indicated that ATIC release kinetics could be controlled effectively by modifying the polymer composition of the electrospun fibers, i.e., by blending 9% PLA and 3% PEO at various ratios. The addition of PEO to PLA polymers significantly increased (p< 0.05) the AITC release rate, with the highest AITC release rate (0.38 ± 0.01 h-1) attained from the 50PLA50PEO MSMP-containing fiber membranes. Yet, the highest amount of AITC was released from 50PLA50PEO and

! +-! ! 25PLA75PEO fiber membranes at 8.88 ± 0.05 and 8.37 ± 0.30 µL/L, respectively, when the release amount was normalized per one gram of MSMP. On the other hand, fiber membranes that were spun from pure PLA polymers had the slowest AITC release rate at 0.09 ± 0.00 h-1 with the lowest quantities released of only 4.6 ± 0.33 µL/L of AITC per gram of MSMP.

FTIR results indicated that upon blending PLA and PEO, these two polymers interacted with each other, the extent of which depended on the particular fiber formulation. Specific chemical interaction between PLA, PEO polymers and MSMP were not detected from the FTIR analyses. DSC analysis revealed that when MSMP was incorporated into PLA, PEO, and their blended polymers, considerable decreases in the melting temperature of PLA and PEO were observed. Moreover, a noticeable increase in melting enthalpy of MSMP occurred. It is therefore possible that besides physical entrapment, the MSMP also has intermolecular interactions with the PLA and PEO fiber mats that could not be detected by the infrared analysis.

In summary, this study shows that various AITC release behaviors could be achieved via preparing electrospun membrane carriers with different PLA and PEO contents. The appropriate selection of the fiber polymer composition is dependent on the properties, end use target applications, and food products. In view of the antimicrobial efficiency of AITC in food systems, practical use of the MSMP-containing electrospun fibers in active packaging appears to be feasible.

! ,.! ! 6. Thesis Conclusions and Future Directions

6.1 Conclusions

AITC is well known as an antimicrobial agent. While many studies have investigated the use of pure and synthetic AITC for food preservation purposes, this research focused on studying a natural source of AITC – MSMP.

In this thesis, Chapters 1 and 2 presented background information on active packaging with an emphasis on antimicrobial packaging, the antimicrobial properties of

AITC, and the concepts of encapsulation. Information on using electrospun fiber membranes as carriers for controlled release purposes was also reviewed and discussed in these two chapters. Chapter 3 highlighted some of the challenges on applying AITC in food antimicrobial packaging applications, and reviewed methodologies to overcome AITC’s volatility and strong odor issues. Here, polymer carriers to encapsulate AITC are discussed.

Two main topics were examined in this research: (1) the investigation of AITC release kinetics from modified MSMP as affected by RH and temperature; and (2) the development of a methodology for controlling the release of AITC from MSMP by encapsulating it in electrospun PLA, PEO, and PLA-PEO fibers. Regarding the first topic

(Chapter 4), mustard seed meal was ground into powders with particle sizes ranging from 5 to 300 !m and placed in hermetically sealed glass jars wherein the headspace was adjusted to different initial RH (85 to 100%) and temperatures (5, 20 and 35°C).

Kinetic data showed that both the release rate and the maximum amount of AITC released increased with elevated RH, while temperature significantly enhanced AITC

! ,%! ! release rate from MSMP. Moreover, the smaller the particle size of the MSMP, the faster the AITC release rate when the powder was exposed to excess moisture. The results suggested that the RH-dependent AITC release phenomenon of MSMP could be exploited for triggering the release of the antimicrobial volatile for extending the shelf-life of food products.

For the second topic (Chapter 5), Ground-defatted MSMP samples were encapsulated in ultrafine electrospun fiber membranes. Here, the polymers selected for electrospinning were PLA and PEO, which were dissolved in a chloroform: DMF mixture

(9:1 w/w) to form 9 and 3% stock solutions, respectively. The MSMP samples were then dispersed in the fiber-forming solutions prepared by blending PLA and PEO solutions at different polymer ratios. The AITC release kinetic data showed that both the release rate and the maximum amount of AITC could be controlled by adjusting the proportion of PLA and PEO. Specifically, the addition of PEO into PLA increased the AITC release rate and quantity released by enhancing the hydrophilicity of the fiber. Although MSMP could be encapsulated in pure PEO fibers, the resulting membrane carrier tended to shrink upon exposure to elevated RH environment due to the decomposition of the fiber. The addition of PLA into PEO was favorable as the former stabilized the structural integrity of the composite membrane carrier. FTIR and DSC results indicated that MSMP particles were not only physically entrapped with the polymer matrices, but also interacted with the polymers at the molecular level. Since the AITC release behavior from MSMP-containing fiber membranes could be adjusted by changing the formulation of fiber-forming solutions. This method of producing electrospun composite membrane carriers is very versatile, and can accommodate different end use requirements for various food

! ,&! ! systems. In conclusion, this thesis research adds to the existing knowledge of using

MSMP in antimicrobial food packaging applications. It illustrates the feasibility of encapsulating MSMP in electrospun fibers for controlled release of AITC. In view of the antimicrobial properties of AITC, the MSMP-containing composite fibers warrant further study for shelf-life extension of food products.

6.2 Future Directions

There are several outstanding questions that are yet to be addressed. These are:

• Evaluation of antimicrobial efficacy of the MSMP and MSMP-containing fiber

membranes in model food systems, including the determination of MIC for different

microorganisms (e.g., bacteria, yeasts, and molds).

• Validation of antimicrobial efficacy of MSMP and MSMP-containing fiber

membranes for shelf-life extension in actual food products; development of a better

understanding of the influence of food constituents on AITC efficacy (e.g., water

activity, lipid content, pH).

• Determination of the enzymatic activity of myrosinase and the stability of sinigrin in

MSMP after electrospinning, as well as during extended storage.

• Development of methods to encapsulate MSMP in food-grade polymers (e.g.,

proteins and polysaccharides) using solvents that possess GRAS status suitable

for food applications.

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! -)! ! 8. Appendices

8.1 Surface-to-volume ratio distribution of MSMP particle samples as examined in

Chapter 4.

(A) APR-MSMP

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! -*! ! (B) Ground-MSMP

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(C) Defatted-MSMP

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! -+! ! (D) Ground-defatted-MSMP

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Figure A.1- Particle surface-to-volume ratio distribution of mustard seed samples: (A) APR-MSMP; (B) Ground-MSMP; (C) Defatted-MSMP; and (D) Ground-defatted-MSMP.

! -,! ! 8.2 RH building up curves under various experiment conditions used in Chapters

4 and 5.

Figure A.2 - Building up of relative humidity (with 6 and 12 µL distilled water respectively) in the headspace of 1 L hermetic sealed bottle at 5°C in Chapter 4.

! --! !

Figure A.3 - Building up of relative humidity (with 15 and 30 µL distilled water respectively) in the headspace of 1 L hermetic sealed bottle at 20°C in Chapter 4.

Figure A.4 - Building up of relative humidity (35 and 70 µL distilled water respectively) in the headspace of 1 L hermetic sealed bottle at 35°C in Chapter 4.

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Figure A.5 - Building up of relative humidity (with 1000 µL distilled water) in the headspace of 1 L hermetic sealed bottle at 20°C in Chapter 5.

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