The Effect of Different Concentrations of Hydroxypropyl Methylcellulose on the Morphology

and Mechanical Properties of Whey Protein Isolate Edible Films

Thesis

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the

Graduate School of The Ohio State University

Cyd Marie Quiñones Pérez, B.S.

Graduate Program in Food Science and Technology

The Ohio State University

2019

Thesis Committee

Dr. Melvin Pascall, Advisor

Dr. Gireesh Rajashekara

Dr. Hua Wang

Copyrighted by

Cyd Marie Quiñones Pérez

2019

Abstract

A container is a barrier between a packaged product and its external environment, and it protects the product from interaction between water, oxygen, dirt, light in some cases and microorganisms. The main types of food packaging materials are glass, plastic, metal, paper board and composites. Plastics are made from polymers. A polymer is made of a large number of monomeric units connected by covalent chemical bonds to make a large molecular weight structure. Some polymers are petroleum based while others are made from plants, animals or microorganisms. Due to increased environmental concerns about plastic waste, many researchers are currently focusing on the synthesis of bio-based sustainable plastic packaging materials.

Polysaccharides such as Hydroxypropyl Methylcellulose (HPMC), and proteins such as Whey

Protein Isolate (WPI) are being used in the food industry because they are widely available, nontoxic and have the capacity to produce edible films with acceptable properties. The objectives of this study were to compare the ratio of blending HPMC with WPI on physical, mechanical, thermal and permeability properties of films made with the blends. Five different blends were tested HPMC-WPI (100:0), HPMC-WPI (75:25), HPMC-WPI (50:50), HPMC-WPI (25:75) and

HPMC-WPI (0:100). The impact of these blends on thickness, moisture content, water activity, homogeneity, oxygen transmission, water vapor permeability (WVP), glass transition temperature

ii and morphology were determined. The results for moisture content showed that there was no significant difference between the films made with the different blends. However, HPMC-WPI

(75:25) had a higher moisture content than the other blended films. The results for water activity showed that significant differences existed between some samples. The oxygen transmission analysis showed that there were no significant differences between the samples tested. HPMC-

WPI (25:75) had the highest oxygen transmission rate and HPMC-WPI (50:50) had the lowest, but modifications such as increasing the relative humidity during the analysis are still necessary to confirm these results. There were no significant differences between the different permeabilities of the samples tested for water vapor permeability. The glass transition temperatures of the films shifted from 135oC to 95oC as a result of increased addition of the HPMC fraction. This indicated that the thermal transitions of the blends shifted to higher temperatures by the HPMC. The thermo-

Gravimetric Analysis showed that the degradation peaks shifted to a lower temperature as the WPI concentration increased in the samples. The scanning electron micrographs of the films at different

HPMC/WPI concentrations revealed homogeneously blended materials. The X-ray diffraction patterns that the blends developed higher and more intense peaks when the HPMC fraction increased. This indicated that HPMC had a higher degree of crystals when compared with WPI.

This research showed that blended HPMC/WPI film could be made but more research is needed for their optimization.

iii Dedication

To my mom, dad and my sister, I could not have done this without you. Thank you for letting

me fly.

iv Acknowledgment

I would like to thank Dr. Melvin Pascall for the opportunity of being part of his laboratory and for his patience during the last three years. I am grateful to the members of my committee Dr.

Gireesh Rajashekara and Dr. Hua Wang for their support and advice. In addition, I would like to express my gratitude to the members of my laboratory for sharing their knowledge specially to

Elliot Dhuey for his help and support in overcoming the obstacles I faced during my research. My gratitude goes to the faculty, staff and students that wake up every day with the intention of making the world a better place.

My sincere thanks go to Sandy Puckett, for listening when I could barely speak. Thank you for supporting me during the lows and highs of graduate school. Thanks for creating a safe space in your office and in your heart for me. The word thank you will never be enough. I would like to thank Gabriel Fernandez Pedrosa, Paola Judith Padilla Vazquez, Evemarie Bracetti

Resto and Carlos Alberto Rodríguez Santiago for simply making everything brighter. This would not have been possible without you.

I thank my coworkers from The College of Nursing – Office of Student Affairs Equity and Inclusion. Thank you for being by my side during the most exciting moments and during the

v most difficult ones. You have been my family, my support and my home during the last three years. You will always be in my heart and in my thoughts

Finally, thank you Benedicto Quiñones Torres, Noemí Pérez Bocanegra and Hilda

María Quiñones Pérez. Thank you for always understanding, for never judging, for always accepting and for letting me fly. Thank you for having faith in me, for teaching me how to be a better student, professional, and a better human. There are not enough words to describe how thankful I am. Thank you, my love.

vi Vita

July 1993 Born – San Juan, Puerto Rico

2016 B.S. Industrial Microbiology, University of Puerto Rico, Mayagüez Campus

2019 M.S. Food Science and Technology, The Ohio State University

Field of Study

Major Field: Food Science and Technology

vii Table of Contents

Abstract ……………………………...... …………………………………………………...... ii

Dedication ………………………………………………………………………………………. iv

Acknowledgment ………………………………………………………………………………... v

Vita …………………………………………………………………………………………….. vii

List of Tables ………………………………………………………………………………….... xi

List of Figures ………………………………………………………...……………………….. xiii

1 Literature Review ...... 1

1.1 Packaging and it functions ...... 1

1.2 Packaging categories ...... 1

1.3 Packaging materials ...... 2

1.3.1 Glass ...... 2

1.3.2 Metal ...... 5

1.3.3 Paper board ...... 7

1.3.4 Polymers ...... 10

1.4 Types of Edible Films ...... 15

viii 1.4.1 Polysaccharide-based edible films and coatings ...... 15

1.4.2 Protein-based edible films and coatings ...... 31

1.4.3 Lipid-based edible films and coatings ...... 40

1.5 The methods of characterizing edible films ...... 43

1.5.1 Thickness ...... 43

1.5.2 Tensile strength, elongation and elastic modulus ...... 45

1.5.3 Permeability properties ...... 46

2 The Incorporation of Selected Small Molecule Compounds into Whey Protein Isolate –

Hydroxypropyl Methylcellulose and their effect on Physical, Permeability, Thermal, Mechanical and Chemical Properties ...... 66

2.1 Introduction ...... 66

2.2 Experimental Design ...... 68

2.3 Materials and Methods ...... 69

2.3.1 Preparation of HPMC-WPI at different concentrations ...... 69

2.3.2 Film Moisture Content and Water Activity ...... 70

2.3.3 Oxygen Transmission Rate ...... 71

2.3.4 Water Vapor Permeability ...... 72

2.3.5 Thermal Analysis by Differential Scanning Calorimetry ...... 73

2.3.6 Thermo-Gravimetric Analysis ...... 73

2.3.7 Scanning Electron Microscope ...... 74

2.3.8 X-ray Diffraction ...... 74

2.3.9 Statistical Analysis …………………………………………….………………….. 75

2.4 Results and Discussion ...... 75

2.4.1 Film Moisture Content and Water Activity ...... 75

2.4.2 Oxygen Transmission Rate ...... 80

2.4.3 Water Vapor Permeability ...... 84

2.4.4 Thermal Analysis by Differential Scanning Calorimetry (DSC) ...... 88

2.4.5 Thermal Analysis by Thermo-Gravimetric Analysis (TGA) ...... 93

2.4.6 Scanning Electron Microscopy ...... 96

2.4.7 X-ray Diffraction ...... 99

2.5 Conclusions ...... 101

References …………………………………………………………………………………….. 104

Appendix A ………………………………………………………………………………….... 112

List of Tables

Table 1 Polysaccharides-based edible films and their significant characteristics...... 17

Table 2 Protein-based edible films and their significant characteristics...... 32

Table 3 Lipid-based edible films and their significant characteristics...... 41

Table 4 A Summary of Moisture Contents and Water Activity of Hydroxypropyl

Methylcellulose and Whey Protein Isolate films at different concentrations ...... 76

Table 5 The summary of OTR calculations for Hydroxypropyl Methylcellulose and Whey

Protein Isolate Films ...... 82

Table 6 Summary of WVP calculations for Hydroxypropyl Methylcellulose and Whey Protein

Isolate Films ...... 85

Table 7 HPMC-WPI Edible Films Summary of Results ...... 103

Table A.1 Water Activity Multiple Comparison …………………....………………………... 113

Table A.2 Moisture Content Multiple Comparison ……...………………………………...… 115

Table A.3 Water Vapor Permeability Test Multiple Comparison ……………...……………. 117

Table A.4 Oxygen Transmission Rate Multiple Comparison ……………………………...... 119

Table A.5 Moisture Content Raw Data for HPMC-WPI Edible Films ………………………. 120

Table A.6 Water Activity Raw Data for HPMC-WPI Edible Films …………………………. 121

xi Table A.7 Water Vapor Permeability Raw Data for HPMC-WPI Edible Films ……………... 122

Table A.8 Oxygen Transmission Rate Raw Data for HPMC-WPI Edible Films ……...…...... 123

x ii List of Figures

Figure 1 Blow-and-blow glass forming process used to make narrow neck bottles (Robertson

1998)...... 4

Figure 2 Press-and-blow glass forming process used to make wide mouth jars (Robertson 1998).

...... 5

Figure 3 The double seam first and second operations (21CFR113.60)...... 7

Figure 4 Gable-top cartons: (a) made with high barrier aluminum foil; (b) made with EVOH, and used to package liquid products (Emblem and Emblem 2012)...... 9

Figure 5 The reaction of monomeric ethylene molecules in the presence of an initiator to create the polyethylene (Selke and Culter 2016)...... 11

Figure 6 The synthesis of nylon 6,6 from adipic acid and hexamethylene diamine (Boyd and

Phillips 1996)...... 12

Figure 7 Structure of a repeating unit of cellulose (O’Sullivan 1997)...... 19

Figure 8 Molecular structure of methylcellulose (Huber and Embuscado 2009a)...... 20

Figure 9 The structure of Hydroxypropyl methylcellulose (Quinten et al. 2011)...... 21

Figure 10 The structure of amylose and amylopectin (Huber and Embuscado 2009a)...... 23

Figure 11 Repeating segment of the pectin molecule (Thakur et al. 1997)...... 27

xiii Figure 12 The chemical structure of alginic acid (Szklarska et al. 2014)...... 28

Figure 13 Chemical structure of commercial carrageenan (Tavassoli-Kafrani et al. 2016)...... 30

Figure 14 DSC thermogram of the HPMC-WPI blended films at a ratio of 1:1 with the average of the pure WPI and HPMC (Yoo and M Krochta 2011)...... 38

Figure 15 Stress and strain curve for protein-based film (Wan 2016)...... 46

Figure 16 Measuring system for the Water Vapor Transmission Rate through a plastic film and sheeting using a Modulated Infrared Sensor (ASTM D1249-13) ...... 50

Figure 17 The conditioning System for the Water Vapor Transmission Rate through a plastic film and sheeting using a Modulated Infrared Sensor (ASTM D1249-13)...... 51

Figure 18 Functional Diagram of Diffusometer (ASTM F372 – 99)...... 53

Figure 19 An illustration of the calorimetric scan of a globular protein where Cp,p is the heat capacity of the protein solution and Cp,b is the heat capacity of the buffer solution as the reference (Shirley 1995)...... 57

Figure 20 Glass transition temperature reported using the midpoint of the change in the heat capacity of the samples...... 58

Figure 21 The physical states of a polymer under various temperatures and a given frequency

(Menczel and Bruce Prime 2008)...... 61

xiv Figure 22 X-ray diffraction patterns for films made with starch and chitosan powders (Y.X. et al.

2005) ...... 65

Figure 23 Experimental Design Flowchart for HPMC-WPI Edible Films at Different

Concentrations ...... 68

Figure 24 The Moisture Content of Hydroxypropyl Methylcellulose and Whey Protein Isolate

Films at varying blended ratios ……………………………………………………………….... 77

Figure 25 The summary of Water Activity of Hydroxypropyl Methylcellulose and Whey Protein

Isolate Films ...... 78

Figure 26 The summary of OTR of Hydroxypropyl Methylcellulose and Whey Protein Isolate films ...... 81

Figure 27 The summary of WVP of Hydroxypropyl Methylcellulose and Whey Protein Isolate films ...... 86

Figure 28 Differential Scanning Calorimetry of HPMC-WPI films at different concentrations. 89

Figure 29 Differential Scanning Calorimetry – Second heating curve of HPMC-WPI films at different concentrations ...... 90

Figure 30 Mass loss curve (TGA) and the derivative for the mass loss curve (DTG) of

Hydroxypropyl Methylcellulose and Whey Protein Isolate Edible Films at Different

Concentrations ……………………………………………………………………………...….. 93

Figure 31 Derivatives for the mass loss curve (DTG) of Hydroxypropyl Methylcellulose and

Whey Protein Isolate Films at different concentrations ...... 94

Figure 32 SEM surface morphologies of (a-e) HPMC-WPI (100:0), HPMC-WPI (75:25),

HPMC-WPI (50:50), HPMC-WPI (25:75), HPMC-WPI (0:100) ……………………………... 97

Figure 33 X-ray Diffraction of Hydroxypropyl Methylcellulose and Whey Protein Isolate Films at Different Concentrations …………………………………………………………………….. 99

Figure A.1 Water Activity Statistical Analysis analyzed by a SAS statitistical program ...…. 112

Figure A.2 Moisture Content Analyzed by a SAS statistical program ………………………. 114

Figure A.3 Water Vapor Permeability Analyzed by a SAS Statistical program …………….. 116

Figure A.4 Oxygen Transmission Rate Analyzed by a SAS Statistical program …………..... 118

xvi 1 Literature Review

1.1 Packaging and it functions

A package is intended to contain, protect, preserve, transport, communicate and sell a food product (Han 2005). It is the barrier between the product and the environment and protects the food from interaction with water, oxygen, temperature and microorganisms such as bacteria, viruses and fungi. Since most food products cannot be sold unpacked, the package remains the most important part of retail marketing. A good package informs consumers about the general features of the product. These include the ingredients, net weight, product name, address of the manufacturer, preparation and storage information, and the maximum retail price. Nowadays, consumers are demanding microwavable containers, with easy opening features, tamper-evident devices for safety, ease of transport, smaller sizes, ability to see the product and biodegradability of the material (Chiellini 2008). Glass, plastic, metal, paper board and composites are the main types of food packaging materials (Emblem and Emblem 2012).

1.2 Packaging categories

Primary, secondary and tertiary types are the three main categories of packages. A primary package is one that is in direct contact with the product and offers protection from oxygen, light,

1 moisture, dirt and other types of contaminants. A secondary package is not in direct contact with the product, but it is used for the movement and sale of multiple primary packaged products. A tertiary package is used for transportation and distribution in wholesale trade. It is designed to protect the product from physical damage that could occur during shipping and transportation

(Thomas 2015).

1.3 Packaging materials

1.3.1 Glass

Glass is an inert material that is excellent for preserving the quality of food products. It is also completely recyclable. The main ingredient in commercial glass is silica (quartz) which is obtained from sand. Other ingredients include limestone, soda ash, alumina, cullet (recycled glass) and other trace compounds that are used in less than 4% in proportion. Different coloring agents are used in the glass making process. Chrome oxides is used to make emerald (green) glass. This glass is mostly used in the beverage and pharmaceutical industries. Iron and sulfur are used to make amber (brown) glass that is used for UV sensitive products like beer, for example. Cobalt oxide is used for making blue glass. Decolorizers are used to remove color, and for making clear glass. This is known as flint glass and is used in most packaging applications (Robertson 1998).

During the smelting process, the raw materials are heated to approximately 1500oC in a furnace to produce the glass. Different glass container forming processes exist. These include the blow- and-blow and press-and-blow processes. Figure 1 shows the blow-and-blow process. The process begins when a gob of hot glass drops into a blank mold through a funnel-shaped guide. The funnel is replaced by a baffle that prevents the escaping of the glass and a puff of air from below pushes the glass into the mold and creates the finish section in the process. The baffle is replaced by a solid plate and air is forced through the gob to expand the glass into the pre-form of the bottle.

The pre-form bottle is removed from the blank mold, rotates 180o and transfers to a blow mold where air is blown from above into it and expands the glass against the walls of the mold. The mold then opens, and the bottle is removed. Before the container is safely used it is annealed, inspected and tested. At the end of the glass forming process a surface coating is applied to lubricate the containers. This surface treatment facilitates the smooth flow of the containers through the conveyor system and protects them from abrasion (Emblem and Emblem 2012).

Figure 1 Blow-and-blow glass forming process used to make narrow neck bottles (Robertson

1998).

Figure 2 shows the press-and-blow process. The process is similar to the blow-and-blow process except that the pre-form of the jar is created by pressing it into shape with a metal plunger instead of blown into shape. Glass is 100% recyclable without losing its quality or purity, thus making it a great environmentally friendly material for food.

Figure 2 Press-and-blow glass forming process used to make wide mouth jars (Robertson 1998).

1.3.2 Metal

Metal containers constitute a big part of the food packaging industry since they are cheap, conserve the freshness and preserve the food for a long time. Different materials are used to make metal containers. These are tin free steel, tinplated steel, aluminum and electrolytic chromium coated steel (ECCS). These containers are usually coated with epoxy, polymers and lacquers to prevent certain foods from corroding the base metal (Robertson 2013). In some cases, the metal is left uncoated and these are used for canned milk and some fruit products. Composite containers

5 are usually made with laminated paper, plastic and foil made from aluminum. Metal packaging has been used for many years because it provides excellent barrier to moisture, gases, light and microorganisms. Also, it is ideal for recycling and is being the most recycled food and beverage container in the world. The most commonly used metal containers for food packaging are tin coated steel cans (Marsh and Bugusu 2007). These exist as either two or three-piece cans. Three- piece cans consist of a bottom lid, a cylindrical body and a top lid. The two-piece can is made of the can body with a lid attached to only one end. Two-piece cans are made by stretching one sheet of metal to create the bottom and body of the can. The open top of the can is covered by a lid to give a complete package. The lids in the three-piece and two-piece cans are joined to the body by a method called the double seam (Singh et al. 2017). The double seam is formed during two operations, and these are referred to as the first and second operations. Figure 3 shows the double seam first and second operations. During the first operation, the end curl of the lid is folded around the flange of the body of the can. In this process, the seam should not be too loose or too tight.

Errors in forming the seam during the first operation cannot be corrected during the second operation. The second operation is designed to press the cover hook into the body hook and iron out wrinkles, distribute the sealing compound throughout the seam, and to develop the proper degree of tightness.

Figure 3 The double seam first and second operations (21CFR113.60).

1.3.3 Paper board

The main source for paper is wood, but it can be made from different materials such as rags, cotton, straw and certain plant stems. Wood is composed of carbohydrates, lignin and cellulose.

To create paper, cellulose must be removed from the lignin. This is called the pulping method and it could be done by either mechanical or chemical techniques. Chemical treatment produces fibers with less damage and is used for making paper with good mechanical strength. The mechanical method uses cutting and hammering to remove the cellulose (fiber) from the lignin. This method is faster and cheaper. Since it causes more damage to the fibers, it is used for paper with less

7 mechanical strength. Examples of these would be tissue paper. Different machines are used to make paper. These are the fourdrinier, cylinder and twin-wire machines (Emblem and Emblem

2012). Newsprint, book paper, commercial paper and labels are the commonly used paper types for printing purposes. Paper and paperboard are important in the food packaging industry because of their flexibility, ease of recycling, excellent printability and ability to hold geometric shapes

(Roncarelli and Ellicott 2010). Examples where they are used include folding cartons, setup boxes, tubs, trays and liquid resistant boxes. Liquid resistant boxes are made with a combination of paperboard, aluminum foil and plastic. These are usually coated with wax or polyethylene to increase wetting resistance (Emblem and Emblem 2012). Figure 4 shows a composite gable-top carton made from pliers of different materials and used to package liquid products. Another example is the corrugated fiberboard that is used mainly in tertiary packaging. These types of containers can be single, double or triple lined, depending on the product that is to be transported.

Figure 4 Gable-top cartons: (a) made with high barrier aluminum foil; (b) made with EVOH,

and used to package liquid products (Emblem and Emblem 2012).

1.3.4 Polymers

A polymer is a large number of monomer units (carbohydrates, lipids, nucleic acids or proteins) connected by covalent chemical bonds to make a large molecule that can be natural or synthetic in type. A plastic is made from polymers and is the term used to describe the final product.

Monomers are mostly produced from downstream petrochemical operations. Examples of monomers are acrylamide, mono styrene, ethylene, propylene and vinyl chloride (R. Allafi 2018).

Addition and condensation polymerizations are the two main methods of polymer syntheses.

Addition polymerization has no by-products, but condensation polymerization produces by- products such as H2O, HCl, NaCl, or CH2O (Selke and Culter 2016). Figure 5 shows the addition polymerization of polyethylene, a commonly used polymer for plastic bags and bottles. Addition polymerization consists of three steps: initiation, propagation and termination. For the initiation step, the creation of a free radicals is done on the monomer using a radical initiator such as an organic peroxide. This radicalized monomer then takes an electron from a stable monomer and continues with the propagation, which creates the repetitive growing chain. The termination step occurs when two radicals combine, thus paring their unpaired electrons and forming a covalent bond that links the chains together (R. Allafi 2018).

Figure 5 The reaction of monomeric ethylene molecules in the presence of an initiator to create

the polyethylene (Selke and Culter 2016).

Nylon 6,6 is an example of a condensation polymer that is synthesized by the polycondensation of hexamethylene diamine and adipic acid. Figure 6 shows the condensation reaction that creates nylon 6,6. The condensation reaction occurs when two compounds combine to form a single molecule and producing the loss of the small molecules mentioned earlier. High temperatures around 150-350oC are needed for the condensation polymerization to occur, but the temperature should not be close to the thermal degradation temperature of the monomers present in the reaction.

The monomers involved in condensation polymerization have functional groups such as amino alcohols or carboxylic acids. These functional groups would react to form an ester or amide linkage and the by product is released (Pethrick et al. 2011).

Figure 6 The synthesis of nylon 6,6 from adipic acid and hexamethylene diamine (Boyd and

Phillips 1996).

Polymers are characterized by studying their morphology, molecular weight and molecular weight distribution, thermal properties, chemical composition and microstructure of their chemical

12 backbone. Polymer are classified depending on the source from which they are made. Polymers made from hydrocarbon sources, renewable resources or polysaccharides are some examples (R.

Allafi 2018). Polymers are also divided into thermoplastics and thermosets. This is based on how they behave at high temperatures. Thermoplastics can be formed, melted back into a liquid, and reformed again. On the other hand, thermosets are irreparably damaged when melted (Ghosh

2015). Polymers are also classified as being either homopolymers or heteropolymers.

Homopolymers have the same repeating building-block throughout their molecule.

Heteropolymers have two or more different building-block units. Common plastic shaping methods are extrusion cast film and sheets making, injection molding, extrusion blow molding, compression molding, injection blow molding and co-extrusion. Synthetic polymers commonly used in the food packaging industry include low-density polyethylene (LDPE), high-density polyethylene (HDPE), polystyrene (PS), polypropylene (PP), polyethylene terephthalate (PET), nylons, polyvinylidene chloride (PVDC), ethylene vinyl alcohol (EVOH) and polyvinyl chloride

(PVC). Plastic is commonly used for food packaging applications because it is widely available, easy to process, light weight, unbreakable, relatively inert and low cost. Since there has been an increase in the environmental concern about plastic waste, many researchers are focusing on the creation of bio-based sustainable plastic packaging for the future. These bio-based sustainable plastics can be made from polysaccharides, proteins, lipids or a combination of these (Wan 2016).

1.3.4.1 Petroleum based polymers

Petroleum based polymers are made from non-renewable resources such as crude oil and natural gas and are used in greater quantities in the food packaging industry because of their low price and good mechanical characteristics. Petrochemical derived plastic materials could be harmful to the environment because they add greenhouse gases, and efforts to recycle them are not as effective as it should be. These are the main reasons why research has focused on biodegradable, compostable and bio-based plastics (Muhammad Shamsuddin 2017).

1.3.4.2 Bio-based polymers

Bio-based polymers are derived from annually renewable sources. These polymers are called bio-based because they are derived from biomass or microorganisms (Rudin and P.Eng 2012).

Biologically derived resources were used before petroleum-derived chemicals for products like clothing, synthetic fibers and packaging. Replacing bio-based materials with petroleum-derived ones was due to the lower cost of production and the superior properties of petroleum-derived plastics (Chiellini 2008). In addition to this, many governments are enacting legislations that are encouraging the use of bio-based packaging. Consumers are also more educated about the problems of solid waste disposal and are demanding more bio-based materials (Song et al. 2009).

Bio-based packaging materials are divided into three different categories depending on their development. The first category includes polymers extracted from biomass such as polysaccharides and proteins. Proteins from milk, soy, chickpea, gluten, corn or collagen can also be used to make plastics. The second category includes polymers that are synthesized from bioderived monomers such as polylactates or polyesters. The third category includes polymers produced from natural or genetically modified organisms. Examples of these include polyhydroxyalkanoates (PHAs), polyhydroxybutyrate (PHB) or bacterial cellulose (Chiellini

2008). These materials can be used alone or in combination with other polymers to improve or influence the overall properties of the composite material (Han 2005).

1.4 Types of Edible Films

1.4.1 Polysaccharide-based edible films and coatings

Polysaccharides are being increasingly used in the food packaging industry because they are widely available, nontoxic, low cost and have the capacity to produce edible films with great characteristics. They are used in different food products to extend shelf life and improve the quality of the food (Baldwin et al. 2011). Table 1 shows simple generalized characteristics of each category of polysaccharide-based edible films. Polysaccharides can be obtained from plants in the form of starch, galactomannans, lectin and cellulose or from the exoskeleton of shellfish and

15 crustaceans. Carrageenan and alginate are polysaccharides obtained from algae and pullulan, while gellan gum and xanthan gum are obtained from microorganisms (Ferreira et al. 2016).

Starch is a natural polymer that is considered to be the most promising polysaccharide for edible film formation because it has several desirable characteristics. Polysaccharide-based edible films can be used to develop materials that could be used to package products such as poultry, meat, fruits, nuts, vegetables and confectionaries (Debeaufort et al. 1998). They could be used to reduce the respiration of fruits, to provide partial barrier to moisture and oxygen and be carriers of antimicrobials or other additives (Baldwin et al. 2011). Moreover, they can be combined with other polysaccharides, proteins or lipids to enhance properties such as poor moisture barrier or oxygen permeability (Su et al. 2010).

Table 1 Polysaccharides-based edible films and their significant characteristics.

Polysaccharide-based Edible Films Characteristics

Cellulose and Derivatives - Most abundant polysaccharide

- Major building block of plant cell

walls

- Good films-forming performance

Starches and Derivatives - Low cost

- Good component for edible film

formation

- Good oxygen-barrier properties

Pectin and Derivatives - Main commercially available form

- Main component in plant cell walls

- Generally recognized as safe

Seaweed Extracts - Retain water

- Good compound for industrial use

- Good for adding antimicrobial

compounds

1.4.1.1 Cellulose and derivatives

Cellulose is an abundant polysaccharide and the major building block of plant cell walls.

Figure 7 shows the composition of cellulose. It is composed of a linear chain of (1® 4)-b-D- glucopyranolsyl molecules. The beta molecules of cellulose link together using 1,4 glycosidic bonds to form the structure of cellulose (Conn and Stumpf 1976). This reaction produces water since it is a condensation reaction. Cellulose has an abundance of hydroxyl groups (OH-) that can form intramolecular and intermolecular hydrogen bonds. A variety of characteristics like biocompatibility, good films-forming performance and biodegradability make it a polymer of great interest for food packaging, without negative environmental impact (Ferreira et al. 2016).

Different cellulose derivatives can be formed in the presence of alkaline solutions (Dinand et al.

2002). Examples of these derivatives are methylcellulose (MC), ethyl methylcellulose (EMC), hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), and sodium carboxymethylcellulose (CMC).

Figure 7 Structure of a repeating unit of cellulose (O’Sullivan 1997).

Methylcellulose is synthetically produced from the substitution of the hydroxyl groups of cellulose for methyl groups, thus decreasing the polarity of the cellulose molecule. Additionally, it is non-ionic and the least hydrophilic when considering all other cellulose derivatives such as

EMC, HPMC, HPC and CMC (Yoo and M Krochta 2011). Figure 8 shows the molecular structure of methylcellulose. Krochta and Yoo (2011) created edible films using methylcellulose and reported that the results for oxygen permeability followed the rule of like-dissolves-like, meaning that polar compounds will dissolve in polar solvents and likewise non-polar compounds will dissolve in non-polar solvents. In less polar polymers like methylcellulose, oxygen would dissolve better because it is a non-polar molecule. Methylcellulose could be used to create an edible film with higher oxygen permeability and with high tensile strength and elastic modulus due to the

19 organization of the polymeric chain when compared with EMC, HPMC, HPC and CMC (Yoo and

M Krochta 2011).

Figure 8 Molecular structure of methylcellulose (Huber and Embuscado 2009a).

Hydroxypropyl methylcellulose (HPMC) is produced by the substitution of hydroxyl groups on the polysaccharide molecule with methoxyl and hydroxypropyl groups. Figure 9 shows the molecular structure of hydroxypropyl methylcellulose. It creates tasteless edible films with relatively good mechanical properties, excellent oil barrier and relatively quick solubility in water

(Yoo and M Krochta 2011). Blending HPMC with other polysaccharide is an effective method that can be used to enhance the properties of this cellulose derivative (Su et al. 2010). Blending

HPMC with whey protein isolate, for example, can create a film with a combination of properties or with more superior properties than that of HPMC alone. Hydroxypropyl methylcellulose produces transparent, odorless, low flexibility films with relatively high tensile strength. On the

20 other hand, whey protein isolate (WPI) produces films with good flexibility and elongation but low tensile strength. Blending HPMC with WPI produces films with properties superior to those of the individual compounds. In this blend, HPMC adds hydrogen bonding capacity which helps with cross-linking and a strengthening of the mechanical properties of the blend. Whey protein isolate in the blend adds hydrophobic interactions and disulfide bonding which help with improved moisture and oxygen barrier, and mechanical strength (Pallas and Krochta 2008).

Figure 9 The structure of Hydroxypropyl methylcellulose (Quinten et al. 2011).

Hydroxypropyl cellulose (HPC) is non-ionic and can be used for commercial formulations because it is non-toxic and a non-irritant (Francis et al. 2006). It has characteristics such as lower tensile strength and greater elongation when compared with other cellulose derivatives (Baldwin et al., 2011).

1.4.1.2 Starches and derivatives

Starches are very common in food products because of their low cost and abundance. Some properties that make starches good for edible film formation are their biodegradability, high functionality and the good oxygen-barrier properties (Yoo and M Krochta 2011). Starches contain amylose and amylopectin which are two different polysaccharide fractions that are present in different sizes and shapes (Buléon et al. 1998). Figure 10 shows the structures of amylose and amylopectin. Amylose is a small and linear polysaccharide made from (1®4)a-D-glucopyranosyl units. This starch polymer is mostly used to form films because it has a liner structure (Huber and

Embuscado 2009). Amylopectin also has (1®4)a-D-glucopyranosyl units and has large numbers of branches made of glucose (Yoo and M Krochta 2011).

Figure 10 The structure of amylose and amylopectin (Huber and Embuscado 2009a).

The amount and proportion of amylose and amylopectin in a starch molecule vary depending on the source of the starch. Most starches contain around 18% to 30% amylose, except in the case of waxy corn starch that contains mostly amylopectin. This influences properties such as film formation ability, tensile strength and elongation of edible films made from this polymer.

Amylose has the potential to form strong, odorless, taste and colorless films (Wolff et al. 2002).

On the contrary, amylopectin does not have that film-forming potential that amylose shows.

Blending starch with other polymer like whey protein isolate is an approach that has been used to improve the properties of starch-based films. Almasi et al. (2009) reported that moisture uptake and mechanical properties are example of these. Yoo and Krochta (2011) used two different types of corn starch to create edible films: melojel and amioca. Melojel is a regular corn starch with

23% amylose and amioca is waxy corn starch with 99% amylopectin on a dry weight basis. It was found that blending starch with whey protein isolate created a film with lower oxygen permeability, tensile strength and elastic modulus when compared with films made of whey protein isolate with cellulose derivates such as methylcellulose and hydroxypropyl methylcellulose.

Modified starches have been used in the food industry because they have better overall properties than the unmodified forms. Native starches do not produce good gels because they are weak-bodied. To make good edible films they must be chemically modified (A. Abbas et al. 2010).

A method that is used to modify starches is the starch hydrolysis by acid treatment (Baldwin et al.

2011). Different modification processes create starches with distinctive properties that can be used for different applications such as binders, thickening agents, gel forming or coatings (A. Abbas et al. 2010). Acid hydrolysis is done by exposing starch to different acids such as sulfuric (H2SO4), hydrochloric (HCl), nitric (HNO3) or phosphoric acids (H3PO4). This causes cleavage of the glucosidic linkages (Pratiwi et al. 2018). The acid hydrolysis alters the structure of the starch and

24 has an effect on the physicochemical properties. Bleaching, another method used to modify starches, is an alkaline treatment done by exposing the starch to sodium or potassium hydroxide.

This treatment isolates the starch granules, thus making them high in purity and with well-defined physical properties (Wang and Copeland 2012). Additionally, the pyroconversion of starches is a well-known factor that should be taken into consideration during the formation of films.

Pyroconversion is also known as dextrinization and includes two aspects. The first one is a partial depolymerization that is achieved through hydrolysis. This is accomplished by the addition of water to break the bonds. The second aspect is the recombination of the fragments in a branched structure. These modified starches are identified as dextrin (Taggart 2004). In the food industry the chemical cross-linking of starch polymer is used to stabilize and texturize food systems since cross-linked starches have better properties than natural or unmodified starches (Stephen and

Phillips 2016).

1.4.1.3 Pectin and derivatives

Edible films created from pectin are usually made from apple pomace and citrus peel because they are the principal commercially available forms (Baldwin et al. 2011). The soluble form of pectin is obtained using an extraction process in acidic conditions where the pectin is separated from the citrus peel. At this point it is known as the soluble pectin. Pectin is considered

25 a main component in plant cell walls. Figure 11 shows the repeating segment of the pectin molecule. The backbone of the pectin molecular chain contains of (1®4)-a-D-galacturonic acid units, including a varying degree of methylation of its carboxylic acid and/or amidated polygalacturonic acids, depending on its source. This can give rise to high methoxyl or low methoxyl pectins (Espitia et al. 2014). High methoxyl pectin refers to those in which greater than

50% of the carboxyl groups are methylated, while low methoxyl pectin has less than 50% percent methylation (Voragen et al. 1986). Pectin has received a lot of attention as a film formulation component because it is considered generally recognized as safe (GRAS) by the FDA. In solution, it is used by the food industry as a stabilizer, gelling and thickening agent (Espitia et al. 2014).

Figure 11 Repeating segment of the pectin molecule (Thakur et al. 1997).

Galus and Lenart (2013) combined different polysaccharides to improve the functionality of each and found that blending sodium alginate with pectin created a homogeneous and transparent edible film. The transparency of the film is important when sensory aspects, such as consumers acceptability is taken into consideration. These researchers also found that the pectin film network was not as organized when compared with that of alginate films (Galus Kokoszka and Lenart

2013). Other studies showed that films made from pectin have the ability to be used for antimicrobial activity, thus protecting the food from foodborne pathogens (Espitia et al. 2014).

1.4.1.4 Seaweed extracts

Three of the most common seaweed extracts used in the food industry are alginates, carrageenan and agar. Alginates are salts derivatives from alginic acid. Alginic acid is a polysaccharide with a high molecular weight that contains D-manuronic and L-guluronic acids

(Ertesvåg and Valla 1998). Figure 12 shows the chemical structure of alginic acid. Its capacity to bind calcium and other divalent ions gives it the ability to form good gels.

Figure 12 The chemical structure of alginic acid (Szklarska et al. 2014).

Carrageenan is another commonly used seaweed extract and it is a mixture of polysaccharides.

It is isolated from red seaweeds. There are three types of carrageenan: i-, k-, and l-, and each has special properties because of the substitution patterns present in the polymers (van de Velde 2008).

Kappa carrageenan has the capacity to form strong and rigid gels in the presence of potassium ions. Soft gels are formed when iota carrageenan is in the presence of calcium ions. Lambda carrageenan does not have the capacity to form gels. Figure 13 shows the different structures of commercial carrageenan. Kappa and iota carrageenan are used as gel forming compounds in the food industry and they have been investigated in combination with starch. Abdou and Sorour

(2014) found that, as the concentration of carrageenan increased, the water vapor permeability, the elongation and the tensile strength of the films made with it, also increased. Carrageenan is more hygroscopic than starch and it causes more water uptake than starch and this explains the increase elongation at break when carrageenan was added in higher quantities in the starch/carrageen blend.

This water uptake will increase the plasticization of the films, causing a higher elongation (Abdou and Sorour 2014).

Figure 13 Chemical structure of commercial carrageenan (Tavassoli-Kafrani et al. 2016).

Agar is a galactan polysaccharide extracted from the intercellular matrix of red seaweeds. It is composed of b(1,3) and a(1,4)-linked galactose residues. It does not have good film making properties or uses in food applications because it tends to separate into water over time, but polyols have been found to increase the gel strength when added into a multicomponent gel system

(Whistler 2012). Films made of agar have been investigated by D. Phan and colleagues (2005) for food quality preservation. It was found that when plasticized with glycerin, agar can form a

30 transparent, homogeneous and clear film. This film can be used in some foods, mostly for short- term storage, because of their hygroscopicity (D Phan et al. 2005).

1.4.2 Protein-based edible films and coatings

Proteins can be divided into two different groups: fibrous and globular proteins. Fibrous proteins are insoluble in water, associated with each other via hydrogen bonding and fully extended

(Hassan et al. 2018). Globular proteins are soluble in solutions such as acids, bases or salts and they fold into spherical structures in which ionic, covalent and hydrogen bonds hold together the structure (Wan 2016). Soy and whey protein are examples of globular proteins that have received attention in the food packaging industry because they are capable of forming films with favorable characteristics. Table 2 shows important characteristics of different protein-based edible films.

Characteristics such as transparency, barrier against oxygen and carbon dioxide make them valuable for food packaging and coating applications (Chiralt et al. 2018).

Table 2 Protein-based edible films and their significant characteristics.

Protein-based Edible Films and Coatings Characteristics

Soy Films and Coatings - Natural low oxygen permeability

- Low moisture barrier

- High flexibility

Wheat Gluten Proteins - Low tensile strength

- Low oxygen permeability

- High elasticity

Whey Proteins - Tasteless

- Flexible

- Good oxygen permeability and

impermeability to oil

Casein Films and Coatings - Low gas barrier and oil migration

- Low water vapor transmission rate

- High tensile strength

1.4.2.1 Soy films and coatings

Soy is a protein classified as globulin and it has the capacity to create films or serve as a coating agent for different food products. It has been used for many years in the Orient to wrap different meats and vegetables (Brandenburg et al. 1993). This protein is high in asparagine and glutamine residues and can be classified into groups depending on the protein content. Defatted soy flour

(DSF) belongs in a group with a protein content ranging from 50% to 59%. Soy protein concentrate belongs in a group having a protein content of 65% to 72%, while soy protein isolate has a 90% protein content (Baldwin et al. 2011). Soy protein edible films can be created by using different methods such as cast film-forming, extrusion and compression molding, among others.

Some of the characteristics of soy protein films are high transparency, good flexibility, great oxygen barrier, but low moisture barrier (Cho et al. 2007). Although soy protein isolate can form clear films, some particles can be insoluble, and this could give rise to a film that cannot be commercialized. The low oxygen permeability of soy protein isolate films makes them a good choices for preserving products that are susceptible to oxidative deterioration (Brandenburg et al.

1993).

Different methods used to improve the water vapor permeability and the tensile strength of soy protein films have been studied. An interaction between soy protein isolate and cellulose has been shown to create molecular interactions and hydrogen bonding between the two polymers. The

33 combination of soy protein isolate with other components such as lipids can also improve the water barrier properties of films made from these blends (Sessa and Willett 1998). The functionality of soy protein concentrate has not been studied as much as soy protein isolate, but comparable characteristics with soy protein isolate were found. In using soy protein isolate to make films, a pH adjustment step is required in order to increase the solubility of the protein (Cho et al. 2007).

However, more transparent film with higher solubility and uniform tensile strength can be created using soy protein concentrate without the need for a pH adjustment.

1.4.2.2 Wheat gluten proteins

The residual that results from washing away the starch from wheat flour dough is known as wheat gluten (WG). The composition of wheat gluten include protein, traces of starch, nonstarch polysaccharides and minerals. This protein is classified into four different categories: albumins, globulins, gliadins, and glutenins. The solubility of the protein in each group is different. The protein network of gluten is made of extensive covalent and noncovalent bonding.

Edible films made from wheat gluten can be made by cast film-forming or extrusion molding (J.a and J.s 1980). It is believed that intermolecular disulfide, hydrophobic and hydrogen bonding occur during the film formation process (Tanada-Palmu et al. 2000). The mechanical and barrier properties of these films have been studied for applications such as the coating of fruits and

34 vegetables. When wheat gluten films are produced in alkaline conditions, they appear yellowish in color, and films made under acidic conditions tend to be more brittle and are prone to develop flex cracks (Tanada-Palmu et al. 2000). Even though these films have these characteristics, in general, they tend to show low tensile strength and high elasticity when compared with films made with zein, soy protein isolate or sodium caseinate (Gennadios et al. 1993). Edible films made from wheat gluten have low oxygen permeability due to their polar nature and their linear structure.

This allows them to form cross-linking bonds with adjacent chain, especially when they interact with certain polar molecules, and under specific conditions. This low oxygen permeability could change in the presence of glycerol or plasticizers such as fatty acids or polyols. The amount of plasticizer added to these films could potentially change their properties. For example, it could reduce brittleness and increase the flexibility of the films (Tanada-Palmu et al. 2000). Other options such as combining wheat gluten with other proteins or polysaccharides can be done to reduce this brittleness and change other physical and chemical characteristics of the films.

Antimicrobial compounds can also be added to the film and this could help to reduce microbial contamination or to extend the shelf-life of some food products.

1.4.2.3 Whey proteins

Films made from whey proteins could help to decrease the use of synthetic polymers, and thus create a better environmental impact because whey protein is edible and biodegradable. This protein can be used to create a transparent, tasteless and flexible edible film with good oxygen permeability and impermeability to oils. Although these films show poor mechanical strength and high water vapor permeability, these properties could be improved by blending whey protein with another protein or a polysaccharide (Yoo and Krochta 2011). Figure 14 presents a DSC thermogram of the HPMC-WPI blended films at a ratio of 1:1 with the average of the pure WPI and HPMC.

In characterizing polymeric films, factors influencing the mechanical properties are investigated. Mechanical properties include tensile strength, elastic modulus and percent of elongation at break. Tensile strength is the ability of the film to resist being pulled apart by tensile forces. Films made from whey proteins generally tend to have low tensile strength, but this can be modified by combining them with other biopolymers such as methylcellulose or hydroxypropyl methylcellulose (Yoo and Krochta 2011). Elastic modulus is an important property because it measures the resistance of the film to be deformed. Blending whey proteins with the other biopolymers can work to increase the elastic modulus of the film. The elongation at break is also reported as a percent and this is the strain on a sample when it breaks.

Ramos and colleagues (2013) investigated the influence of glycerol in WPI and WPC on the barrier properties of films made from these proteins. Whey protein isolate and concentrate differ in their protein, lipids, minerals and lactose contents. These differences can influence properties such as oxygen and water permeabilities, tensile strength and thermal properties of films made from WPI and WPC. This is because of the difference in the intermolecular bond between WPI and WPC. Published research showed that the oxygen and water vapor permeabilities of WPC were higher than those of WPI when the same glycerol content was added to both compounds.

This indicated that films made from WPI were more stable and the network was more strongly crosslinked when compared with WPC (Ramos et al. 2013a). Whey proteins generally has a lower oxygen permeability when compared with films made from wheat gluten and soy proteins. This is because of the polar nature of whey protein and the linearity of its polymeric arrangements. In general, increasing concentrations of a plasticizer in whey protein causes the interchain attractive forces to decrease and this causes the energy of activation for diffusion to also decrease. This in turn makes it easier for the diffusion of moisture though the material and this can increase the water vapor permeability of films made from whey protein isolate and glycerol (Zinoviadou et al.

2009, p.).

Figure 14 DSC thermogram of the HPMC-WPI blended films at a ratio of 1:1 with the average

of the pure WPI and HPMC (Yoo and M Krochta 2011).

1.4.2.4 Casein films and coatings

Casein is a major dairy protein group found in milk and synthesized in the mammary gland of bovine, goats and other ruminants. Casein is a family of phosphoproteins and has a large number of proline amino acids with a hydrophobic nature. The phosphorus in casein can be esterified to form phosphate centers that can bind to calcium cations and form strong hydrophobic interactions

(Fox et al. 2015). There are four subunits of casein: alpha s1-casein, alpha s2-casein, beta-casein and kappa-casein. Each sub-unit exhibits different properties when used to form films. The molecule is a “random-coil” structure that provides an open and flexible conformation. The amino acid composition of casein has high content of proline and low content of cysteine. Since the

38 molecule is a random coil and it has the ability to form hydrogen bonds, no further treatment is needed if casein is to be used for the formation of a film (Gennadios 2002).

The addition of lipids to casein base films was studied by Chick and Hernandez (2006). In that study carnauba and candelilla waxes were used because they have good water barrier properties.

Scanning electron microscopy was used to determine the microstructure of the film samples. The results showed that no difference between them after different quantities of the waxes were blended with the casein. However, continuous and homogeneous phases were observed after analyzing cross-sectional areas of the blended films using electron microscopy. Chick and Hernandez (2006) also studied the tensile strength and elongation of films with and without carnauba and candelilla wax and found that at a higher relative humidity, films with either wax presented higher tensile strength and lower elongation when compared with the films without any wax. This showed that water plasticized the films without wax and exposed to a high relative humidity. Other tests such as the thermal characteristics and barrier properties were also conducted. The results showed lower water vapor permeability when compared with the films without wax, concluding that these films had the potential to be used in the food industry (Chick and Hernandez 2006).

1.4.3 Lipid-based edible films and coatings

Lipid-based edible films can be used to prevent or limit the migration of moisture between different food components, and to coat products such as fresh fruits and vegetables, to prevent desiccation. Substances such as oils, fats, waxes, paraffin, resins, shellac and essential oils have been used in making films (Huber and Embuscado 2009a). Table 3 has important characteristics of different lipid-based edible films. Excellent moisture barrier but poor mechanical properties are some of the characteristics that are seen in lipid-based edible films (Wan et al. 2007).

Table 3 Lipid-based edible films and their significant characteristics.

Lipid-based Edible Films and Coatings Characteristics

Waxes and Paraffin - Can limit the migration of moisture in

food products

- Generally recognized as safe

- Colorless, odorless and tasteless

Resins - The color goes from clear to

translucent yellow depending on the

resin

- Viscosity goes from high to semisolid

depending on the resin

- Soluble in acidic and alkaline solutions

- Not a GRAS substance

1.4.3.1 Waxes and paraffin

Different formulations and compounds are used to create edible films and coatings, but waxes are most commonly used. Examples of waxes that are used to coat fruits and vegetables

41 are carnauba, polyethylene, paraffin, bees and candelilla waxes. Carnauba wax has a relatively high melting point and is used in combination with other waxes to improve characteristics such as the melting point and toughness of films and coatings. This wax is generally recognized as safe

(GRAS) and it can be added to different products without limitations other than following good manufacturing practices (GMP’s) (21CFR184.1978). To substitute carnauba wax in the process of microemulsions, polyethylene wax can be used. Carnauba wax can be used in products that must be peel before consumption. Examples of these are avocado or bananas. Beeswax is a product of honeybees and it is considered generally recognized as safe. One limitation of bees wax is that it is plastic at room temperature but at lower temperatures it is brittle. Candelilla wax is another generally recognized as safe (GRAS) product that is obtained from the candelilla plant and it is commonly used in chewing gums and hard candies (21CFR184.1976). Products like cheese and yucca are commonly coated with paraffin wax obtained from the refining of crude petroleum. The refining process of crude petroleum includes vaporization and fractionation.

Being colorless, odorless and tasteless are some of the characteristics that make paraffin wax a good option for the coating different food products.

1.4.3.2 Resins

Resin is an acidic compound that is secreted by plant cells in response to an injury and it can be used as an edible coating on certain foods (Baldwin et al. 2011). This coating has some interesting characteristics. For example, its color can change from clear to translucent yellow and the viscosity can be relatively high, and it could even become a semisolid. Those characteristics allow the resins to be used in synthetic plastics and in other products such as pharmaceuticals.

Shellac is also used for pharmaceuticals as well as in food processing. Resin is used to coat apples, chocolates and citrus fruits. Since resin is not considered a GRAS substance, it is added as an indirect additive in food (21CFR177.2260).

1.5 The methods of characterizing edible films

1.5.1 Thickness

Factors such as the processing parameters, environmental conditions and the nature of the additives (including the type of plasticizers) used to make an edible film can affect the water vapor permeability, oxygen transmission rate and the mechanical properties of the material (Galdeano et al. 2013). The thickness of a film is the distance between the opposite faces of the material. This measurement can be taken by using a micrometer, thickness gauge, ultra violet imaging, Scanning

Electron Microscopy or Atomic Force Microscopy. The thickness of films can be influenced by

43 the relationship between the mass of the dry matter in a film forming solution and the molding area of a casting plate in the batch method of forming the film. This is so because heat is used to evaporate the solvents thus leaving the dissolved solids to form the film. According to Park and

Chinnan (1995) a relationship exists between the permeability of gases and the thickness of a given film (J. Park and S. Chinnan 1995). The gas permeability is described mathematically by Fick’s

First and Second Laws. Fick’s First Law explains how a solute will move from a region of high concentration to a region of low concentration across a concentration gradient. Fick’s first law of diffusion states that “the rate of transfer of diffusing substances through unit area of a section is proportional to the concentration gradient measured normal to the section” (Crank 1980).

Where:

�ϕ � = −� (1.1) ��

Fick’s Second Law predicts how diffusion influences concentration changes with time. Where:

�∅ �∅ = � (1.2) ��

Where D is the diffusion coefficient (m2/s), � is the rate of transfer per unit area of the film, f is concentration (mol/m3), x is the distance traveled (m) and t is time (s) (Robertson 1998).

1.5.2 Tensile strength, elongation and elastic modulus

The ability of an edible packaging to maintain its integrity is crucial when it is to be used as a food coating or as a stand-alone film. Thickness, tensile strength, elongation and elastic modulus are important characteristics of a packaging material. The ratio of the stress-strain curve and the slope varies depending on the deformation of the film when force is applied. For testing materials like edible films, the samples must be conditioned around room temperature and 50% relative humidity for at least 48 hours. The sample can be tested by mounting rectangular portions of the material into two separate grips that are located on the equipment. Tension will be applied and the tensile strength and elongation at break point can be obtained from the stress-strain curve. Figure

15 illustrates the tensile strength and the elongation curve of a protein-based film. Elongation indicates the change of the material original length and it can be expressed as a percentage. Tensile strength is the ability of the film to resist being pulled apart by tensile forces.

Figure 15 Stress and strain curve for protein-based film (Wan 2016).

1.5.3 Permeability properties

1.5.3.1 Oxygen transmission

The gas permeability, particularly oxygen and carbon dioxide, of edible films is often studied for their applications on products such as fruits, vegetables, dairy, meats and foods with a high fat content. The oxygen transmission rate of the package plays an important role in the shelf life determination of those products. To study the oxygen transmission rate of edible films, the ASTM

D3985 standard could be referenced. The test method is used to determine the oxygen transmission through the film after the sample is conditioned in an environment, having less than 1% relative humidity. During the test, the sample is mounted between two chambers. One chamber is purged

46 with nitrogen and the other with oxygen. As the oxygen permeates across the film it is sweeped towards a detector by a stream of nitrogen gas. This causes the detector to produce an electrical current that is proportional to the amount of oxygen that flows into it. This information is used by the software of the system to calculate the oxygen transmission rate through the sample.

The oxygen permeability coefficient is the result of the permeance and the thickness of the film. The ASTM standard recommends studying the relationship between permeance and thickness with several different thicknesses of the material before using this quantity. The oxygen permeance is defined as “the ratio of the oxygen transmission rate to the difference between the partial pressure of oxygen on the two sides of the film” (ASTM D3985-17). The oxygen transmission rate is defined by the ASTM standard as “the quantity of oxygen gas passing through a unit area of the parallel surfaces of the film per unit time under the conditions of the test” (ASTM

D3985-17). The oxygen permeability coefficient (OPC) quantifies the oxygen barrier property of a material and the units are �� × ��/(� × � × ��). The oxygen transmission rate describes the quantity of oxygen gas that pass through the area of the parallel surfaces of a plastic film per unit time under the conditions of the test (�� × �/(� × �). The oxygen permeability coefficient is correlated to the oxygen transmission rate by the following equation:

� �� = �� × (1.3) ∆�

Where 1 is the thickness of the film (m) and DP is the oxygen partial pressure (Pa), which is the mol fraction of oxygen multiplied by the total pressure (ASTM D3985-17).

1.5.3.2 Water Vapor Transmission Rate

The water vapor barrier properties of edible films and other packaging materials are known to significantly influence the shelf life and quality of moisture sensitive food products. The ASTM

E96/E96M-16 standard defines water vapor permeability as “the rate of water vapor transmission

(WVT) through a unit area of a flat material of unit thickness by unit vapor pressure difference between two specific surfaces, under specified temperature and humidity conditions”. Several methods are currently used to test WVT of films used for food packaging. This include the gravimetric method, using an electrolytic detection sensor, using a modulated infrared sensor and using an infrared detection technique. The gravimetric method is done by sealing the film or the material to a test dish containing a desiccant or distilled water. This dish must be impermeable to moisture. The dish is then placed in a controlled environment of temperature and relative humidity. At given times, its weight changes are recorded until reaching equilibrium. The water vapor permeability (WVP) can then be calculated using the following equation:

(� − � )ℎ �� (� ∙ ��⁄�� ∙ � ∙ ℎ) = (1.4) (� − �)��

Where h is the film thickness (mm), PA1 and PA2 are the partial pressure (kPa) of water vapor at

100%RH for the dish containing the distilled water and 50% RH for the controlled environment,

2 respectively, A1 is the area of the exposed film (m ) and T1 is the time (hour) during which the changes in weight (g) had taken place.

The Standard Test Method for Water Vapor Transmission Rate through plastic film and sheeting using a Modulated Infrared Sensor (ASTM F1249 – 13) is done by placing the material to be tested between a dry and a wet chamber at pre-determined temperature and humidity. The water vapor that diffuses through the film mixes with the gas that is present in the dry chamber and it is swept towards a pressure-modulated infrared sensor that measures the infrared energy absorbed by the water and produces a proportional signal. This signal is compared with a signal produced by the equipment when a calibration film is tested. The software uses the data to calculate the water vapor transmission rate of the test film (ASTM F1249 – 13). Figure 16 and

Figure 17 shows the measuring and the calibration systems for the modulated infrared sensor method.

Figure 16 Measuring system for the Water Vapor Transmission Rate through a plastic film and

sheeting using a Modulated Infrared Sensor (ASTM D1249-13)

Figure 17 The conditioning System for the Water Vapor Transmission Rate through a plastic

film and sheeting using a Modulated Infrared Sensor (ASTM D1249-13).

The Infrared Detection Technique is another Standard Test Method used to calculate the Water

Vapor Transmission Rate of flexible materials. Figure 18 shows the functional diagram of the diffusometer. This test method uses a film to separate a dry chamber from a wet chamber where

51 the temperature and humidity are known. The differential between two bands in the infrared spectral region is monitored to measure the time for a given increase in water vapor concentration to develop in the dry chamber. The information that is obtained is used to calculate the water vapor movement through the test material. This study provides a rapid and reliable method for the

Water Vapor Transmission Rate testing of flexible materials (ASTM F372-99).

Figure 18 Functional Diagram of Diffusometer (ASTM F372 – 99).

1.5.3.2.1 Moisture content

The diffusion of moisture into a food product increases its water activity (aw). This increase in water activity can decrease the shelf life of the food product and also increases microbial growth, texture degradation and deteriorative chemical reactions. The migration of additives such as plasticizers or antimicrobials is also affected by the moisture content of the film. This could be beneficial for materials containing additives such as antioxidant and antimicrobial agents that are intended to migrate from the package to the food product (Anker et al. 2001). Other properties like tensile strength, tearing resistance, dimensional instability and flexibility are also affected by the moisture content. Moisture content can be determined by oven drying, which is a gravimetric method. For this test the samples should be conditioned at 23oC and 50% relative humidity for around 24 hours. The samples are then dried at 105±2oC inside the oven until a constant weight is achieved. Moisture content is calculated using the following equation:

(� − � ) ��, % = × 100 (1.5) �2

In where W1 is the original weight (g) of the specimen and W2 is the weight (g) of the specimen after oven drying.

1.5.3.2.2 Water activity

Water activity is defined as “the ratio of the vapor pressure of water in food to the vapor pressure of pure water at the same temperature” (Sancho-Madriz 2003). This represents the water in the food product that is available to react with another material. When the relative humidity in the food is in equilibrium with its environment, the water activity will be equal to the relative humidity divided by 100. The water activity result ranges from zero to 1.0 which indicates pure water and it can increase as the temperature increases. Specific water activity values in certain food products are important since it impacts microbial outgrowth and chemical and enzymatic reactions (Hettiarachchy and Ziegler 1994). Unlike moisture content, which is the quantity of water contained in a material, water activity is more relevant to the growth of microorganisms, chemical processes, enzyme activity and physical parameters. For microorganisms to grow, a minimum level of water activity is needed. Microorganisms such as bacteria and fungi require high water activity to grow. The water activity (aw) is commonly determined using a water activity meter (AquaLab Ò, Decagon Devices, Inc., Pullman, Washington, USA).

1.5.3.3 Thermal properties

1.5.3.3.1 Differential scanning calorimetry

Differential Scanning Calorimetry (DSC) is a useful tool that is used to determine the thermodynamic properties of different materials like proteins, polynucleotides and lipid assemblies. During this test, the phase transition of the ingredients can be determined and details about the unfolding process and the intermediate state characteristics during the melting of a protein material can be obtained. This instrument can also provide information about the interaction between proteins and polysaccharides and between other materials (Spink 2008). Other information that can be obtained using DSC is the absolute partial heat capacity of a molecule, crystallinity, melting and the thermodynamic parameters that are associated with transitions that are induced by heat. Examples of these transitions include glass transition temperature, enthalpy, entropy and heat capacity change (Shirley 1995).

The measurement is made by comparing two identical cells. One cell contains the sample under investigation and the other one contains a reference sample that could be a buffer or an empty aluminum DSC pan. The difference in heat capacities between both of the cells is what the machine measures. The heat capacity is the amount of energy the sample can hold, and it is measured by the heat required to raise the temperature of the sample by one degree (oC). Figure

19 shows the calorimetric scan of a globular protein.

Figure 19 An illustration of the calorimetric scan of a globular protein where Cp,p is the heat

capacity of the protein solution and Cp,b is the heat capacity of the buffer solution as the

reference (Shirley 1995).

Edible films can be tested using DSC and the results commonly obtained are the glass transition temperature (Tg), temperature of melting (Tm), percent of crystallinity and the associated enthalpy

(DHm). When tested by DSC, edible films made from WPI and WPC display two thermal transition temperatures. The first transition temperature is associated with the amorphous fraction and the

57 second thermal transition is the melting transition for the crystalline region (Ramos et al. 2013b).

The glass transition temperature could be reported using the midpoint of the change in the heat capacity of the samples as shown in Figure 20. Yoo and Krochta used the midpoint of the change in the heat capacity of the samples to report the miscibility of films made using whey protein isolate and different polysaccharides (Yoo and M Krochta 2011). If the blend is immiscible, the thermograms will show two different peaks due to two different behaviors in the sample.

Figure 20 Glass transition temperature reported using the midpoint of the change in the heat

capacity of the samples (Spink 2008).

1.5.3.3.2 Thermo-gravimetric analysis

The thermal stability of edible films and other materials can be investigated using a thermogravimetric analysis (TGA) method. This technique measures the mass of a polymer as a function of temperature and time while the sample is exposed to heat over a given range (Menczel and Bruce Prime 2008). The study is done using a TGA-apparatus in which a sample is placed in the balance system and heated in a nitrogen atmosphere from room to a predetermined temperature, that in many cases is above 500oC. During the test, the sample is placed in an aluminum pan while an empty aluminum pan is used as a reference. As the test proceeds, a descending thermal curve shows the weight loss of the sample and the stages of degradation of the material can be seen from transitions in the peaks. The software of the equipment is also capable of plotting a derivative weight curve of the transitions (Kadam et al. 2013). Factors that could affect the TGA results are atmospheric turbulence, heating rate, thermal conductivity and enthalpy of the processes (Menczel and Bruce Prime 2008). As an example, sample with cross-linking will show less weight loss when compared with one that has no cross-linking at the same heating conditions. This is so because the cross-linked sample will withstand more heat before deterioration.

1.5.3.4 Mechanical properties

1.5.3.4.1 Dynamic mechanical thermal analysis

Dynamic Mechanical Thermal Analysis (DMA) is a technique in which deformation at a given frequency is applied to a film or material and its response to the deformation over a temperature range is measured. The information obtained reveals the mechanical properties of the film or material (Menczel and Bruce Prime 2008). The storage modulus, loss modulus and tan d of the films are provided as part of the results. The modulus is the elastic behavior of the edible film or the sample. How the energy dissipates in the material is known as the tan delta and what it expresses is how the material absorbs energy. Other properties of the polymer that can be tested using DMA are the glass transition temperature, secondary transitions, crystallinity, aging and phase separation. Figure 21 shows the thermal transitions and the multiple physical states of a polymer when tested by DMA analysis. The multiple physical states start with the bending and stretching of bonds which is known as glass. That continues with the segmental motion of the backbone and the gradual slippage of the main backbone. The last segment of the graph shows the free chain movement and interchain slipping which is known as liquid flow. A Dynamic

Mechanical Analyzer (DMA 2980, TA Instruments, Surrey, England) is an example of a machine that is used for this type of experiment.

Figure 21 The physical states of a polymer under various temperatures and a given frequency

(Menczel and Bruce Prime 2008).

Dynamic Mechanical Thermal Analysis has been used to study the aging of whey protein films with glycerol or sorbitol as a plasticizer and how it affects the mechanical and barrier properties of the film. Anker and colleagues used DMA to determine that films made with glycerol

61 became harder, or the storage modulus increased and the film became less extendable with the time, when compared with films made with sorbitol as a plasticizer (Anker et al. 2001).

1.5.3.5 Chemical structure and interaction

1.5.3.5.1 Scanning electron microscopy

Electron microscopy works by focusing a beam of electrons into a specimen. To create a good image with electron microscopy, the specimen has to be chemical treated (Carter and Shieh

2015). For example, whey protein isolate blended with hydroxypropyl methylcellulose as an edible film needs to be coated with a thin layer of gold in order to improve the contrast of the material. This is essential for easy visibility of the different components in the sample. Scanning electron microscopy (SEM) is commonly used to study the surface of specimens or materials like edible films. After collecting, processing and translating to pixels, the result of the interaction between the sample and the electron beam is an imagine of the specimen in what appears to be a three dimensional picture (Carter and Shieh 2015). As an example, scanning electron microscopy has been used to study the homogeneity of a sample made from blending whey protein isolate with titanium dioxide (TiO2). In this example, SEM was able to show if the TiO2 was dispersed in the

WPI or if it agglomerated (Li et al. 2011). It was also used to study the surface morphology of carboxymethyl cellulose/soy protein isolate blend edible films crosslinked by Maillard reactions.

In this example, SEM showed the presence of cracks and micro-holes and the general porosity of the sample (Su et al. 2010).

1.5.3.5.2 X-ray diffraction (XRD)

X-ray diffraction is used to determine the positions or the arrangement of atoms in a crystal.

It is a non-destructive technique that can analyze materials like fluids, powders and crystals. In edible films, it is used to determine the crystalline areas based on the main peaks. A peak that is not well defined means a low crystallinity of the material and multiple amorphous regions (Wan et al. 2017). On the contrary, a well-defined peak means high crystallinity and this result has a correlation with the barrier properties of the material. XRD works by shooting x-ray beams through a crystal of atoms and that crystal causes the beams to diffract in patterns that are predictable. With X-ray diffraction the stereochemistry, bond length and distance between the atoms can be determined. This test is usually done using a diffractometer with a vertical goniometer. The X-ray diffraction patterns of edible films are amorphous-crystalline. In edible films made with whey protein and cellulose, Le et al. (2000) showed that XRD peaks got sharper when there was more crosslinking of the chains in the sample (Le et al. 2000). In the case of composite edible films, they commonly present two diffraction peaks, meaning that the structure is amorphous-crystalline (Y.X. et al. 2005). The crystalline zone is seen as sharper and more

63 defined peaks. The amorphous zone is less defined near to the baseline and low in intensity.

Figure 22 shows X-ray diffractograms for films made with chitosan and starch and blends of these two polysaccharides. Two diffraction peaks were obtained for the film made with chitosan powder showing the crystallinity of the material. The starch film had an amorphous structure completely different to the structure of the starch powder. The starch powder was gelatinized and showed distinctive crystalline peaks. A combination of both structures, the crystalline and the amorphous, was also obtained after the blended starch-chitosan film was tested (Y.X. et al. 2005).

Intensity

2q (degree) Figure 22 X-ray diffraction patterns for films made with starch and chitosan powders (Y.X. et al.

2005)

2 The Incorporation of Selected Small Molecule Compounds into Whey Protein Isolate –

Hydroxypropyl Methylcellulose and their effect on Physical, Permeability, Thermal,

Mechanical and Chemical Properties

2.1 Introduction

Environmental-friendly packaging materials are increasing in popularity due to media reports of large stock piles of indiscriminately discarded plastic waste (Hao et al. 2019). Synthetic polymers are more harmful to the environment because they add greenhouse gases and efforts to recycle them are not as effective as bio-based materials (Rubilar et al. 2015). Biopolymer-based edible films have the potential to serve as food packaging and must protect the product from interaction with water, oxygen, temperature and microorganisms such as bacteria, viruses and fungi. Polysaccharides, proteins and lipids can be used to develop biopolymer-based edible films

(Su et al. 2010). Whey protein isolate (WPI) and cellulose derivatives are examples of biopolymers used to develop biodegradable films (Ferreira et al. 2016).

Whey protein isolate is a by-product generated when milk is separated by acid or rennet coagulation. Whey protein isolate is a versatile film former, and is colorless, tasteless, odorless, transparent, flexible and has excellent oxygen barrier. However, these films show relatively poor mechanical strength and high water vapor permeability (Fernández-Pan et al. 2012). Since films

66 developed with WPI are relatively brittle, the incorporation of a plasticizer such as glycerol, or blending it with other polysaccharides is needed in order to improve its properties. Cellulose derivatives such as Hydroxypropyl Methylcellulose (HPMC) have been used to improve the characteristics of WPI. Hydroxypropyl Methylcellulose is produced by substitution of the hydroxyl groups on the cellulose molecule with methoxyl and hydroxypropyl groups. It creates films with good oil barrier, excellent mechanical properties, tastelessness, but a relative poor oxygen barrier (Rubilar et al. 2015). It was hypothesized that with the incorporation of the hydrogen-bonding capacity of HPMC and the hydrophobic interactions and disulfide bonds of cross-linked WPI, HPMC-WPI blended films would have properties that would be superior to those of WPI and HPMC films alone.

The objectives of this study were to compare the physical, mechanical, thermal and permeability properties, and the chemical structure and interaction of films made with different concentrations of WPI and HPMC.

2.2 Experimental Design

Film formulation HPMC-WPI (100:0, 75:25, 50:50, 25:75, 0:100) + Glycerol

Morphology / Thermal Physical Properties Permeability Mechanical Properties Properties

Oxygen Transmission Differential Scanning Dynamic Mechanical Thickness Rate Calorimetry Analysis

Water Vapor Thermo-Gravimetric Moisture Content Tensile Strenght Permeability Analysis

Water Activity X-ray diffraction Elongation

Scanning Electron Elastic Modulus Microscope

Figure 23 Experimental Design Flowchart for HPMC-WPI Edible Films at Different

Concentrations

2.3 Materials and Methods

2.3.1 Preparation of HPMC-WPI at different concentrations

BiPRO whey protein isolate (WPI) powder (Davisco Foods International, Inc., Eden

Prairie, MN) weighing 6g was dissolved in 141.5g of distilled water and heated at 90oC for 30 minutes. This was done to break internal disulfide bonds and hydrophobic interactions and allow for the formation of inter-molecular interactions. The solution was then cooled in an ice bath to rapidly reduce the temperature. This was necessary to allow the formation of a crosslinked polymeric network structure when the WPI was blended with HPMC (Rubilar et al. 2015).

Hydroxypropyl methylcellulose powder (Methocelä F4M Food Grade Modified Cellulose, The

Dow Chemical Company, New Milford, CT), weighing 6g, was dissolved in 141.5g of distilled water and stirred on a magnetic stirrer/hot plate overnight (12 hours) after adding 2.5g of glycerol

(Fisher Scientific Inc., Fair Lawn, NJ, USA). The glycerol served as a plasticizer. A series of

WPI and HPMC solutions were prepared in ratios of 4:0, 3:1, 2:2, 1:3 and 0:4, respectively, to compare the properties of the blended films with the WPI and HPMC alone. The result was a total of 12g of blended HPMC/WPI in 283g of distilled water. Each WPI/HPMC blend was then heated to a temperature of 70oC. Each blend was mixed using a homogenizer (IKA Labortechnik T25

Basic, Wilmington, NC) at five different speeds. Each speed was one minute in duration and increased from: 8000 (min-1) to 9500 (min-1) to 13500 (min-1) to 20500 (min-1) and finally at 24000

(min-1). Each blend was then cooled in an ice bath to room temperature (23oC). Degassing of the blend was done using a KNF Neuberher vacuum pump model UN726 FTP (Trenton, NJ) overnight

(12 hours). Each solution was then poured into casting plates and dried at 40oC for 24 hours in an environmental incubator shaker (New Brunswick Scientific CO., Inc., Edison, NJ). The films thicknesses were measured using a Magna-Mike 8500 Thickness Gage (Olympus, Japan), with a resolution of 0.001mm. A total of 5 measurements per film, at various locations, were taken to determine the average thickness of each film.

2.3.2 Film Moisture Content and Water Activity

The moisture content of each film sample was determined by a gravimetric method according to a modified version of the ASTM D644-99 (2007) Standard. This test was done since moisture uptake can affect the tensile strength, tearing resistance, and flexibility of edible films in given applications. Before performing the test, a small square container made of metal wires and used to house the film samples, was dried at 105oC in a laboratory oven, then equilibrated to the room conditions in a desiccator containing P2O5, until a constant weigh was achieved. Each film sample tested weighed approximately 0.05g and was conditioned at 23oC for 24 hours in a desiccator containing P2O5 to create 51.8% relative humidity, prior to the test. The initial weight of the metal wires, container and each sample was recorded and measured every three hours until

70 a constant weight was achieved. The final moisture content was calculated using the Equation

1.5.

The water activity of each film sample was determined using a water activity meter

(AguaLab ®, Decagon Devices Inc., Pullman, Washington, USA). Each film sample weighed around 2.0g and was placed in a sample cup inside the AquaLab’s sample drawer. The drawer was closed, and the reading obtained. Each test was conducted in triplicate and the average value was recorded.

2.3.3 Oxygen Transmission Rate

The Oxygen Transmission Rates (OTR) of the films were tested using an OX-TRAN®

Model 2/21 Series OTR instrument (Mocon Inc. Minneapolis, MN). Calibration of the instrument was frequently performed with a standard sample obtained from Modern Control Inc. The method of oxygen permeability testing was done using the ASTM D3985-17 Standard with some modifications. The oxygen transmission rates of the film samples were measured at 23oC and 0% relative humidity. An aluminum mask was placed on each film sample to expose a test area of

5cm2. The tests were performed after the films were exposed to 50 hours of conditioning at 23oC and 0% relative humidity inside the machine. During the test, nitrogen carrier gas was used to purge the chamber of the diffusion cell while oxygen gas flowed over one side of the samples.

Oxygen gas that permeated through each film was transported to the detector at a flow rate of 10 cm3/min. To measure the quantity of oxygen that permeated the material, an oxygen-sensitive coulometric sensor was used. Each sample was tested three times and the test repeated twice.

2.3.4 Water Vapor Permeability

The ASTM E96 Standard was used to conduct the water vapor permeation (WVP) test on each film. The films were conditioned for 48 hours at room temperature (23oC) and 50% relative humidity using calcium nitrate (51.8%) in a controlled chamber. The test dishes with 50cm2 open area were filled with 5g of distilled water (DW) that created a relative humidity of 100% on one side of the films. The film samples were placed on top of the dish and sealed with an O-ring and a high vacuum silicone sealant. The dishes with the films and distilled water were weighted to obtain the initial weights. The cups were then placed in a humidity chamber at room temperature

(23±2oC) and a relative humidity of 50±2%. Each sample was weighed at intervals (0, 1, 4, 8, 24,

48, 60 and 72 hours) until a steady state weight was reached. The water vapor transmission rate in g.mm/kPa.m2.h (WVT) was calculated from the quantity of moisture that permeated the films at a given time, and unit area using the Equation 1.4. Three replicates per film were tested and the test repeated three times.

2.3.5 Thermal Analysis by Differential Scanning Calorimetry

A TA Instrument (Q200, USA) Differential Scanning Calorimeter (New Castle, DE) connected to a data collection station was used to determine the thermal transitions of the sample films. Each sample weighted approximately 5 to 7mg using a 5 decimal point (±0.01 mg) Mettler

Toledo Analytical Balance (Toledo, OH). Each sample was placed in an aluminum pan that was sealed with an inverted lid in order to achieve optimum thermal conductivity. The reference was an empty aluminum pan sealed using the same technique. Both pans were equilibrated under the same conditions and heated from 35oC to 250oC at a heating rate of 10oC min-1 for the first and second heating curves. For the cooling curve, each sample was cooled from 250oC to 35oC at a cooling rate of 10oC min-1. The thermograms were recorded and analyzed using the TA Instrument software (Universal Analysis 2000, Version 24.11).

2.3.6 Thermo-Gravimetric Analysis

A TA Instrument (Q50, USA) Thermogravimetric Analyzer with a data collector (New

Castle, DE) was used to determine the thermal transitions of the test films. Thermo-gravimetric analysis is a valuable thermal technique used to determine the temperatures at which the polymeric film samples lose volatile compounds. Each sample was weighed to approximately 20.0mg using a Mettler Toledo Analytical Balance (±0.01 mg) (Toledo, OH). Each sample was placed in an

73 aluminum heating pan. The reference was a similar aluminum pan, but without a film sample. It was also weighed before the test began. The pans were heated from 23oC to 600oC at a heating rate of 10oC min-1. The data were recorded and analyzed with the TA Instrument software.

2.3.7 Scanning Electron Microscope

A Scanning Electron Microscope (SEM) (Quanta 200, Thermo Fisher Scientific, USA) was used to qualitatively characterize the surface microstructure and blending quality of the whey protein isolate – hydroxypropyl methylcellulose edible films. A small amount of each sample was coated with gold and micrographs of each sample were taken at different magnifications (130x,

1000x, 1200x and 2000x) to examine each film microstructure and the homogeneity of the

WPI/HPMC blends. Three replicates per film were tested and the test repeated three times.

2.3.8 X-ray Diffraction

A Rigaku Miniflex 600 (Woodlan, TX) diffractometer with a vertical goniometer (Cu Ka radiation l1.542A was used to obtain X-ray diffraction patterns and determine the morphology of the samples. The tube was set to 40kV and 20mA. Each sample was mounted on a plain microscope slide (Fisher Scientific Inc., Fair Lawn, NJ, USA) and inserted into the equipment.

The X-ray intensities were recorded with a scintillation counter having a scattering angel (2q) at

74 a range of 5-40o and a scanning speed of 2o min-1. The crystalline areas were evaluated based on the areas and locations of the main peak.

2.3.9 Statistical Analysis

All experiments were repeated 3 times and differences in the means analyzed by a SAS statistical program. One-way analysis of variance (ANOVA) was used to evaluate significance in differences (p<0.05) between the WPI edible films with different concentrations of HPMC.

2.4 Results and Discussion

2.4.1 Film Moisture Content and Water Activity

The moisture contents and water activities of the WPI and HPMC films at different concentration ratios are shown in Table 4, Figure 24 and Figure 25, respectively. From the results obtained, the HPMC-WPI (75:25) film has the highest moisture content and the HPMC-WPI

(100:0) had the highest water activity. There are no significant differences (p>0.05) between the different moisture contents of the samples tested. There are significant differences (p<0.05) between the water activities of HPMC-WPI (100:0) and HPMC-WPI (75:25), HPMC-WPI (100:0) and HPMC-WPI (50:50), HPMC-WPI (100:0) and HPMC-WPI (25:75); and HPMC-WPI (50:50)

75 and HPMC-WPI (25:75). Results for HPMC-WPI (0:100) are not shown because the sample could not be investigated due to its brittleness.

Table 4 A Summary of Moisture Contents and Water Activity of Hydroxypropyl

Methylcellulose and Whey Protein Isolate films at different concentrations

Sample ID Moisture Content1(%) Water Activity1

HPMC-WPI (100:0) 2.606 ± 1.021 0.467 ± 0.017

HPMC-WPI (75:25) 2.878 ± 0.990 0.402 ± 0.034

HPMC-WPI (50:50) 2.440 ± 0.418 0.422 ± 0.029

HPMC-WPI (25:75) 2.427 ± 1.606 0.380 ± 0.042

HPMC-WPI (0:100) - -

1Each data point is expressed as the mean of nine measurements

a a a

* Letter “a” means that no significant differences exist.

Figure 24 The Moisture Contents of Hydroxypropyl Methylcellulose and Whey Protein Isolate

Films at varying blended ratios.

a a b b

* Letter “a” and “b” means that no significant differences exist.

Figure 25 The summary of Water Activity of Hydroxypropyl Methylcellulose and Whey Protein

Isolate Films

Moisture content is the quantity of water contained in a material and it can be measured by the loss of weight on drying. The moisture content is known to affect properties such as permanence, flexibility and tensile strength of a polymer ASTM D644-99 (2007). The results for moisture content show that there is no significant difference between the films made with different concentrations of HPMC and WPI. However, HPMC-WPI (75:25) has a higher moisture content than the rest of the other films. It is expected that HPMC will have a higher moisture content than

WPI since it is characterized by having high water affinity due to the large number of hydrophilic groups present in its structure (Fernández-Pan et al. 2012). Thus, a higher moisture content would be expected in films containing HPMC in higher quantities than in blended films with higher concentrations of WPI. The HPMC-WPI (25:75) blend contained a lower moisture content since

WPI was in higher quantities when compared with the HPMC-WPI (75:25) blended film. Whey protein isolate generally has a lower moisture content than HPMC and it is highly influenced by the plasticizer used during its formulation (Regalado et al. 2006).

Water activity is the partial vapor pressure of water in the food divided by the partial vapor pressure of pure water and it ranges between 0 and 1. Water that is bound through weak bonds or surface bonding is considered free water and can be utilized by bacteria or can be exchanged with the environment (Barbosa-Cánovas et al. 2008). The results in Figure 25 show that significant differences (p<0.05) exist between HPMC-WPI (100:0) and HPMC-WPI (75:25), HPMC-WPI

(50:50) and HPMC-WPI (25:75); and HPMC-WPI (50:50) and HPMC-WPI (25:75). These results also show that the HPMC-WPI (25:75) sample has the lowest Aw (0.380 ± 0.042) when compared with all the other samples, while HPMC-WPI (75:25) had the higher Aw (0.467 ± 0.017). The presence of the hydroxyl groups in HPMC and glycerol had an effect on the moisture contents and water activities of the samples due to their hygroscopic character (Sharma et al. 2015). Moreover, glycerol could interact with the molecules, creating more space between the chains and allowing water migration in all the samples. As a result of this, higher water activities and moisture contents were obtained when compared with previous studies.

2.4.2 Oxygen Transmission Rate

The Oxygen Transmission Rates (OTR) of the films are shown in Figure 26 and Table 5.

There were no significant differences (p>0.05) between the different oxygen transmission rates of the samples tested. From the results obtained, the HPMC-WPI (25:75) film had the highest oxygen transmission rate and HPMC-WPI (50:50) had the lowest. Results for HPMC-WPI (0:100) are not presented due to the brittleness of the sample and the amount needed for this experiment.

a a

* Letter “a” means that no significant differences exist.

Figure 26 The summary of OTR of Hydroxypropyl Methylcellulose and Whey Protein Isolate

films

Table 5 The summary of OTR calculations for Hydroxypropyl Methylcellulose and Whey

Protein Isolate Films

Sample ID OTR (ml/[m2-day]) Permeant Conc. (%) Ambient Temp. (oC)

HPMC-WPI (100:0) 79.2033 ± 8.183 100 23.0

HPMC-WPI (75:25) 49.7951 ± 15.514 100 23.0

HPMC-WPI (50:50) 25.3892 ± 0.598 100 23.0

HPMC-WPI (25:75) 84.8917 ± 106.458 100 23.0

As for the oxygen transmission rate, there were no significant differences between the films with different concentrations of HPMC and WPI. The average oxygen transmission rates of

HPMC-WPI (100:0), HPMC-WPI (75:25) and HPMC-WPI (50:50) are 79.2033, 49.7951 and

25.3892 (ml / [m2 – day]), respectively. The average oxygen transmission rates of HPMC-WPI

(25:75) is 84.8917 (ml / [m2 – day]), exhibiting a higher oxygen transmission rate when compared with the other blended films. The films with higher concentration of WPI are expected to show high barrier to oxygen because of its polar nature and the linearity of its polymeric arrangements.

Hydroxypropyl methylcellulose (Figure 9) is a cellulose derivative that is formed by the substitution of hydroxyl groups with methoxyl and hydroxypropyl groups and thus resulting in a

82 low barrier against oxygen. The results obtained followed a trend, when the concentrations of

WPI increased, the oxygen transmission rates decreased (Table 5). Krochta and Yoo (2011) found similar results when testing polar compounds dissolved in polar solvents and likewise non-polar compounds dissolved better in non-polar solvents, meaning that oxygen would dissolve better in a less polar compound such as HPMC. This could be also explained by Henry’s Law which describes the relationship between solubility and partial pressure (Selke 1997). Where:

� = � � (1.6)

Where S is the Henry’s law constant (solubility constant) for the polymer and p is the partial pressure of the permeant. In the study made by Krochta and Yoo, they blended whey protein isolate with different polysaccharides such as methylcellulose, HPMC, sodium alginate and two different starches. They found that HPMC had the highest oxygen permeability and sodium alginate presented the lowest when compared with the different blended films (Regalado et al.

2006). Ramos and colleagues (2013) also investigated the oxygen permeability of films made using different whey protein (WPI and WPC) with glycerol as a plasticizer. They found that films made from WPI had a lower oxygen transmission rate than films made from WPC alone, concluding that WPI films have a higher oxygen barrier than WPC. This means that the WPI

83 network is more strongly crosslinked via covalent and non-covalent bonds when compared with the WPC network. Ramos and colleagues also studied the oxygen permeability when different quantities of glycerol were added to WPC or WPI edible films. They found that the oxygen transmission rate was higher when the concentration of glycerol increased from 40 to 60%(w/w).

Since WPI edible films are extremely brittle at low % RH, analyzing the oxygen transmission rate using the OX-TRAN® Model 2/21 Series OTR instrument could become a challenge, creating results that may not follow previously published results. An outlier was found in the HPMC-WPI (25:75) blended film, creating a high average oxygen transmission rate.

Additional oxygen transmission studies were conducted on the HPMC-WPI (25:75) sample at a relative humidity of 30%. This was done to optimize the relative humidity for testing the sample and thus minimize cracking of the sample inside the machine. The result obtained showed that the test was successful at 30% relative humidity and the average oxygen transmission rate obtained was 8.90 (ml / [m2 – day]). This showed that further research is needed to in order to optimize the test conditions for oxygen transmission analysis of HPMC/WPI blended films.

2.4.3 Water Vapor Permeability

The water vapor permeabilities of the films are shown in Table 6 and Figure 27. From the results obtained, the HPMC-WPI (75:25) film had the highest water vapor permeability. There

84 was no significant difference (p>0.05) between the different permeabilities of the samples tested.

As a result of the brittleness of the sample data for HPMC-WPI (0:100) could not be collected.

Table 6 Summary of WVP calculations for Hydroxypropyl Methylcellulose and Whey Protein

Isolate Films

Sample ID WVP Relative DP (kPa)

(� ⋅ ��/�� ⋅ �� ⋅ �) Humidity (%)

HPMC-WPI (100:0) 3.40 × 10 ± 0.107 51.8 1.40

HPMC-WPI (75:25) 4.18 × 10 ± 0.109 51.8 1.40

HPMC-WPI (50:50) 4.11 × 10 ± 0.103 51.8 1.40

HPMC-WPI (25:75) 3.37 × 10 ± 0.060 51.8 1.40

a a

* Letter “a” means that no significant differences exist.

Figure 27 The summary of WVP of Hydroxypropyl Methylcellulose and Whey Protein Isolate

films

The results for Water Vapor Permeability show no trend and also no significant difference

(p>0.05) between the different samples tested. This in accordance with other findings and additional investigation is required to determine the effect of glycerol as a plasticizer in the results of WVP since it was not possible to reach conclusions with the results obtained. HPMC-WPI

(25:75) shows the lowest water vapor permeability when compared with the rest of the samples.

X-ray diffraction confirmed low crystallinity and high percentage of amorphous areas in WPI. The crystallinity of the material has a correlation with the barrier properties of the film. Since WPI is less crystal the permeability of the material is less creating a film with unfavorable characteristics for water vapor permeability. It is also possible that the plasticizer had an effect on moisture sorption and diffusion, affecting the results for WVP. Glycerol has a high affinity for water and that affects the diffusion of water, creating a higher WVP in edible films since it competes for the active sites on the polymer. Lower diffusion rates were found for films made with WPI by Ramos and colleagues (2013) due to the compact matrix in the films. It was also found that with a higher glycerol content, the test films showed a higher permeability due to reductions in internal hydrogen bonding (Ramos et al. 2013b).

2.4.4 Thermal Analysis by Differential Scanning Calorimetry (DSC)

The results for the thermal analyses by differential scanning calorimetry of the films are shown in Figure 28 and Figure 29.

Figure 28 Differential Scanning Calorimetry of HPMC-WPI films at different concentrations.

Figure 29 Differential Scanning Calorimetry – Second heating curve of HPMC-WPI films at

different concentrations

Different methods such as Differential Scanning Calorimetry (DSC) and Thermo-Gravimetric

Analysis are used to characterized edible films made from different polysaccharides, protein or lipids. Differential Scanning Calorimetry can be used to determine the glass transition temperature

(Tg) of polymeric samples (Su et al. 2010). The differential scanning calorimetry curves in Figure

29 display the second heating curve for the HPMC-WPI (100:0), HPMC-WPI (75:25), HPMC-

WPI (50:50), HPMC-WPI (25:75) and HPMC-WPI (0:100) edible films. For the analysis of the glass transition temperature (Tg), the second heating curve was used. The Tg of HPMC-WPI

(100:0), HPMC-WPI (75:25), HPMC-WPI (50:50), HPMC-WPI (25:75) and HPMC-WPI (0:100) edible films were found to be 135oC, 130oC, 115oC, 100oC and 95oC, respectively. These results

o o show that the addition of HPMC caused a shift in the Tg of WPI from 95 C to 130 C. This was an indication that the lower crystallinity shown by WPI was affected by the addition of a more crystallinity molecule (Greener Donhowe and Fennema 2007). This was confirmed by the results shown in Figure 33, in which the diffraction patterns obtained from the x-ray diffractometer showed that WPI had a lower crystallinity when compared with HPMC.

Yoo and Krochta (2011) found similar results when they determined the Tg of whey protein- polysaccharide blends using the midpoint of the change in the heat capacity of the blended edible films to indicate polymer miscibility. The Tg values of WPI, HPMC and HPMC-WPI blended films were 62oC, 99oC and 75oC respectively (Yoo and M Krochta 2011). The differences in the temperature could be attributed to the quantity of glycerol used during the film formulation since glycerol can attract water and reduce the stability of the samples. Dynamic Mechanical Thermal

Analysis (DMA) is another useful instrument for a more accurate measurement of the glass transmission temperature of edible films. Dynamic mechanical analysis (DMA) is used to measure the mechanical properties of polymers as a function of time and temperature (Chartoff et al. 2008).

DMA was used by Anker and colleagues to study the aging of whey protein films prepared with two different plasticizers, sorbitol and glycerol. The glass transition temperature of the films

91 plasticized with glycerol was lower when compared with the sorbitol plasticized films. Glycerol is hygroscopic and has a tendency to absorb moisture and this reduces the glass transition temperature of the polymer due to water acting as a plasticizer (Anker et al. 2001). A lower glass transition temperature indicates that films plasticized with glycerol have more pore spaces when stored at a constant temperature and relative humidity. This will result in property changes such as low barrier to gases and moisture. Further testing such as DMA is needed in order to obtain more information about thermal transitions with the HPMC-WPI blended films created in this study.

The color of the film could be used to determine the miscibility as well. A low transparency indicates some degree of immiscibility. Blended films made from whey protein isolate and other polysaccharide like methylcellulose, hydroxypropyl methylcellulose and sodium alginates tend to have higher transparency (Yoo and M Krochta 2011). Differential Scanning Calorimetry can also be used to show the melting transition of polymers. As an example, Ramos and colleagues (2013) studied how the glycerol contents affected the Tg and the temperature of melting (Tm) of WPC and

WPI films. The thermograms obtained showed two thermal transitions for WPI and WPC.

2.4.5 Thermal Analysis by Thermo-Gravimetric Analysis (TGA)

The thermo-gravimetrical analysis in Figure 30 and Figure 31 displays the mass loss curves and the derivatives for the HPMC-WPI blended films.

120 2.0 HPMCWPI (25:75)––––––– HPMCWPI (50:50)· · · · HPMCWPI (75:25)–– –– – HPMCWPI (100:0)––––– · HPMCWPI (0:100)––– ––– 100 1.5

1.0

0.5 ––––––– [ ] Weight (%) 40 ––––––– 0.0 [ ] Deriv. Weight Change (%/°C) 20

0 0.5 0 100 200 300 400 500 600 Temperature (°C) Universal V4.5A TA Instruments

Figure 30 Mass loss curve (TGA) and the derivatives for the mass loss curve (DTG) of

Hydroxypropyl Methylcellulose and Whey Protein Isolate Films at different concentrations.

2.0 HPMCWPI (0:100)––––––– HPMCWPI (25:75)ꞏ ꞏ ꞏ ꞏ HPMCWPI (50:50)– – – – HPMCWPI (75:25)––––– ꞏ HPMCWPI (100:0)–– –– –

1.5

1.0

0.5 Deriv. Weight (%/°C)

0.0

0.5 0 100 200 300 400 500 600 Temperature (°C) Universal V4.5A TA Instruments

Figure 31 Derivatives for the mass loss curve (DTG) of Hydroxypropyl Methylcellulose and

Whey Protein Isolate Films at different concentrations

Thermogravimetric analysis is done to measure the weight changes in a polymer as a function of temperature and time. This is used to determine the stability of the material under high

94 temperatures. The descending TGA thermal curves (temperature vs weight) in Figure 30 indicate weight losses of the sample as temperatures increases from 0oC to 600oC. As components within the blends vaporize, the weight losses are recorded by the equipment. The first transition relates to the loss of water from the films and can be found between 50 to 150 oC. The second transition relates to the decomposition of the protein and the loss of the plasticizer which in this case is glycerol. This second transition can be found around 150 to 300 oC. The third transition shows the oxidation of partially decomposed proteins and can be found around 450 to 550 oC (Kadam et al. 2013). It can be seen from Figure 30 that the films experienced these thermal degradations.

The derivatives for the mass loss curve (DTG) for the HPMC and WPI films at different concentrations are presented to enhance the ease of quantifying the thermal degradations. The derivatives represent the rates of change of the weight losses with respect to the corresponding temperatures. The results in Figure 30 show that second degradation peaks between 300-400oC tended to shift to a lower temperature as the WPI concentration increased. This could be due the higher crystallinity and heat stability of HPMC that was confirmed previously during the DSC and

XRD analyses.

Ramos and colleagues found similar results when they studied the effect of protein purity and glycerol upon the physical properties of edible films. Whey protein isolate and WPC showed decomposition around 180ºC and 230ºC and a sharp weight loss around 280ºC and 500ºC,

95 respectively. This sharp weight loss was associated with the degradation of the major protein in the film (Ramos et al. 2013). According to Su and colleagues, a single decomposition temperature shows good compatibility between the protein and the plasticizer. Similar results were found when they studied the interaction between carboxymethyl cellulose and soy protein isolate blends to create edible films. A lower decomposition temperature was found for the protein being used when compared with the decomposition of the cellulose. The cellulose derivative decomposed in

- a two-stage process, in which moisture was lost first and CO2 and COO groups were lost next. As they increased the carboxymethyl cellulose contents, a shift to a lower temperature occurred, indicating that cellulose had a greater heat stability than the pure protein (Su et al. 2010). These results are similar to the results obtained in the study and shown in Figure 30.

2.4.6 Scanning Electron Microscopy

The scanning electron micrographs in Figure 32 display the microstructure of the HPMC-

WPI films.

a. b.

c. d.

Figure 32 SEM surface morphologies of (a-e) HPMC-WPI (100:0), HPMC-WPI (75:25),

HPMC-WPI (50:50), HPMC-WPI (25:75), HPMC-WPI (0:100).

Electron Microscopy is used to study the surface and the homogeneity of materials such as edible films. The scanning electron micrographs of the HPMC-WPI (100:0), HPMC-WPI (75:25),

HPMC-WPI (50:50), HPMC-WPI (25:75) and HPMC-WPI (0:100) films revealed homogeneously blended materials. These results show excellent miscibility and no phase separation of the HPMC from the WPI. The miscibility of the sample can affect properties such as oxygen transmission rate, water vapor permeability and the glass transition temperature. Another way to study the homogeneity of the samples is using Differential Scanning Calorimetry in which the glass transition temperature (Tg) can be determined and compared. A single Tg indicates compatibility between polymers. The lack of compatibility will be shown with two Tg indicating the immiscibility of the polymers (Ghanbarzadeh et al. 2010). The process of creating a composite edible film include steps such as the heating of WPI to break internal disulfide bonds and allow the formation of inter-molecular interaction, the blend of WPI and HPMC using a homogenizer and the drying of the film. Blending the film using a homogenizer is a crucial step in creating a homogeneous film with no phase separation. Studies such as Transmission Electron Microscopy

(TEM) would be required to understand the blending properties between the protein and the cellulose derivative (Reimer 2013).

2.4.7 X-ray Diffraction

The X-ray diffraction patterns of the HPMC-WPI films are shown in Figure 33. Each pattern shows the main peak positions which represents the crystalline or amorphous regions of the material.

Figure 33 X-ray Diffraction of Hydroxypropyl Methylcellulose and Whey Protein Isolate Films

at different concentrations.

In the X-ray diffraction patterns, peaks that are not well defined represent the low crystallinity regions of the material. On the contrary, well-defined peaks are indicative of the high crystallinity regions. The crystallinity of the material has a correlation with the barrier properties of the film.

The diffraction pattern of the edible films in this study is amorphous-crystalline. The addition of a plasticizer, such as glycerol to the blended films, would be expected to influence a decrease in the crystallinity because it would decrease the interaction between the molecules, increasing the spaces between the chains. Donhowe and Fennema (2007) studied the effect of different plasticizers on the crystallinity of methylcellulose films. They found a significant change in the position of the peak but not in its intensity. It was concluded that the plasticizers interacted with the amorphous region of the cellulose, increasing the mobility of the methylcellulose segments and affecting the crystallinity of the films (Greener Donhowe and Fennema 2007). The diffraction patterns for HPMC showed a well-defined peak when compared with the film made from WPI alone, indicating a higher crystallinity and lower percentage of amorphous regions in the HPMC polymer. The shaper peaks in the diffraction patterns decreased in intensity with the addition of

WPI, suggesting that the crystalline structure of HPMC was reduced by the addition of the WPI.

An intermediate result was found in HPMC-WPI (50:50) suggesting that the sample was blended homogeneously. Whey protein isolate is a globular protein that, in some cases, can be crystallized if it is separated from the mixtures in which it is found, but in its natural state, it does not have

100 high crystallinity (Mateus 2011). On the other hand, HPMC is a derivative from cellulose that has a crystalline structure. During the substitution process of cellulose to create HPMC, the addition of a base and water disrupts the crystalline structure, but it still keeps a higher crystallinity when compare with WPI (Huber and Embuscado 2009b). In addition to X-ray diffraction, Fourier- transform infrared spectroscopy (FTIR) can be useful to understanding and explanation of the blending properties of WPI and HPMC.

2.5 Conclusions

In this study, different concentrations of HPMC and WPI were used for the creation of an edible film, and their effects on the film’s moisture content, water activity, microscopy, oxygen transmission rate, water vapor permeability, glass transition temperature and X-ray diffraction patters were investigated. Table 7 shows a summary of the results. It is concluded that HPMC-

WPI (75:25) had the highest moisture content and water vapor permeability. HPMC-WPI (100:0) had the highest water activity and HPMC-WPI (25:75) had the highest oxygen transmission rate.

The addition of HPMC caused a shift in the Tg of WPI, indicating that the lower crystallinity shown by WPI was affected by the addition of a more crystalline polymer. The degradation peaks in the thermal analysis by TGA tended to shift to a lower temperature as the WPI concentration increased indicating that the higher crystallinity and heat stability of HPMC had an effect on the thermal

101 properties of WPI. Homogeneous films were confirmed by the scanning electron microscopy images. The diffraction patterns for HPMC obtained from the X-ray diffraction analysis showed a well-defined peak when compared with the film made from WPI alone, indicating a higher crystallinity and lower percentage of amorphous regions in the HPMC polymer. As a conclusion,

HPMC-WPI edible films have great potential to be used as a package, but some alterations are still required for their optimization depending on the use that will be given to the film.

102

Table 7 HPMC-WPI Edible Films Summary of Results Experiment Results HPMC-WPI HPMC-WPI HPMC-WPI HPMC-WPI HPMC-WPI (100:0) (75:25) (50:50) (25:75) (0:100) Moisture Highest MC Lowest MC Content Water Highest Aw Lowest Aw Activity Oxygen Lowest OTR Highest OTR Transmissio n Rate Water Vapor Highest WVP Lowest WVP Permeability o o o o o Differential 135 C Tg 130 C Tg 115 C Tg 100 C Tg 95 C Tg Scanning Calorimetry Thermo- High Intermediate Lowest Gravimetric Temperature Temperature Temperature Analysis of of of Degradation Degradation Degradation Scanning Homogeneou Homogeneou Homogeneou Homogeneou Homogeneou Electron s and good s and good s and good s and good s and good Microscopy miscibility miscibility miscibility miscibility miscibility X-ray Highest Intermediate Lowest Diffraction crystallinity Crystallinity Crystallinity

103

References

Abbas, K., Khalil S.K., Hussin, ASM (2010) Modified Starches and Their Usages in Selected Food Products: A Review Study. Journal of Agricultural Science 2:p90. doi: 10.5539/jas.v2n2p90

Abdou E., Sorour M. (2014) Preparation and characterization of starch/carrageenan edible films. International Food Research Journal 21:189–193

Allafi AR (2008) Effect of Different Percent Loadings of Nanoparticles and Food Processing Conditions on the Properties of Nylon 6 Films. The Ohio State University.

Anker M., Stading M., Hermansson A., (2001) Aging of Whey Protein Films and the Effect on Mechanical and Barrier Properties. Journal of Agricultural and Food Chemistry 49:989– 995. doi: 10.1021/jf000730q

Baldwin E.A., Hagenmaier R., Bai J. (2011) Edible Coatings and Films to Improve Food Quality, Second Edition, 2 edition. CRC Press, Boca Raton, FL p.29-34

Barbosa-Cánovas G.V., Fontana J., Schmidt S.J., Labuza T.P. (2008) Water Activity in Foods: Fundamentals and Applications. John Wiley & Sons p.1-11

Bietz J., Wall J. (1980) Identity of high molecular weight gliadin and ethanol-soluble glutenin subunits of wheat: relation to gluten structure. Cereal Chemistry p.415-421

Brandenburg A.H., Weller C.L., Testin R.F. (1993) Edible Films and Coatings from Soy Protein. Journal of Food Science 58:1086–1089. doi: 10.1111/j.1365-2621.1993.tb06120.x

Buléon A., Colonna P., Planchot V., Ball S. (1998) Starch granules: structure and biosynthesis. International Journal of Biological Macromolecules 23:85–112. doi: 10.1016/S0141- 8130(98)00040-3

Carter M., Shieh J. (2015) Chapter 5 - Microscopy. Guide to Research Techniques in Neuroscience (Second Edition). Academic Press, San Diego, pp 117–144

104

Chartoff R.P., Menczel J.D., Dillman S.H. (2008) Dynamic Mechanical Analysis (DMA). In: Thermal Analysis of Polymers. Wiley-Blackwell, pp 387–495

Chick J., Hernandez R.J. (2006) Physical, Thermal, and Barrier Characterization of Casein‐Wax‐ Based Edible Films. Journal Food Science 67:1073–1079. doi: 10.1111/j.1365- 2621.2002.tb09455.x

Chiellini E. (2008) Environmentally compatible food packaging. CRC ; Woodhead, Boca Raton; Cambridge p.5-16

Chiralt A., González-Martínez C., Vargas M., Atarés L. (2018) 18 - Edible films and coatings from proteins. In: Yada RY (ed) Proteins in Food Processing (Second Edition). Woodhead Publishing, pp 477–500

Cho S., Park J.W., Batt H., Thomas R.L. (2007) Edible films made from membrane processed soy protein concentrates. LWT - Food Science Technology 40:418–423. doi: 10.1016/j.lwt.2006.02.003

Conn E.E., Stumpf P.K. (1976) Outlines of Biochemistry, 4th edition. John Wiley & Sons Inc, New York p.87-92

Crank J. (1980) The Mathematics of Diffusion. Clarendon Press, U.S.A, p.38-42

D Phan T., Debeaufort F., Luu D., Voilley A. (2005) Functional Properties of Edible Agar-Based and Starch-Based Films for Food Quality Preservation. Journal of Agricultural and Food Chemistry 53:973–981. doi: 10.1021/jf040309s

Debeaufort F., Quezada-Gallo J.A., Voilley A. (1998) Edible films and coatings: tomorrow’s packagings: a review. Critical Review Food Science Nutrition 38:299–313. doi: 10.1080/10408699891274219

Dinand E., Vignon M., Chanzy H., Heux L. (2002) Mercerization of primary wall cellulose and its implication for the conversion of cellulose I→cellulose II. Cellulose 9:7–18. doi: 10.1023/A:1015877021688

Emblem A., Emblem H. (2012) Packaging technology: fundamentals, materials and processes. Woodhead Pub., Cambridge; Philadelphia p.65-68

105

Ertesvåg H., Valla S. (1998) Biosynthesis and applications of alginates. Polymer Degradation and Stability 59:85–91. doi: 10.1016/S0141-3910(97)00179-1

Espitia P., Du W., Avena-Bustillos R., et al (2014) Edible films from pectin: Physical- mechanical and antimicrobial properties - A review. Food Hydrocolloids 35:287–296. doi: 10.1016/j.foodhyd.2013.06.005

Fernández-Pan I., Royo M., Ignacio Maté J. (2012) Antimicrobial activity of whey protein isolate edible films with essential oils against food spoilers and foodborne pathogens. Journal of Food Science 77:M383-390. doi: 10.1111/j.1750-3841.2012.02752.x

Ferreira A.R.V., Alves V.D., Coelhoso I.M. (2016) Polysaccharide-Based Membranes in Food Packaging Applications. Membranes 6:22-25. doi: 10.3390/membranes6020022

Fox P.F., Uniacke-Lowe T., McSweeney P.L.H., O’Mahony J.A. (2015) Dairy Chemistry and Biochemistry, 2nd edn. Springer International Publishing p.3-15

Francis M.F., Piredda M., Winnik F.M. (2006) Hydroxypropylcellulose in Oral Drug Delivery. In: Marchessault RH, Ravenelle F, Zhu XX (eds) Polysaccharides for Drug Delivery and Pharmaceutical Applications. American Chemical Society, Washington, DC, pp 57–75

Galdeano M., Wilhelm A., Mali S., Grossmann M. (2013) Influence of Thickness on Properties of Plasticized Oat Starch Films. Brazilian Archives of Biology and Technology 56:637– 644. doi: 10.1590/S1516-89132013000400014

Galus Kokoszka S., Lenart A. (2013) Development and characterization of composite edible films based on sodium alginate and pectin. Journal of Food Engineering 115:459–465. doi: 10.1016/j.jfoodeng.2012.03.006

Gennadios A. (2002) Protein-Based Films and Coatings. CRC Press p.2-25

Gennadios A., Brandenburg A.H., Weller C.L., Testin R.F. (1993) Effect of pH on properties of wheat gluten and soy protein isolate films. Journal of Agricultural and Food Chemistry 41:1835–1839. doi: 10.1021/jf00035a006

106

Ghanbarzadeh B., Almasi H., Entezami A. (2010) Physical properties of edible modified starch/carboxymethyl cellulose films. Innovative Food Science Emerging Technologies 11:697–702. doi: 10.1016/j.ifset.2010.06.001

Ghosh A (2015) Technology of Polymer Packaging, First edition. Hanser, Munich Cincinnati. p.83-114

Greener Donhowe I., Fennema O.R. (2007) The effects of plasticizers on crystallinity, permeability, and mechanical properties of methylcellulose films. Journal of Food Processing and Preservatives 17:247–257. doi: 10.1111/j.1745-4549.1993.tb00729.x

Han J.H. (2005) Innovations in food packaging. Elsevier Academic ; Elsevier Science, distributor, San Diego, Calif.; Oxford p.54-67

Hao Y., Liu H., Chen H., et al (2019) What affect consumers’ willingness to pay for green packaging? Evidence from China. Resources, Conservation and Recycling 141:21–29. doi: 10.1016/j.resconrec.2018.10.001

Hassan B., Chatha S.A.S., Hussain A.I., et al (2018) Recent advances on polysaccharides, lipids and protein based edible films and coatings: A review. International Journal of Biological Macromolecules 109:1095–1107. doi: 10.1016/j.ijbiomac.2017.11.097

Hettiarachchy NS, Ziegler GR (1994) Protein Functionality in Food Systems, 1 edition. CRC Press, New York p.39-79

Huber KC, Embuscado ME (eds) (2009a) Edible Films and Coatings for Food Applications. Springer New York, New York, NY p.160-210

Kadam D., Thunga M., Wang S., et al (2013) Preparation and characterization of whey protein isolate films reinforced with porous silica coated titania nanoparticles. Journal of Food Engineering 117:133–140. doi: 10.1016/j.jfoodeng.2013.01.046

Le T., Letendre M., Ispas-Szabo P., et al (2000) Development of Biodegradable Films from Whey Proteins by Cross-Linking and Entrapment in Cellulose. Journal of Agricultural and Food Chemistry 48:5566–75. doi: 10.1021/jf0002241

107

Li Y., Jiang Y., Liu F., et al (2011) Fabrication and characterization of TiO 2/whey protein isolate nanocomposite film. Food Hydrocolloid 25:1098–1104. doi: 10.1016/j.foodhyd.2010.10.006

Marsh K., Bugusu B. (2007) Food Packaging: Roles, Materials, and Environmental Issues. Journal of Food Science 72:R39–R55. doi: 10.1111/j.1750-3841.2007.00301.x

Mateus M. (2011) Controlled and tailored denaturation and aggregation of whey proteins.

Menczel J.D., Judovits L., Prime R.B., et al (2008) Differential Scanning Calorimetry (DSC). In: Thermal Analysis of Polymers. Wiley-Blackwell, pp 7–239

Muhammad Shamsuddin I. (2017) Bioplastics as Better Alternative to Petroplastics and Their Role in National Sustainability: A Review. ABB Advances in Bioscience and Bioengineering 5:63

Pallas L., Krochta J.M. (2008) Physical Properties of Whey Protein- Hydroxypropylmethylcellulose Blend Edible Films. Journal of Food Science 73:E446-54. doi: 10.1111/j.1750-3841.2008.00941.x

Park H., Chinnan M. (1995) Gas and water barrier properties of edible films from protein and cellulosic materials. Journal of Food Engineering 25:497–507. doi: 10.1016/0260- 8774(94)00029-9

Pethrick R.A., Amornsaichai T., North A.M. (2011) Introduction to Molecular Motion in Polymers, 1 edition. Whittles Publishing, Dunbeath p.5-17

Pratiwi M., Faridah D.N., Lioe H.N. (2018) Structural changes to starch after acid hydrolysis, debranching, autoclaving-cooling cycles, and heat moisture treatment (HMT): A review: Structural changes to modified starch. Starch - Stärke 70:1700028. doi: 10.1002/star.201700028

Ramos O., Reinas I., Baptista da Silva S., et al (2013) Effect of whey protein purity and glycerol content upon physical properties of edible films manufactured therefrom. Food Hydrocolloids 30:110–122. doi: 10.1016/j.foodhyd.2012.05.001

108

Regalado C., Perez-Perez C., Lara-Cortes E., Garcia-Almedarez B. (2006) Whey protein based edible food packaging films and coating p.237-261

Reimer L. (2013) Transmission Electron Microscopy: Physics of Image Formation and Microanalysis. Springer p.1-14

Robertson G.L. (1998) Food Packaging: Principles and Practice. CRC Press p.3-24

Robertson G.L. (2012) Food Packaging: Principles and Practice, Third Edition, 3 edition. CRC Press, Boca Raton, FL p.1-31

Roncarelli S., Ellicott C. (2010) Packaging essentials: 100 design principles for creating packages. Rockport Publishers, Beverly, Mass. p.22-34

Rubilar J., Zúñiga R., Osorio F., Pedreschi F. (2015) Physical properties of emulsion-based hydroxypropyl methylcellulose/whey protein isolate (HPMC/WPI) edible films. Carbohydrate Polymers 123:. doi: 10.1016/j.carbpol.2015.01.010

Rudin A., Choi F. (2012) The Elements of Polymer Science and Engineering, 3 edition. Academic Press, San Diego p.1-27

Sancho-Madriz M.F. (2003) Preservation of Food. In: Caballero B (ed) Encyclopedia of Food Sciences and Nutrition (Second Edition). Academic Press, Oxford, pp 4766–4772

Selke S. (1997) Understanding Plastics Packaging Technology, First edition. Hanser, Munich; Cincinnati p.2-30

Selke S., Culter J.D. (2016) Plastics packaging: properties, processing, applications, and regulations p.-56-87

Sessa D.J., Willett J.L. (1998) Paradigm for Successful Utilization of Renewable Resources. The American Oil Chemists Society p.78-88

Sharma P., Modi S.R., Bansal A.K. (2015) Co-processing of hydroxypropyl methylcellulose (HPMC) for improved aqueous dispersibility. International Journal of Pharmaceutics 485:348–356. doi: 10.1016/j.ijpharm.2015.03.036

109

Shirley B.A. (1995) Protein Stability and Folding: Theory and Practice. Humana Press p.35-65

Singh P., Wani A.A., Langowski H.C. (2017) Food Packing Materials: Testing and Quality Assurance. Chapman and Hall/CRC, Bosa Roca, United States p.251-303

Song J.H., Murphy R.J., Narayan R., Davies G.B.H. (2009) Biodegradable and compostable alternatives to conventional plastics. Philosophical Transaction of the Royal Society B: Biological Sciences 364:2127–2139. doi: 10.1098/rstb.2008.0289

Spink C.H. (2008) Differential Scanning Calorimetry. In: Methods in Cell Biology. Academic Press, pp 115–141

Stephen A.M., Phillips G.O. (2016) Food Polysaccharides and Their Applications. CRC Press p.589-629

Su J.F., Huang Z., Yuan X.Y., et al (2010) Structure and properties of carboxymethyl cellulose/soy protein isolate blend edible films crosslinked by Maillard reactions. Carbohydrate Polymers 145–153. doi: 10.1016/j.carbpol.2009.07.035

Taggart P. (2004) 12 - Starch as an ingredient: manufacture and applications. In: Eliasson A-C (ed) Starch in Food. Woodhead Publishing, pp 363–392

Tanada-Palmu P., Helén H., Hyvönen L. (2000) Preparation, properties and applications of wheat gluten edible films. Agricultural and Food Science in Finland 9:1

Thomas S., Alavi S., Sandeep K.P., et al (eds) (2014) Polymers for Packaging Applications, 1 edition. Apple Academic Press, Toronto p.3-39

Van de Velde F. (2008) Structure and function of hybrid carrageenans. Food Hydrocolloids 22:727–734. doi: 10.1016/j.foodhyd.2007.05.013

Villalobos R., Hernández-Muñoz P., Chiralt A. (2006) Effect of surfactants on water sorption and barrier properties of hydroxypropyl methylcellulose films. Food Hydrocolloids 20:502–509. doi: 10.1016/j.foodhyd.2005.04.006

110

Voragen A., Schols H.A., Pilnik W. (1986) Determination of the Degree of Methylation and Acetylation of Pectins by HPLC. Food Hydrocolloid 1:65–70. doi: 10.1016/S0268- 005X(86)80008-X

Wan Z. (2016) The Antibacterial Activity of Selected Small Molecule Compounds and Their Effect On The Morphology And Mechanical Properties of Tapioca Starch Films. The Ohio State University

Wan Z., Rajashekara G., Fuchs J., et al (2018) Incorporation of Selected Antimicrobial Small Molecule Compounds into Tapioca Starch and the Effects on Thickness, Moisture, and Oxygen Mass Transfer, and Mechanical Properties of the Films. Starch - Stärke 70:1700060. doi: 10.1002/star.201700060

Wang S., Copeland L. (2012) Effect of alkali treatment on structure and function of pea starch granules. Food Chemistry 135:1635–1642

Whistler R. (2012) Industrial Gums: Polysaccharides and Their Derivatives. Elsevier p.49-83

Wolff I.A., Davis H.A., Cluskey J.E., et al (1951) Preparation of Films from Amylose. Ind Eng Chem Industrial & Engineering Chemistry 43:915–919

Yoo S., M Krochta J. (2011) Whey protein-polysaccharide blended edible film formation and barrier, tensile, thermal and transparency properties. Journal of Science Food and Agriculture 91:2628–36. doi: 10.1002/jsfa.4502

Y.X. X, K.M. K, M.A. H, D. N (2005) Chitosan–starch composite film: preparation and characterization. Industrial Crops Products 21:185–192. doi: 10.1016/j.indcrop.2004.03.002

Zinoviadou K.G., Koutsoumanis K.P., Biliaderis C.G. (2009) Physico-chemical properties of whey protein isolate films containing oregano oil and their antimicrobial action against spoilage flora of fresh beef. Meat Science 82:338–345. doi: 10.1016/j.meatsci.2009.02.004

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Appendix A

Figure A.1 Water Activity Statistical Analysis analyzed by a SAS statistical program.

112

Table A.1 Water Activity Multiple Comparison

113

Figure A.2 Moisture Content Analyzed by a SAS Statistical Program.

114

Table A.2 Moisture Content Multiple Comparison

115

Figure A.3 Water Vapor Permeability Test Analyzed by a SAS Statistical Program.

116

Table A.3 Water Vapor Permeability Test Multiple Comparison

117

Figure A.4 Oxygen Transmission Rate Analyzed by a SAS Statistical Program.

118

Table A.4 Oxygen Transmission Rate Multiple Comparison

119

Table A.5 Moisture Content Raw Data for HPMC-WPI Edible Films

Sample Results

Replicate 1 Replicate 2 Replicate 3

HPMC-WPI Sample 1 2.756 3.695 1.203

(100:0) Sample 2 2.352 3.687 1.302

Sample 3 3.015 3.729 1.715

HPMC-WPI Sample 1 2.027 2.328 3.941

(75:25) Sample 2 1.933 2.132 4.249

Sample 3 2.591 2.391 4.306

HPMC-WPI Sample 1 1.747 2.545 2.263

(50:50) Sample 2 1.882 2.475 2.505

Sample 3 2.654 2.926 2.967

HPMC-WPI Sample 1 1.431 1.948 5.485

(25:75) Sample 2 1.456 1.761 0.412

Sample 3 2.422 2.354 4.576

HPMC-WPI Sample 1

(0:100) Sample 2

Sample 3

120

Table A.6 Water Activity Raw Data for HPMC-WPI Edible Films

Sample Results

Replicate 1 Replicate 2 Replicate 3

HPMC-WPI Sample 1 0.488 0.455 0.459

(100:0) Sample 2 0.49 0.454 0.457

Sample 3 0.49 0.453 0.457

HPMC-WPI Sample 1 0.405 0.373 0.445

(75:25) Sample 2 0.398 0.362 0.441

Sample 3 0.396 0.360 0.440

HPMC-WPI Sample 1 0.388 0.450 0.446

(50:50) Sample 2 0.380 0.441 0.439

Sample 3 0.383 0.439 0.436

HPMC-WPI Sample 1 0.372 0.346 0.438

(25:75) Sample 2 0.364 0.340 0.433

Sample 3 0.367 0.335 0.432

HPMC-WPI Sample 1

(0:100) Sample 2

Sample 3

121

Table A.7 Water Vapor Permeability Raw Data for HPMC-WPI Edible Films

Sample Results

Replicate 1 Replicate 2 Replicate 3

HPMC-WPI Sample 1 0.325845714 0.20291425 0.289542991

(100:0) Sample 2 0.375783929 0.266717009 0.39497567

Sample 3 0.442413571 0.233549598 0.534533929

HPMC-WPI Sample 1 0.416174464 0.451246875 0.583712411

(75:25) Sample 2 0.306274107 0.505437321 0.260253616

Sample 3 0.306274107 0.419814375 0.302792946

HPMC-WPI Sample 1 0.274025179 0.299379821 0.48608683

(50:50) Sample 2 0.377484821 0.392566071 0.385110491

Sample 3 0.547942634 0.363565848 0.569543973

HPMC-WPI Sample 1 0.402771429 0.230556027 0.392384643

(25:75) Sample 2 0.333624464 0.320584286 0.374621652

Sample 3 0.301126071

HPMC-WPI Sample 1

(0:100) Sample 2

Sample 3

122

Table A.8 Oxygen Transmission Rate Raw Data for HPMC-WPI Edible Films

Sample Results

Replicate 1

HPMC-WPI Sample 1 74.57051

(100:0) Sample 2 80.1928

Sample 3 86.84681

HPMC-WPI Sample 1 57.45305

(75:25) Sample 2 59.99213

Sample 3 31.94017

HPMC-WPI Sample 1 25.81182

(50:50) Sample 2 24.9667

Sample 3

HPMC-WPI Sample 1 160.1689

(25:75) Sample 2 9.6124608

Sample 3

HPMC-WPI Sample 1

(0:100) Sample 2

Sample 3

123