STRATEGIES TO IMPROVE THE AGING, BARRIER AND MECHANICAL PROPERTIES OF CHITOSAN, WHEY AND WHEAT GLUTEN PROTEIN FILMS

Idoia Olabarrieta

AKADEMISK AVHANDLING Som med tillstånd av Kungliga Tekniska Högskolan framlägges till offentlig granskning för avläggande av teknisk doktorsexamen fredagen den 27 maj 2005, kl 10.00 I sal K2, Teknikringen 28, KTH, Stockholm. Avhandlingen försvaras på engelska.

STRATEGIES TO IMPROVE THE AGING, BARRIER AND MECHANICAL PROPERTIES OF CHITOSAN, WHEY AND WHEAT GLUTEN PROTEIN FILMS

ABSTRACT
Chitosan, Whey Protein Isolate (WPI) and vital wheat gluten (WG) are three biomaterials that have quite promising properties for packaging purposes. They have good film forming properties and good gas barrier properties in dry conditions. Moreover, because they are produced from industrial waste of food processing, they offer an ecological advantage over polymers made from petroleum. However, their physicochemical characteristics still must be improved for them to be of commercial interest for the food packaging industry. The purpose of this work was to study different strategies aiming to improve the water resistance and aging properties of these polymers, which are some of the key disadvantages of these materials. The produced solution cast chitosan and WPI films were characterised with scanning electron microscopy (SEM), density measurements and thermogravimetry. The water vapour transmission rate was determined at a relative humidity of 11%. In the first part, mechanical properties of solid films and seals were assessed by tensile testing. WG film’s tensile properties and oxygen and water vapour permeabilities were measured as a function of aging time. The changes in the protein structure were determined by infrared spectroscopy and size-exclusion high-performance liquid chromatography and the film structure was revealed by optical and scanning electron microscopy. Gluten-clay nanocomposites were characterised by tensile testing, X-ray diffraction and transmission electron microscopy. The incorporation of a hydrophobic biodegradable polymer, poly (ε-caprolactone), PCL, in both chitosan and whey protein, yielded a significant decrease in water vapour transmission rate. It was observed that a certain amount of the PCL particles were ellipsoidal in chitosan and fibrous in WPI. The obtained data also indicated that the particle shape had an important influence in the water vapour transmission rate. In the second part, the aging properties of WG films, plasticized with glycerol and cast from water/ethanol solutions with pH=4 or pH=11 were investigated. WG films made from alkaline solutions were mechanically more time-stable than the acidic ones, the latter being initially very ductile but turning brittle towards the end of the aging period. The protein solubility measurements indicated that the protein structure of the acidic films was initially significantly less aggregated than the in basic films. During aging the acidic films lost more mass than the basic films through slow evaporation of volatiles (water/ethanol) and through migration of glycerol to the paper support. The oxygen permeability was also lower for the basic films. In the last part, the properties of new and aged glycerol-plasticized WG films at acidic and basic conditions containing ≤4.5 wt% natural or quaternary-ammonium-salt-modified montmorillonite were studied. Films of WG with montmorillonite were possible to produce by solution casting. The aging rate of acidic and basic films was unaffected by the incorporation of clay. However, the large reduction in water vapour permeability for most systems suggested that the clay sheets were evenly distributed within the films. The film prepared from basic solution and containing natural clay was almost completely exfoliated as revealed by transmission electron microscopy and X-ray diffraction. The best water vapour barrier properties were obtained by using modified clay.
Keywords: biodegradable polymers, chitosan, whey protein, wheat gluten, poly(ε-caprolactone), montmorillonite, food packaging, permeability, mechanical properties, aging, pH, solubility, migration, solution casting.

STRATEGIES TO IMPROVE THE AGING, BARRIER AND MECHANICAL PROPERTIES OF CHITOSAN, WHEY AND WHEAT GLUTEN PROTEIN FILMS

SAMMANFATTNING
Kitosan, WPI (Whey Protein Isolate / vassleproteinisolat) och vitalt vetegluten (vital wheat gluten, WG) är tre biomaterial med ganska lovande förpackningsegenskaper. De har goda egenskaper för filmformning och de är utmärkta gasbarriärer i torrt tillstånd. Dessutom tillverkas de av biprodukter från livsmedelsindustrin, vilket ger en ekologisk fördel jämfört med petroliumbaserade plaster. Deras fysikokemiska karakteristika måste emellertid förbättras innan de blir kommersiellt intressanta som livsmedelsförpackningar. Syftet med det här arbetet var att studera olika strategier för att förbättra egenskaperna avseende resistans mot vattenånga och åldring, vilka är de huvudsakliga svagheterna hos de här polymererna. De tillverkade filmerna av kitosan och WPI karakteriserades med hjälp av svepelektronmikroskop (SEM), densitetsmätningar och termogravimetri. Transmissionsgraden för vattenånga bestämdes vid en relativ fukthalt av 11%. I den första delen fastställdes mekaniska egenskaper för filmer och förseglingar med dragprov. Veteglutenfilmernas mekaniska egenskaper och syre- respektive vattenångpermeabilitet uppmättes som en funktion av åldringstiden. Förändringarna i proteinstrukturen fastställdes med infraröd spektroskopi och vätskekromatografi och filmstrukturen studerades med optisk- och svepelektronmikroskopi. Gluten-lera nanokompositer karakteriserades med dragprovning, röntgendiffraktionsmätning och transmissionselektronmikroskopi. Tillsatsen av en hydrofob bionedbrytbar polymer, poly (ε-kaprolakton), PCL, i både kitosan och vassle, medförde en märkbar minskning av transmissionsgraden för vattenånga. Experimenten klargjorde att en viss mängd av PCL-partiklarna var ellipsoidformade i kitosan och hade en mer avlång struktur i vassle. Erhållna data visade också att partikelformen hade en betydande påverkan på transmissionsgraden för vattenånga. I den andra delen undersöktes åldringsegenskaperna för veteglutenfilmer, med glycerol som mjukgörare, gjutna från vatten/etanollösningar med pH=4 eller pH=11. Veteglutenfilmer från alkaliska lösningar var mekaniskt mer tidsstabila än de från sura, de senare var initialt mycket elastiska men blev mot slutet av åldringsperioden spröda. Mätningar av proteinlösligheten indikerade att proteinstrukturen för de sura filmerna initialt var signifikant mindre aggregerade än de basiska filmerna. Under åldring förlorade de sura filmerna mer massa än de basiska filmerna genom långsam evaporering av flyktiga ämnen (vatten/etanol) och genom migration av glycerol till pappersunderlaget. Syrepermeabiliteten var också lägre för de basiska filmerna. I den sista delen studerades egenskaperna hos nya och åldrade glycerol-mjukgjorda veteglutenfilmer, vid basiska och sura förhållanden, innehållande ≤4.5 vikt% naturliga eller kvartenär ammoniiumsalt-modifierad montmorillonit. Det var möjligt att tillverka veteglutenfilmer med montmorillonit genom gjutning. Åldringshastigheten för sura och basiska filmer var opåverkad av inblandingen av lera. Den stora reduktionen, med avseende på vattenångpermeabilitet för de flesta systemen påvisade emellertid att ”lerflaken” var jämt fördelade i filmerna. Filmen som preparerades från basisk lösning och innehöll naturlig lera var nästan fullständigt exfolierad såsom visats med transmissionselektronmikroskopi och röntgendiffraktionsstudier. De bästa vattenångbarriäregenskaperna erhölls med modifierad lera.
Nyckelord: bionedbrytbara polymerer, kitosan, vassleprotein, vetegluten, poly(ε-kaprolakton), montmorillonit, livsmedelsförpackningar, permeabilitet, mekaniska egenskaper, åldring, pH, löslighet, migration, filmgjutning

I. W. IV. Hedenqvist. I. J. M. Olabarrieta. I. J. Sarasua.LIST OF PUBLICATIONS This thesis is a summary of the following publications: I. U.42. ‘Properties of new and aged montmorillonite-wheat gluten composite films’. Forsström. S. Hedenqvist. A. Olabarrieta. Jansson. 4401-4408 (2001). S. Gedde. M. Gedde. ‘Transport properties of chitosan and whey blended with poly(ε-caprolactone) assessed by standard permeability measurements and microcalorimetry’. caprolactone). 51: 275-289. Gällstedt. M. R. Submitted to Biomacromolecules. Gällstedt. Vol. Olabarrieta. Sarasua. I. Olabarrieta. E. Hedenqvist.R. (2002). To be submitted to Journal of Agricultural and Food Chemistry. M. ‘Aging properties of films of plasticized vital wheat gluten cast from acidic and basic solutions’. S. Polymer. U. Hedenqvist. Johansson and M. M. D. . ‘Mechanical and physical properties of chitosan and whey blended with poly(ε- III. II. W. Ispizua. International Journal of Polymeric Materials. I. S.

................................ 2 1.................. 7 1......3.............................. 17 1.....................................................................................2............... 16 1....2 Water vapour permeability..................................3...............................1 Permeability .1 1... 12 1................................................................ 19 1........................2 Chitosan films ............ 23 2......................................................................................... 16 1.......................................................................... 29 I ....................1 Water sensitivity....................4 Content of volatile mass......................... 25 2.............................................................................................................................. 5 1.......................................2 Aging of biodegradable polymers .......................................................................................................2.................... 9 1...................4 PCL/WPI films.1 MATERIALS ......................... 15 1................................ 28 2..8... 19 1................................................................3........................3 PCL/Chitosan films ........................3...................................................................................................... 10 1.......1 Poly(ε-caprolactone) composites ............................... 23 2..... 26 2..........................................2...............................................3 Tensile test ..................1 Biodegradable polymers.................................... 3 1..........6 CHITOSAN.4 BARRIER PROPERTIES ................................ 27 2..................................................................2 Biopolymer films ......2 Nanoclay nanocomposites..3........ 24 2............................ 6 1.................................................................. 20 2 EXPERIMENTAL........1 Wheat gluten films ..........3...................................1 BACKGROUND.........................PERMEABILITY......................... 4 1..11 STRATEGIES TO ENHANCE FILM PROPERTIES AND LIFE TIME................ 27 2........................................................3....1 Oxygen permeability .......................4..2............................. 16 1.................................................... 28 2.................................. 12 1.............3 EDIBLE AND BIODEGRADABLE MATERIALS IN FOOD PACKAGING..................................10.................1 Whey Protein Isolate films.....................................5 Microscopy....9 PLASTICIZERS IN PROTEIN FILMS..................... 23 2...............3......2 Microcalorimetry............................................................................................................................................................................................ 28 2................................................4.......................................................1 Chitosan films .........................10 Packaging related tests ............................................................................3..................................................................................... 29 2..................12 HYBRID COMPOSITE STRUCTURES..........................................................2............................................................................. 23 2.12....1 WPI films .....................................................................................................................................................3...............................3.3 METHODS and INSTRUMENTS .....................................................................8 Protein solubility .....TABLE OF CONTENTS 1 INTRODUCTION ................................................6 Water uptake ......... 8 1...............................................................12.........................9 Characterization of Polymer-Clay Nanocomposites ...........................................................................................................................................................................................................................................7 Spectroscopy ............................................................................................. 1 1..7 WHEY PROTEIN ........6.................................. 11 1.................................................................5 MECHANICAL PROPERTIES... 27 2..................2 PURPOSE AND SCOPE OF THE STUDY .................. 24 2.............................. 2 1.................... 14 1..............................................................23 2..............................8 WHEAT GLUTEN......................................6 Gluten/MMT films .................................5 Gluten films...2.............2 FILM FORMATION......................................................................................10.................. 25 2.....................3........................................................................................10 DRAWBACKS OF BIOPOLYMER FILMS.................7........ 25 2............................................ 24 2..................................................

.................................................3 RESULTS AND DISCUSSION .............2.3......... 50 3.........3.................2.................1............................ 42 3......................................59 II ...................................................................3 Mechanical properties ...............................2 Water vapour permeability................................ 32 3.............. 48 3...........1...........................1...........55 ACKNOWLEDGEMENTS ............4 Other packaging related properties.....................................................................................1 Mechanical properties ...................................................................................................................................1 Oxygen permeability ........ 45 3..5 Mass loss ................................................................................................................................................................ 50 3...........3 Permeability .............................................................2.........57 REFERENCES .................................................................2 Permeability .......31 3....................................... 40 3.......................... 31 3..................................................3........................................................................................................... 46 3...........................................1 BIOPOLYMER/PCL BLENDS .........................................2................................................... 41 3....1 General film characteristics....... 31 3........................................3 X-ray and TEM ......................................................................... 51 3...............................2 Mechanical properties of unaged and aged WG films .. 53 4 5 6 CONCLUSIONS ......... 37 3...............................4 Protein solubility of unaged and aged WG films ............................................2 AGING OF WHEAT GLUTEN FILMS..................................3 GLUTEN /MONTMORILLONITE FILMS .............1........................................ 41 3......................................2.........................

have been under scrutiny for many years as heavy contributors to the plastic waste-management problems. the pollution from incineration and the cost and limitations of recycling. In this case. together with more restrictive customer demands and government legislation about plastics and waste materials drives the plastics industry to search for other solutions. The limited availability of landfills. processability and low price [1]. These polymers quickly found wide acceptance. whether fresh or processed. but also because its conversion contributes to solve the disposal problem by turning waste into useful products [10]. Almost all foods. The use of commodity plastics such as polyolefins provides many conveniences including light weight and desirable physical and mechanical properties with a favourable cost performance for the food industry and the consumer. Packaging protects food from the outside environment. are enclosed in some form of packaging. poly(ethylene terephtalate) and poly (vinyl-chloride). due to their attractive properties such as flexibility. low weight.1 BACKGROUND Synthetic polymers have become very important since their development and huge boost under the World War II. During the last two decades the interest in edible and biodegradable materials has increased due to their environmental benefits [4-9]. However. The three main strategies available for the management of plastic waste are: incineration. This gave cause to the first important commercial modern plastic appearances in the 1940s. The use of these materials. Packaging materials for foodstuff. The property of durability which makes these materials so useful also ensures their persistence in the environment and complicates their disposal which is becoming an ever growing problem for many of the plastics now in use. could at least to some extent solve the waste problem. in different packaging applications. Indeed. polystyrene. due to their biodegradable nature. toughness. agro-industrial waste offers a great promise as feedstock because of the low price. 1 . recycling and landfill [3]. Interest to maintain or enhance food quality while reducing packaging waste has encouraged the exploration of new packaging materials. both renewable biomass feedstock (crops) and agro-industrial waste emerge as the key alternatives to produce new biodegradable materials. such as edible and biodegradable films from renewable resources. like any other short-term storage packaging material.Introduction 1 INTRODUCTION 1. Packaging is one application area that was revolutionised by oil-based polymers such as polyethylene. the growing dependence on synthetic polymers has raised a number of environmental concerns. polypropylene. Most plastic materials are not biodegradable and are derived from non-renewable resources. from processing and manufacturing through handling and storage all the way to the consumer [2].

1. This study is a contribution to the ongoing research with aim to improve the properties of these potential food packaging materials. Growing environmental awareness and consumer power coupled with the inexorable rise in pre-packaged disposable meals means that food manufacturers and packagers are increasingly being targeted to improve their environmental performances. and sophisticated polymerization and blending techniques are improving the material’s mechanical properties. food and beverage producers.2 PURPOSE AND SCOPE OF THE STUDY The present thesis focuses on the potential of three biopolymers to be used in food packaging. poly(εcaprolactone).Introduction 1. But nowadays. however. the potential of these blends as food packaging films is analysed. In addition. • • The final aim is to find combinations and methods for these biopolymers that make these renewable materials a commercially interesting alternative to materials commonly used today. PCL. New large-scale production systems are bringing down the costs of biodegradable polymers. Things are changing. seeking goodwill from an increasingly environmentally conscious public. with the exception of cellulose. In addition. The scope of the thesis can be divided in three different parts: • The first part deals with the improvement of water vapour permeability of whey and chitosan by mixing them with a hydrophobic biodegradable polymer. Indeed. That is. the influence of different parameters in the long term properties of this material. due to the big dominancy of synthetic plastics. many researchers believe that biodegradable packaging has a bright future. have begun to employ biodegradable plastics for a variety of 2 . biopolymers have been slow to reach commercial maturity.3 EDIBLE AND BIODEGRADABLE MATERIALS IN FOOD PACKAGING Certain newly discovered characteristics of natural biopolymers could make them the choice for different types of wrappings and films [11-13]. as well as water and gas permeability. The purpose of the study is based in finding strategies to improve the properties of whey and wheat gluten (proteins) and chitosan (polysaccharide) films. By blending these polymers and testing the packaging related properties. The third part consists in the blending of wheat gluten with nanoclays to study the nanoparticles’ impact on the mechanical properties and the permeability of the films. The use of natural polymers dates back to the time of the ancient Egyptians. there have not been sufficient incentives for downstream processors to incorporate the biodegradable materials into their products. That is because they have generally had higher costs and less optimal physical properties than conventional plastics. The second part is focused on the aging problems of the wheat gluten protein.

To date. Examples are polysaccharides such as starch and cellulose and proteins like casein and gluten.1 Biodegradable polymers Unlike most other plastics. but developments with bacterial cellulose are in progress. In some cases. • Figure 1: Schematic presentation of types of biobased polymers and origin of the three materials used in this work.3.Introduction packaging applications. Biobased polymers may be divided into three main categories based on their origin and production [13]: • • Category 1: Polymers directly extracted/removed from biomass. Materials used to make biodegradable plastics fall into two broad categories [6]: • • Natural polymers (biopolymers): made from renewable or natural resources Fossil or non-renewable: made synthetically from oil A schematic diagram of the types of biobased polymers or biopolymers is showed in Figure 1. The monomers themselves may be produced via fermentation of carbohydrate feedstock. 15]. this group of biobased polymers consists mainly of the polyhydroxyalkonoates. A good example is polylactic acid. Category 3: Polymers produced by microorganisms or genetically modified bacteria. a biopolyester polymerised from lactic acid monomers. A polymer is referred to as biodegradable when after the degradationprocess it is fully reduced to natural products (such as carbon dioxide. methane or biomass) by micro-organisms (bacteria or fungi) and/or enzymes [14. 1. biodegradable polymers can be broken down in the environment. Category 2: Polymers produced by classical chemical synthesis using renewable biobased monomers. 3 . water. local and national laws are also encouraging the use of degradable materials.

(see Figure 1) which are presented in this report. The basic materials researched to use as edible and biodegradable films and coatings in food packaging are the ones directly extracted from biomass (category 1). polysaccharides and lipids. these polymers produce materials with excellent gas barriers [18]. gluten and chitosan. Even if they are used in several applications there is still a great disposal of these by-products. Current research in the area cover modified biopolymers and blends. The side chains of these amino acids are highly suitable for chemical modification which is helpful to the material engineer when tailoring the required properties of the packaging material [17]. in applications for food packaging. or have structural roles in the plant cell wall (cellulose. soy. All these biopolymers are by nature hydrophilic and somewhat crystalline. especially in relation to packaging of moist products. gluten. The excess of disulphide cross-linking and thermal degradation of these polymers are also two main problems in their processing and performance. non biodegradable products. nature. and keratin) [16]. which are produced in large amounts as by-products of agro-industrial processes.g. whey. The interest in these materials and the development of their applications in food packaging have increased due to large surpluses of the raw materials. and chitosan [13]. The polysaccharides are natural polymers. factors causing problems. compared to conventional plastics derived from mineral oil. proteins have considerable potential for the formation of linkages that differ with respect to their position. Proteins.g. Proteins can be divided into proteins from plant origin (e. But these approaches still require further research and development before they become competitive to the commonly used synthetic polymers [3]. collagen. casein. The most commonly available. gums.Introduction In general. the principal polysaccharides of interest for material production have been cellulose. biobased polymers have more diverse chemistry and architecture of the side chains giving the material scientist unique possibilities to tailor the properties of the final package [13]. and/or energy. They either act as energy-rich food stores in plants (starch) and animals (glycogen). are extracted from marine and agricultural animals and plants and they are based in: proteins. e. On the other hand. contrary to homopolymers or copolymers in which one or two monomers are repeated. To date.g. each with specific sequences and structures. pectin) or the tough outer skeleton of insects and similar creatures (chitin) [16]. are heteropolymers comprised of more than 20 different amino acids. starch. 4 . Three interesting materials in this category are whey. pea and potato) and proteins from animal origin (e.3. Due to this molecular diversity. 1.2 Biopolymer films The purpose with developing biopolymer films is to offer commercially interesting replacement alternatives to existing synthetic.

The films may be used in multi-layer packaging. provided that various components (flavouring. colouring. This could be important since it represents an environmentally ideal package (no dispose). Many of these materials have good film-forming properties. but their moisture barrier properties are poor. mechanical and aesthetic properties as well as economic and recycling considerations. since they are produced from renewable and biodegradable ingredients.g. e.4 BARRIER PROPERTIES . The films can supplement the nutritional value of foods. they do allow the transport of gases and vapours to some extent. the additives used and on the (chemical) modifications during production. as they confer more potential functional properties. Many lipid compounds such as animal and vegetable fats have been used to make edible films and coatings. proteins and lipid compounds.Introduction Advantages of edible and biodegradable films compared to other polymeric materials are [4]: • • • • They contribute to the reduction of environmental pollution. through interaction with the outside environment. with cellulosic material resulting in a totally biodegradable package. oxygen. and aroma and flavour compounds in food systems to increase product shelf life and improve quality. Protein based films are highly interesting. toughness. The selection of a barrier polymer for a particular application typically involves tradeoffs between permeation. lightweight. The films can regulate transfer of moisture. This is particularly true for protein films. Properties of biopolymers and biodegradable polymer films depend on the raw material that they are based on. Some biopolymers are edible. Lipid films have excellent moisture barrier properties but they can cause textural and organoleptical problems due to their oxidation and their waxy taste [5]. carbon dioxide. formability and printability. 1.PERMEABILITY A key characteristic of glass and metals as packaging materials is their high barrier properties to gases and vapours. Water vapour. lipid. gains or loses moisture or aroma. or becomes contaminated with micro-organisms [12].and oxygen permeability and tensile test are the most frequently used methods to determinate film characteristics for food packaging films [4]. The films can enhance the organoleptic properties of packaged foods. and sweeteners) are incorporated to them. There is an ongoing 5 . • • • The most common materials used to form edible films and coatings are polysaccharides. so these kinds of films could be consumed with the product. The films can function as carriers for antimicrobial and antioxidant agents. While polymers can provide an attractive balance of properties such as flexibility. Films formed from polysaccharides are hydrophilic and provide efficient barriers to oils and lipids. Quality and shelf life are reduced when food. takes up oxygen (leading to oxidative rancidity).

P. generally conducted at fixed temperature and dry conditions have shown high oxygen barrier properties [23-25]. while achieving other desirable properties as film flexibility and strength. such as oxygen and some aroma compounds [22]. moisture sensitivity and temperature. That is why the gas permeability of these materials may increase manifold when humidity increases. Barrier properties are determined by the steady-state rate of mass transport through the films [20. film properties should be compared at as near identical testing conditions as possible as the conditions at which the test or analysis is carried out affect the results. The challenge in use of the barrier properties of proteins is to select biopolymer. which makes them excellent barriers to non-polar substances. The development of edible films and coatings with selective gas permeability is potentially of great interest for controlling respiratory exchange. at low to intermediate RH. leading to sensory and nutrient changes [2]. as fruits and vegetables [26-29]. crystallinity. as well as many organic vapours and odours [30]. it also depends on many physical factors such as density.4. 1. plasticizers. In most cases a biopolymer showing a good O2 permeation. modified atmosphere packaging (MAP) [25. Oxygen causes food deterioration such as lipid and vitamin oxidation. This hydrophilicity makes their gas barrier properties very much dependent on the humidity conditions for the measurements. Many biopolymer film research studies are focused in oxygen and carbon dioxide permeability since these two gases influence the rates of oxidation and respiration in the enclosed food. Modification of polymer structure combined with optimised selection of plasticizer may produce. orientation. cross-linking. Gas permeability measurements of wheat gluten films. will also be a good barrier to CO2. Oxygen Transmission Rate (OTR) and Water Vapour Transmission Rate (WVTR) are two of the most important parameters that affect food. 28. biodegradable films with oxygen barrier properties that are as good as those of poly(vinylidene chloride) (PVDC) and ethylene vinyl alcohol copolymer (EVOH) films (see Figure 2). 31]. 6 .1 Oxygen permeability The transfer of oxygen from the environment to food has an important effect on food quality and shelf life. plasticizer and film-formation conditions that optimize the desired barrier properties. The permeability coefficient. can be defined by: P= (volume of permeant) ⋅ (film thickness) (area ) ⋅ (time) ⋅ (pressure drop in film) The permeability coefficient is not only a function of the chemical structure of the polymer. achieving a modified atmosphere effect. Due to the large amount of hydrogen bonds biopolymer films are hydrophilic. Thus.Introduction interest in optimising property sets of barrier polymers to provide an efficient and economical method for packaging and for extending the shelf life of packaged foods and beverages [19]. 21].

HDPE=high-density polyethylene.2 Water vapour permeability As mentioned before natural biodegradable materials show a considerable potential as good barrier polymers.11% RH) WG:gly (5:1) (30 °C.Introduction LDPE (23 °C. WG=wheat gluten. PVC=polyvinyl chloride.3 ).5:1) (23 °C. 78% RH) MC (30 °C. WG=wheat gluten. because they are too water sensitive (see Fig. 90% RH) PVDC (27 °C. 100% RH) EVOH (70% VOH) (38 °C. 50% RH) PP (23 °C. 100% RH) EVOH (70% VOH) (23 °C. 50% RH) PET (23 °C. EVOH=ethylene vinyl alcohol. 11% RH) WPI:gly(2:1) (23 °C. 50% RH) EVOH (70% VOH) (23 °C.75% RH) SPI:gly(4:1) (28 °C. EVOH=ethylene vinyl alcohol. 75% RH) PVC (23 °C. PET=polyethylene terephtalate.1 1 10 100 1000 WVP ⎜ ⎛ g ⋅ mm ⎞ ⎟ 2 ⎝ m ⋅ d ⋅ kPa ⎠ ⎛ g ⋅ mm ⎞ ⎜ 2 ⎟ ⎝ m ⋅ d ⋅ kPa ⎠ Figure 3: Water Vapour Permeability (WVP) of biobased polymers compared to conventional synthetic packaging materials. 95% RH) WG:gly (2. 100% RH) 0. 11% RH) PVC (28 °C. 75% RH) HDPE (23 °C. PVDC=polyvinylidene chloride) 1. WPI:gly(2:1) (23 °C. 0% RH) 0. PVDC=polyvinylidene chloride 7 .3:1) (23 °C.4. 0% RH) PVDC (23 °C.01 0. WPI=whey protein isolate. 0% RH) WPI:gly(2.1 1 10 100 1000 10000 OP ⎜ ⎛ cm 3 ⋅ µm ⎞ ⎟ ⎜ m 2 ⋅ d ⋅ kPa ⎟ ⎝ ⎠ Figure 2: Comparison of oxygen Permeability of biobased materials compared to conventional synthetic polymers. gly= glycerol. 95% RH) Chitosan (25 °C. (LDPE=low-density polyethylene. 0% RH) LDPE (27 °C. PET=polyethylene terephtalate. 100% RH) Chitosan (25 °C. 0% RH) EVOH (70% VOH) (23 °C. 100% RH) HDPE (38 °C. PVC=polyvinyl chloride. WPI=whey protein isolate. LDPE=low-density polyethylene.01 0. 50% RH) WPI:gly (2. gly= glycerol. HDPE=high-density polyethylene. MC= Methyl cellulose.3:1) (23 °C. but their hydrophilic state makes them unsuitable for food packaging application. PP=polypropylene. SPI= soy protein isolate. 90% RH) PET (25 °C.

while the solubility coefficient gives the amount of the permeant taken (absorbed) by the polymer from a contacting phase [34]. tensile properties are the most frequently considered. deviate from this ideal behaviour due to interactions of permeating water molecules with polar groups in the film structure [22. 37]. However. which are often used as moisture barrier coatings on fruits. similar to other hydrophilic films. 39]. Among the many mechanical properties of plastic materials. The permeability coefficient (P) is the product of the diffusion (D) and solubility (S) coefficients: P (mol/m⋅s⋅Pa) = D (m2/s) x S (mol/m3⋅Pa) The diffusion coefficient indicates how fast a permeant will move within the polymer. and used throughout the industry. and elongation at yield and 8 . modulus of elasticity (Young's modulus). are independent of the water vapour pressure. contribute to the mechanical behaviour of films. Hydrophilic biopolymer materials. confections and drugs and low density polyethylene (LDPE) packaging film. which commonly is used to protect food and drugs from moisture [32]. Water vapour permeability measurements of WG films have been reported [23. Interactions between hydrocolloids and small molecules. vegetables. Ideally. Understanding the way of transport of water vapour through the film is important for improving the moisture barrier properties of WG films. plasticizers. such as polyethylene. Water vapour transport through polymer films proceeds through [33]: (i) (ii) (iii) (iv) absorption of water vapour on to the polymer surface solution of water vapour into the polymer matrix diffusion of water vapour through the polymer desorption of water vapour from the other surface of the polymer Permeability is the rate of transport of a molecule through a polymer as a result of the combined effects of diffusion and solubility. including water. Tensile testing provides these useful data: yield strength. Having adequate mechanical strength and being free of minor defects ensure the integrity of a film. when no interaction occurs between a polymer film and the permeating water vapour. P is independent of the apparent equilibrium water vapour pressure corresponding to the water activity (aw) of the film.Introduction Normally protein films have quite high water vapour permeability compared to waxes. and other additives dispersed in the space of the matrix. protein-based films. exhibit water vapour pressure-dependent permeability [35]. 24. lipids. 36. 1. evaluated. The poor resistance of WG films to water vapour is due to the hydrophilic nature of the protein and to the substantial amount of hydrophilic plasticizer added to impart adequate film flexibility. such as protein films. 38. fracture strength (ultimate tensile strength). Water vapour permeabilities of hydrophobic films.5 MECHANICAL PROPERTIES Mechanical properties are as important to edible and biodegradable films as barrier properties are. Quantitative information on the mechanical parameters of edible films is also essential for the packaging design process [4].

Norway and Island. the elongation at yield and break tend to increase. among others. but the only practical and considerable source is the exoskeleton of commercially harvested shellfish [44]. Additives and plasticizers also affect the mechanical properties. Pakistan. The biodegradation of chitin is very slow in crustacean shell waste. Chitosan versatility depends mainly on these high degree chemical reactive amino groups. chitin with a degree of deacetylation of 75% or above is generally known as chitosan [53]. which could influence the performance of chitosan in many of its applications [50-52]. 9 .6 CHITOSAN Chitosan is a derivative of chitin. Chitosan is commercially available from a number of suppliers in various grades of purity. molecular weight and degree of deacetylation [48]. 42]. Films conditioned in the environment of 50 %RH are weaker than films equilibrated with less RH because of the plasticizing effect of the water molecules [4]. the production volumes are relatively much lower than cellulose [46]. leaving behind a complete amino group (-NH2). The main reasons are the high costs for the extraction of chitin and differences between batches depending on the source due to the difficulties in preparing uniformly reproducible charges in bulk quantities from various marine organisms [47]. shrimps. 41. etc) and the cell walls of e. can be employed to differentiate between chitin and chitosan. In addition. are now manufacturing chitin as a by-product from their growing seafood industries. which determines the content of free amino groups in the polysaccharides [48]. Chitin is a naturally occurring polysaccharide found in the major structural component of the exoskeleton of invertebrates (crab. Maximum tensile strength is the maximum tensile stress that a film can sustain. As the plasticizer content increases. The process of deacetylation involves the removal of acetyl groups from the molecular chain of chitin. The test conditions will be important since the mechanical properties of hydrophilic films are also affected by the interactions with the surrounding environment. 1. However. Even though chitin is the second most abundant natural polymer after cellulose. China. tensile strength decreases and elongation increases [4. Accumulation of large quantities of discards from processing of crustaceans has become a major concern in the food processing industry [45]. the degree of deacetylation.Introduction break[40]. Strain is the maximum change in length of the test specimen before breaking. squid. As temperature increases. krill. Japan and USA are the main producers while India. The stress-strain behaviour of these polymers is strongly dependent on temperature and relative humidity. For instance. Elastic modulus is the fundamental measure of film stiffness [41]. It was reported that the degree of deacetylation is one of the more important chemical characteristics [49]. many fungi. Young’s modulus and the tensile yield strength as well as maximum tensile strength generally decrease. The seafood industry produces large amounts of chitin and most of it is wasted.g. which is the second most abundant polymer on earth after cellulose [43]. The natural occurrence of chitin is widespread.

additives for shampoos and cosmetics. in particular chitosan. Chitosan based coatings can protect foods from fungal decay and modify the atmospheres of fresh fruits and vegetables [12]. which makes chitin and chitosan ideal for decreasing the environmental impact of packaging and other products. are currently used only in small quantities as coagulants for water treatment. which makes chitin and chitosan potent bacteria and virus fighters. The chemically active groups in chitosan structure are the free amino groups and the hydroxyl Figure 4: Molecular structures of chitin. The predomination of partially deacetylized groups makes this polymer soluble in dilute organic acids. 1. Their chemical structures are very similar to that of cellulose. providing clear. So the difference is that instead of the -OH group bounded at C-2 in each glucose unit of cellulose. Chitin and the derivatives.Introduction The structure of chitin and chitosan are shown in Figure 4. textiles and dietary fibres [43. Highly positively charged. with both being susceptible to chitosan and cellulose. consumer and pharmaceutical products [54]: • • • • Anti-microbial. groups. thus being ideal for food coatings [47. Biodegradable. macromolecules and cells. Biocompatible. and flexible with good oxygen barrier properties [58. which makes chitosan an effective binder to negatively charged metals. 51].1 Chitosan films Chitosan films are clear. 54. biochemical. Chitin is composed of repeating units of 1. Chitin and chitosan biopolymers possess some distinct properties that give them extraordinary potential in a broad spectrum of commercial.6. non-toxic naturally occurred polymer. 43. 10 . homogenous. 59]. 46. and viscous solutions or gels with a consistent structure. [57] modifications [56]. and most work on chitin and chitosan is performed in Japan [31.4 linked 2 deoxy-2acetoamido-β-D-glucose. 47]. which is biodegradable to normal body constituents. 56]. 55]. medical materials such as artificial skin and sutures. Chitosan consists of repeating units of (2 amino-2deoxy-D-glucose). Several papers have been written about the film properties of chitosan. tough. or acetylated amino group (-NHCOCH3) in chitin there is an amino group (-NH2) in chitosan [50.

67]. Liquid whey is the thin. The major protein fraction is β-lactoglobulin [65. The high content of hydrogen bonds and the crystallinity give these films low oxygen permeability. immunoglobulins. Whey protein accounts for approximately 5% of total solids of the milk (see Figure 6) [65]. 66. clinical nutrition products etc.Introduction Figure 5:Molecular structure of chitosan. poly(ethylene-co-vinyl alcohol). much of this whey is not utilised. a procedure that is very costly. which are α-lactalbumins. compared to the more conventional materials and the majority of the applications are still in a developing stage [63]. Even if it has generally been disposed of as animal feed or used in infant formulas. fats and inorganic minerals [2. 60-62]. is produced in large quantities and its annual production is continuously rising. 11 . they are divided according to their protein content. In particular. The wide range of protein concentration in these products depends on the membrane used and the pre-treatment prior to membrane separation. The main components of liquid whey are whey proteins. and proteose-peptones. water. sport food. Like cellulose. bovine serum albumin. Usually the whey is purified by membrane filtration to concentrate the protein. The properties of chitosan makes it very interesting for applications as a packaging material and for edible coatings [55. both chitin and chitosan are crystalline due to their linear linkages. However. watery portion of milk that is obtained by coagulation and removal of the curd (casein) during cheese production.7 WHEY PROTEIN Liquid whey. Whey protein concentrate (WPC) contains 25–80% protein. Whey protein products can be classified according to their composition. creating serious waste disposal problems [64]. 68]. 1. Purification with respect to the whey protein is performed to enhance its film forming properties. Processing of chitosan is a relatively unexplored field. replacing e.g. Whey protein isolate (WPI) is nearly all protein (>90%) [2]. The whey protein used in this study (WPI) is refined from liquid whey and purified. Whey protein contains five principal proteins. Makino and Hirata [31] have shown that a biodegradable laminate consisting of chitosan-cellulose and polycaprolactone can be used in modified atmosphere packaging of fresh produce. many problems still have to be solved before chitosan can be of commercial interest. a by-product from cheese manufacturing. lactose. β-lactoglobulins. Typically milk contains around 87% water and 13% solids.

fermentation to ethanol and sweeteners. and will probably increase in the near future [81]. Commercially. Since β-lactoglobulin is the dominating protein in whey its properties tend to dominate as well [63].7.1 Whey Protein Isolate films The WPI films are excellent barriers against oxygen [41. 79]. 12 . The fractionated wheat starch is removed for e. exposing internal -SH and hydrophobic groups. Dry wheat flour comprises 9–13% protein and 75–80% starch [80]. Consequently.8 WHEAT GLUTEN Gluten is the main storage protein in wheat and corn. the interest in the study of edible and biodegradable films made from milk proteins has been outlined in the literature [69-77]. Because of the globular nature of these proteins. break existing disulphide bonds and form new intermolecular disulphide bonds.Introduction Figure 6: Bovine milk fractions 1. 1. which promote intermolecular disulfide bonding and hydrophobic interactions upon drying [77. These proteins undergo temperature denaturation reactions at temperatures above 73°C [78]. 69] and since the uptake of oxygen in food causes rancidity they would be suitable for biodegradable food packaging in multi-component foods. it is an industrial by-product of wheat starch production via wet milling. The production of WG has increased in the last decade due to the increased usage of ethanol for fuel. Native whey proteins are globular proteins containing most of the hydrophobic and sulfhydryl groups hidden in the interior of the molecule. Formation of whey protein films has mainly involved heat denaturation of whey proteins in aqueous solutions. production of films requires pH/heat-denaturation to open the globular structure. The films could function as controlled-release carriers for antioxidants and antimicrobial agents. Wheat gluten (WG) is the cohesive and elastic mass that is leftover after starch is washed away from wheat flour dough. Heating modifies the three-dimensional structure of the protein. as well as supplementing the nutritional value of foods.g.

39. Gluten plastics do not dissolve in water. and minerals (0.30 3.99 1. 85].57 0. Figure 7: Typical amino acid profile of wheat gluten (amylum group)[82]. the alcohol-soluble proteins. Gliadins show a maximum solubility in solutions containing from fifty to seventy percent of alcohol volume [85]. β and γ-gliadins [91. Only two classes of protein are considered: linear glutenin proteins and globular gliadin proteins.77 3. Wheat gluten is unique among cereal and other plant proteins in its ability to form a cohesive blend with viscoelastic properties once plasticized.41 6. The number and distribution of cross-links and the intrinsic properties of the central spiral may contribute to the elastic mechanism [89]. to the least hydrophilic proteins due to the high content of nonpolar amino acids [23. During formation of a dough. Although cystine residues are absent in ω-gliadins (so-called sulphur poor gliadins). together with corn zein. The linear proteins interact with each other via the loop and train mechanism 13 .38 3. Gliadins. 83]. Gluten proteins may be classed as prolamines because of their high proportion of prolamine and gluteamine. 94]. linked through intermolecular disulfide bonds to form polymeric proteins which have one of the highest molecular weights among proteins in nature [86-88].76 11. alcohol-insoluble components. The gluten proteins can be subdivided into two approximately equal groups based on their extractability (gliadins) or inextricability (glutenin) in aqueous alcohols [84. lipids (6–8%). 80]. these residues undergo sulfhydril-disulfide interchange reactions resulting in extensive polymerisation of gluten proteins which leads to the creation of a strong. 93].03 2.8– 1.31 2.77 3. WG belongs.57 1. A very simplified model of gluten at the molecular level is shown in Figure 8. visco-elastic and voluminous dough [80]. γ and ω-gliadins) based on electrophoresis mobility under acidic conditions and increasing order of relative molecular mass [83. % OF PROTEIN Ala Arg Asn/Asp Cys Gln/Glu Gly His Ile Leu Met Phe Pro Ser Thr Try Tyr Val 2.48 3.94 5. are monomeric proteins with intra-molecular disulphide bonds with a low or medium molecular weight [90-92]. but they do absorb water during immersion. 38. Cystine amino acids account for 2-3% of gluten’s total amino acid residues (Figure 7).4%) [80].15 36. β. they are involved in intramolecular disulfide bonds in α.Introduction WG consists mainly of wheat storage protein (70–80%. The gliadins are classified into 4 groups (α.76 The low water solubility of wheat gluten has been attributed to the low content of ionized polar amino acids (14%) to numerous hydrophobic interactions between non-polar amino acids (39%) and to the presence of covalent disulfide bonds [17. Glutenins.82 5.09 2. dry matter basis) with traces of starch and non-starch polysaccharides (10–14%). consists of a number of subunits.

adjusting the solution to acidic or basic conditions (above and below this pH) will favour the protein solubility and the film forming process [103-105]. Temperature has an aggregating effect on wheat gluten inducing the formation of covalent bonds between proteins. 79. It is believed that glutenins contribute to the elastic properties of dough. Gennadios et al. for simplicity. Rising the temperature can denaturate globular proteins. 95. As a first approximation. 98-101]. 63. the chains are imagined to interact with the globular proteins by non-bonding forces such as Van der Waals interactions. They are biodegradable and a priori biocompatible.1 Wheat gluten films WG proteins can be utilized to make films with novel functional properties. The isoelectric point for WG was estimated to 7.5 [102]. It is well-known that heat treatment affects protein conformation and improves some film properties depending on heating conditions [106]. resulting in films of uneven thickness and containing big particles of coagulated protein particles. apart from some WGspecific characteristics such as allergenicity.8. mechanically strong. Generally. 14 . 37. The gliadin proteins fit in the spaces of the glutenin network acting as a plasticizer and giving to the compound the viscoelastic properties [89]. Other polymers (such as gliadins) are approximated by spheres [89].Introduction and by disulphide bonding. whereas gliadins contribute to the viscous properties [9597]. WG-based materials are homogeneous. Several studies have been done on gluten films obtained by casting technique or thermal film forming processes [24. HMW subunits (glutenin) are approximated by linear polymers. Also WG dispersion at pH 5-6 is very poor. transparent. 39. They are also edible when food-grade additives are used. which were unsuitable for property evaluation. while ethanol disrupts hydrophobic interactions [80]. changing the pH of the medium disrupts hydrogen and ionic interactions. Figure 8: A very simplified model of molecular structure of gluten. WG proteins are insoluble in water and require a complex solvent system with basic or acidic conditions in the presence of alcohol. and relatively water insoluble. most likely impeded by intermolecular protein repulsive forces. In the diagram. exposing previously unexposed intramolecular disulfide bonds which now can be reduced and form other new intermolecular disulfide bonds [69]. 1. such as selective gas barrier properties and rubber-like mechanical properties. and the presence of impurities is avoided. 80. This means that gliadin proteins when denaturated lose the native conformation and they open. [37] showed that WG films did not form in the protein’s isoelectric region (pH 7-8). disulphide bonds are not shown. interchain disulphide links are not shown. However.

such as polyethylene-glycol. Water is the most ubiquitous and uncontrollable plasticizer for hydrophilic biopolymer material films. This increase in permeability is undesirable for food quality. additives. During drying. 73. It is water soluble. 22.Introduction The drying step is also a critical part of the film forming process which can affect the final film properties [106]. It is also harmless as plasticizer for films in contact with foodstuff. glycerol and sorbitol [72. 107]. 1. 109-111]. 38]. Functional properties of gluten films are highly dependent on WG concentration in solution. This brittleness makes these films useless in packaging film applications. temperature. There are many parameters which must be controlled when making WG films. The most commonly used plasticizers are poly-functional alcohols. 77].9 PLASTICIZERS IN PROTEIN FILMS Proteins by themselves. 42]. hydrophobic bonding. hydrophobic interactions and disulfide bonds contribute to formation of a good oxygen barrier three-dimensional network [5. 112]. These properties make glycerol a suitable plasticizer to use with a compatible water-soluble polymer [75]. active sites for bond formation become free and close enough to each other to create new interactions. and/or disulfide cross-linking [27]. which depends on the type and amount of plasticizer [41. In this study glycerol was used. The usual approach to enhance film forming properties is to add a plasticizer. solvent polarity. 36. Gluten protein plasticization is a complex phenomenon that can be primarily explained by the ability of the hydrophilic plasticizer molecules to share hydrogen bonds with the protein network [108]. pH. including whey and wheat gluten. 15 . The result is an increase of the free volume and the mobility of polymer chains. and drying rate [37. 104. form brittle films. nonvolatile. due to extensive interactions between protein chains through hydrogen bonding. Plasticizers are hydrophilic low molecular weight liquids that reduce protein chain-to-chain interaction. thus the surrounding relative humidity has a large effect on the film properties [100]. 42. 23. and protein miscible. Glycerol is a high boiling point plasticizer. 80. 17. 27. 111. electrostatic forces. so there is a need to optimise the use of plasticizer to offer the right balance of desired mechanical and barrier properties for the desired application [27. The increase in film flexibility is also accompanied by an increase in film permeability. polar. 69. 41. all volatile disruptive agents are progressively eliminated. lower glass transition temperature and more flexible and soft films. and is frequently used as sweetener in foodstuff [41]. Consequently. The plasticizing and/or swelling effect of water on hydrophilic (polar) polymers also result in increased permeability values [25. Solvent removal increases the concentration of WG proteins. 77. New hydrogen bonds.

nutrients. as many food applications demand materials that are resistant to moist conditions. Moreover. Thus. 1. despite the benefits that these polymers offer there are still significant obstacles that must be overcome. but after that they should biodegrade efficiently. The aging of a biodegradable film can be due to physical or chemical reactions in the polymer matrix. Biodegradable polymers suffer a change in their properties during time.10. which may decrease the protective function of the packaging and consequently reduce the shelf life of the packaged food. Some of the main drawbacks and limitations of biopolymer films are listed below. rapid. which is also a great disadvantage. such as retailer and consumer scepticism. Nevertheless. which make them not suitable for commercial applications.1 Water sensitivity As pointed out earlier the major challenge for the material manufacturer is the by nature hydrophilic behaviour of many biobased polymers. non controllable degradation under the influence of bacteria (the disadvantage of biodegradability). concentration and the hydrophobic or hydrophilic character influence the migration. Water. Generally. biopolymer film properties still have some restrictions and drawbacks which limit their use in large-scale applications. biopolymers films are still difficult to process in comparison with synthetic polymers. films made by biological materials do change their mechanical and barrier properties in high moisture conditions. This water sensitivity can develop a more spontaneous. aging. 16 . oxygen levels and time can package integrity and microbial stability be assured. migration of these low molecular compounds leads to stiffer and less extendable polymers. Plasticizer molecular weight. 1. material costs.10. also migrates with time from the matrix to the surface and evaporates leaving a more brittle film [100]. Only by appropriately controlling water activity. Moreover.10 DRAWBACKS OF BIOPOLYMER FILMS During this introduction the potential of biodegradable films for applications in food packaging have been pointed out. as one of the most powerful plasticizer for these types of films. whereas acceptable time of storage in moist environments or time of use with moist foods would be limited [18].Introduction 1. This is a physical process that the films suffer when plasticizers migrate to the surface. pH. Most of the biobased materials are not soluble or are difficult to dissolve in water but they show large water up-take (swelling) and high water permeability. The most common aging processes that films use to go through are: • Physical aging: Migration of additives from the matrix. Apart from not yet competitive mechanical properties.2 Aging of biodegradable polymers One of the challenges for the successful use of biodegradable polymer products is to achieve controlled lifetime. biodegradable polymer films may be safely stored in dry environments and used with dry food products over a relatively long period of time. temperature. the added costs of switching technologies. Products must remain stable and function properly during storage and intended use.

In several studies proteins have been cross-linked with aldehydes. 109]. octanoic and palmitic acids. Most of them apply their modifications as ‘pretreatments’. dibutyl phthalate and tartrate [115]. oxidation of not reacted thiol groups and a reorganisation of the intramolecular disulfide bonds to intermolecular disulfide bonds via thiol-disulfide exchange reactions can occur. Here the most commonly used methods are summarized: Plasticizer • In response to the problems caused by the use of a low molecular weight plasticizer as glycerol (increase in diffusion of gas and water vapour through the film and migration of the plasticizer). 116]. Trying to reduce the plasticizer migration. it is possible to replace glycerol with a higher molecular weight compound that has hydrophobic substituents [108.Introduction • Chemical aging: Oxidation Some studies have shown that formation of disulfide bonds by thiol oxidation occurred during wheat gluten film storage. Cross-linking • Another alternative to improve protein film properties is to modify the polymer network through cross-linking of the polymer chains. Therefore. This may help to decrease WVP and plasticizer migration and hence improve the fracture strain of gluten films [111]. 113]. stearic and oleic acids) [109]. depending on the rate of thiol oxidation during film drying and storage [90. 108. This will induce an increase of protein aggregation and a brittleness of the film structure [88]. enzymatic. Some other attempts are performed using ‘post-treatments’ (applied on the film) [101]. and it is of major importance for achieving good mechanical properties of the final product [114]. Substances studied as possible replacements to glycerol are amphiphilic substances. many other studies have been done comparing the effects of various hydrophilic plasticizers (differing in their chain length) on the mechanical properties of protein films [41. mechanical properties of the films might be changed with film aging. During time of storage. or physical treatments in order to enhance functionality of derived protein films. meaning that the changes occur in the film-forming solution. The sulfhydril in the cystein amino acid is responsible for the formation of disulphide cross links during oxidation [63]. Some different aldehydes have been used for this 17 .11 STRATEGIES TO ENHANCE FILM PROPERTIES AND LIFE TIME The attempts to improve the properties of biopolymer films are made in several ways by different researchers. even in conditions (temperature and relative humidity) under which the mobility of molecules is reduced. including fatty acids (lauric. 1. The presence of reactive side groups in proteins makes it possible to cross-link the polypeptide chains through chemical. This is a process which is accelerated when heating the film solution to make films.

• • Blends • One extensively used method to enhance the water vapour barrier properties of films have been the incorporation of hydrophobic compounds such as lipids into the film-forming solution [60. Preparation of composite films through combined use of compatible polysaccharides and proteins to improve properties of biopolymers is another way of trying to improve film mechanical properties [133-138]. Properties of several proteins have been substantially modified by controlled thermal treatments [22. This must be taken into account when synthesizing biopolymer materials. • • 18 . Gamma irradiation as pre-treatment on the film-forming solution has also been applied. Various investigations have examined and confirmed the effectiveness of composite edible films with fatty acids (stearic or palmitic acids). paraffin or bee wax. Blends with lipids were still brittle so the addition of a plasticizer was needed [111]. fatty acid esters and carnouba wax [130. 36. particularly formaldehyde [117]. This irradiation method has been used by Rhim et al [125] to cross-link protein films. By laminating biopolymer films between other synthetic films their lack of structural integrity and characteristic functionality can be solved [31]. 131]. Thermal treatment of the solution. water vapour transferability. glutaraldehyde and glyoxal [101. 36. These studies have shown that heating at elevated temperatures increase the film strength and water resistant properties due to the formation of intermolecular disulfide bonds necessary to form intact film. and hydrolyzability of the resulting films. 127.Introduction type of reaction. 77. 113. 118-120]. However. However. The incorporation of lipid compounds (waxes and fatty acids) helps to limit the moisture migration since the lipids help lessen water vapour transmission and the proteins or polysaccharides give the films strength and thus help improve structural integrity [129]. although highly reactive. 106. Another weaker form of electromagnetic radiation is ultraviolet (UV) radiation [101]. 71. 121] and wheat gluten [105]. [23. with subsequent analyses of the mechanical properties. 126]. • Enzynmatic cross-linking with transgluminase has been performed with whey protein [75. 114]. 38. 128]. 79. Morillon et al [132] and McHugh et al [128] have published extensive reviews of the factors involved in the water permeability of edible films based on lipid incorporation. the use of these films in multilayer packaging seems to be a good alternative. these components have a major disadvantage: their toxicity [37]. solubility. 74. 41. The still unfavourable properties of biopolymer films prevent any direct use on the market. Cross-linking induced using gamma irradiation was found to be an effective method for the improvement of both barrier and mechanical properties of some edible films and coatings [122-125].

Some examples of nano reinforcements currently coming forward are nano clays. First a composite structure with a high crystalline biodegradable material (PCL) was made. Some of the reasons for choosing this material for blending with chitosan and whey are: • • • • It is highly compatible with a wide range of polymers [143]. Poly(ε-caprolactone). which could help to reduce the water vapour permeability of chitosan and whey films. in combination with acceptable mechanical properties and biodegradability. inexpensive.12 HYBRID COMPOSITE STRUCTURES 1. in which the reinforcing material has dimensions of less than or equal to one nanometer. 1. called nanocomposites. It has good mechanical properties. Natural fibres from grass.12. hemp. cellulose nanowhiskers. have low density. Through the second one a nanocomposites clay-gluten system was tried. In this study two different strategies were followed to try to improve the materials properties.1 Poly(ε-caprolactone) composites Blending is an important method in polymer manufacturing and has received increasing attention because of the strong economic incentives arising form the use of polymer blends [140]. PCL is a linear aliphatic polyester. ultrafine titanium dioxide and carbon nanotubes [136]. These blends may combine the advantages of both components and have better properties than either component [141]. It is completely biodegradable into non-toxic substances so the mixture of this polymer with chitosan and whey biopolymers would still be biodegradable [142]. and ramie have been reported as reinforcements of polymer films [139]. • A fairly new group of composites has emerged. 19 . It is crystalline and hydrophobic. sustainable. Additionally.Introduction • Natural fibre reinforced composites are an attractive research area because natural fibres are eco-friendly. (PCL) is recognised as one of the few synthetic polymers which are biodegradable. these fibres have excellent thermal and sonic insulation properties. which is approximately 50% crystalline [142].

Kojima and co-workers [151] for nylon-clay nanocomposites. The blocking effect. dispersed at nanoscale level. Poly(ε-caprolactone) Density Tensile strength Tensile modulus Ultimate strain Glass transition temperature Melting point ρ (g/cm3) σ (Mpa) 1. If the oxygen diffuse in the high-permeable phase boundary and the montmorillonites are poorly exfoliated. 152].or microscopic dispersions of inorganic fillers [149. Due to the nanometer-size particles obtained by dispersion. The compatibility between the clay nanoparticle and the matrix decides the ability for dispersion and exfoliation (together with the molecular mobility of the matrix). physical and mechanical properties that cannot be obtained with macro. cellulose [145]. that in turn decides the final properties of the film. and poor exfoliation and distribution. 147] and chitin [148].11 – 1. then it will give no improvement [130. 20 . thermal. are most promising materials characterized by a combination of chemical. optical and physicochemical properties when compared with the pure polymer or conventional (microscale) composites as firstly demonstrated by Toyota researchers. The surface of the clay-particles is of major importance. these nanocomposites exhibit distinctly improved mechanical. is important for the final gas transmission properties. Bad compatibility results in a higher free volume in the phase boundary between the montmorillonite particle and the biopolymer film.146 20.Introduction Some properties of PCL are listed in the following table: Table 1: Physical properties of PCL [144].21 – 0. 137.7 – 42 0. 150.44 300 – 1000 -60 – -65 58 – 65 E (Gpa) ε (%) Tg (°C) Tm (°C) The interesting properties of this polyester have made many researches to use it in blends with natural polymers such as protein [133. starch [146. 141]. 1.12. 150].2 Nanoclay nanocomposites Polymers filled with low amounts of layered silicate. which also is a function of the total amount of free volume in the penetrating matrix.

making the entire surface of the layers available for the polymer. forming a monolithic structure on the microscale. alkyl ammonium ions. The layer thickness in momtmorillonite is around 1nm. Montmorillonite is one of the most used clays to produce nanocomposites.g. These polymers can be able to intercalate easier within the galleries to exfoliate the nanoclay layers [150]. Conventional composites or microcomposites are defined when clay acts as a conventional filler. These layers are organized in stacks with periodic Van der Waals gaps in between them called galleries or interlayers. The compatibility between clay mineral and the organic polymer may be substantially enhanced by replacing the charge balancing interlayer cations with e. has lower surface energy and is more compatible with organic polymers. Delaminated or exfoliated nanocomposites represent the situation when 1 nm-thick layers are completely delaminated and dispersed in the matrix. The modified clay then. (c) exfoliated nanocomposite. This should lead to most dramatic changes in mechanical and physical properties [154]. [150] 21 . The latter configuration is of particular interest because it maximises the polymer-clay interactions. Intercalated nanocomposites refer to the state where the gallery regions between the aluminosilicate sheets are swollen by polymer chains while retaining the regular stacking arrangement. Figure 9: Scheme of different types of composite arising from the intercalation of layered silicates and polymers: (a) phase separated microcomposite.Introduction Morphological descriptors such as intercalation and exfoliation are commonly used to describe the state of aggregation of the individual sheets of clay in the polymer matrix (see Figure 9) [153]. (b) intercalated nanocomposite.

Figure 10: Schematic sketch of solvent based synthesis. 155159]. melt intercalation [160-162] and solution method [163.Introduction Several strategies have been considered to prepare polymer-layered silicate nanocomposites whereof three are the most used ones: In-situ polymerisation [151. Layered silicates can be easily dispersed due to the weak forces that stack the layers together. In this study the latter method was used to form gluten-clay nanocomposites. 164]. The polymer is then adsorbed onto the laminated sheets and when the solvent is evaporated an ordered multilayer structure is formed (see Figure 10). Drawn after [149] 22 . Here the organoclay and the polymer are dissolved in a polar organic solvent.

34 wt% fat and 0. Iodine. Cloisite®Na+ is a natural sodium-rich montmorillonite.2 Chitosan films Chitosan. obtained from Southern Clay Products Inc. wheat gluten and composite films were produced by solution casting method (see Figure 11). The solution was decanted into petri dishes covered Solution by Teflon coated aluminium (20g in each) and subsequently dried for 48h at room temperature and 30% relative humidity. 2. whey. PCL grade TONE© P-300 (Mw = 10000 g mol-1 and Mw/Mu = 1. The deionised water was prepared in a Miele Aqua Purificator G7749.2.1 wt% wheat starch. Sweden. was supplied by Merck KGaA.1 MATERIALS MD Foods Ingredients (Denmark) supplied WPI.8 wt% WG proteins. Chloroform (tri-chloromethane stabilised with 1% ethanol) was purchased from Lab Scan (Sweden). So acetic acid was used to protonise the chitosan molecule (-NH2 -NH3+) in order to make the polymer soluble in water and make it 23 . does dissolve in aqueous organic acids to give viscous solutions at a pH below 6.7%) of molar mass Mw ≈ 400 000 g·mol-1 was provided as platelets by Fluka Biochemika (Sweden). was supplied by Norton Performance Plastics Corporation. USA). Sweden. (Texas. Glycerol with a purity of 99.Experimental 2 EXPERIMENTAL 2.5. though insoluble in water. Aldrich Chemie GmbH. Sweden.2 FILM FORMATION All chitosan. The solution was kept at 73°C for 20 min in order to denaturalise the protein. Chitosan (deacetylation degree of 84. Germany. LACPRODAN DI-9224. Acetic acid (glacial 100%) was purchased from MERCK (Germany). Two types of commercial nanoclays were used: Cloisite®Na+ and Cloisite®10A. Sodium hydroxide was supplied by Sigma.5% was supplied by Karlshamns Tefac AB. Lidköping. Cloisite®10A is a natural montmorillonite modified with a quaternary ammonium salt. 1. Karlshamn.2.1 WPI films Whey films were produced by first stirring an aqueous solution of 12%(w/w) whey and 6%(w/w) glycerol at room temperature.76 wt% ash) was kindly supplied by Reppe AB. Germany. The vital wheat gluten (WG) powder (84. 5 wt% water. FILM CASTING Teflon coated aluminium Figure 11: Film casting technique 2. Teflon© coated aluminium foil. 8. 2.7) was supplied as granules from Union Carbide USA. resublimed GR for analysis. The ethanol (95%) was obtained from Kemetyl AB. Bytac© Type AF-21.

5 Gluten films The films were produced by first stirring 30 g WG powder and 9. The solutions were separately stirred until homogeneous solutions were obtained and they were then mixed together in a blender for 4 min. To ensure homogeneity the solution was mixed in a blender for 4 min. The solutions were thereafter heated to 75°C during 20 min (2. Then both solutions were put together and mixed for another 4 min. 90 g of deionised water was added slowly. After being cooled to room temperature. After homogeneity was attained.9 g glycerol in 135 g ethanol. Blends were prepared with 5. Subsequently a layer of foam was removed from the surface and then the solution was heated to 60°C for 20 min. The films were dried for 48 h at room temperature and 30% relative humidity. 10 and 15% by weight of PCL with respect to the chitosan added. 300-400 µmthick films were obtained by pouring the solution into petri dishes and allowing it to dry for 2 days at 23°C and 50%RH. until all the chitosan was dissolved.3 PCL/Chitosan films An aqueous solution containing 1%(w/w) chitosan and 1% acetic acid and a chloroform solution containing 10%(w/w) PCL were each decanted into separate glass tubes. Stirring was continued for approximately 30 min. Norton Performance Plastics Corp. 24 . The chitosan solution was then mixed in a blender for 4 min. Wayne.5 °C/min) and kept at that temperature for 10 minutes under gentle stirring. Films were conditioned in a climate chamber at 23°C and 50% RH for at least 48 h and then placed with the same side facing up on a porous paper support (Blotting Paper 1600. NJ). Solutions with pH=4 or pH=11 was obtained by adding.2. The new solution was cooled to room temperature. 10 and 15% by weight of PCL with respect to the WPI added. respectively. First 1% (w/w) chitosan and 1% acetic acid were dissolved in water during high-speed stirring.Experimental possible to cast and dry the solution into transparent polymer films. Both solutions were mixed with a magnetic stirrer for 20 minutes. The new solution was kept untouched for 30 min.4 PCL/WPI films The WPI and the PCL solutions were made separately as described above. the solution was dried in a vacuum oven for 10 min before it was decanted into petri dishes covered with Teflon© coated aluminium at 23°C and 30%RH. acetic acid or sodium hydroxide. Blends were prepared with 5. 2. The petri dishes were coated with a release agent layer of polytetrafluoroethylene supported by an aluminium foil (Bytac Type AF-21.2. Films were subsequently obtained after the solution had dried for 24h at 23°C and 30%RH. Then it was dried in a vacuum oven for 10 min before it was decanted into petri dishes covered with Teflon© coated aluminium foil. The solution was then kept untouched for 2h before it was decanted into petri dishes covered by Teflon© coated aluminium. 2. 220 diam.. from VWR International).2. Approximately 20 g of solution was poured into each petri dish. 2.

Ethanol solutions with 0.[154] 2.6 Gluten/MMT films The gluten/MMT films were produced following the method shown in Figure 12. The solutions were thereafter heated to 75°C during 20 min (2.% natural and modified montmorillonite were prepared. the specimen was exposed to flowing oxygen gas (99. by using a Mocon OX-TRAN TWIN that was equipped with a coulometric oxygen sensor. acetic acid and sodium hydroxide.5. These were ultrasonicated for three minutes. Figure 12: Flow chart presenting the different steps of the solution approach.2. The petri dishes were coated with a release agent layer of polytetrafluoroethylene supported by an aluminium foil (Bytac Type AF-21. 300-400 µm-thick films were obtained by pouring the solutions into petri dishes and after drying for two days at 23°C and 50%RH. Solutions with pH=4 and pH=11 were obtained by adding. according to ASTM D 3985-95.5. Then 60 g deionised water was added slowly.Experimental 2.3. tightly sandwiched between two pieces of aluminium foil (see Figure 13) had a circular active area of 5 cm2. These films were mounted in isolated diffusion cells and subsequently purged with nitrogen gas (2% hydrogen) to obtain the background oxygen leakage.3 METHODS AND INSTRUMENTS 2. Norton Performance Plastics Corp. 18 g of WG powder and 6 g of glycerol were stirred in 90 g of ethanol until a homogeneous solution was obtained.75. in an Elma Transsonic T420 apparatus.5 wt. respectively. After background correction. NJ).. Consequently the mass ratio of water and ethanol was 1:1.95%) at Figure 13: Polymer sample glued atmospheric pressure. 1. Wayne.5 °C/min). Films were conditioned in an environmental chamber at 23°C and 50% RH for at least 48 h prior to testing. was determined. The specimens.1 Permeability Oxygen permeability test The oxygen permeability of the films. at 23°C. together in between two aluminium foils 25 . 3 and 4. Subsequently the solutions were poured into the 70°C gluten solution and then stirred for 10 minutes.

which in turn was inserted into the perfusion ampoule. A is the exposed surface area of the film (A = 0.3. Water vapour permeability test The water vapour transmission rate (WVTR) of each sample was measured using a Mocon Permatran-W Twin (Minneapolis. The water vapour transmission rate at steady-state through the film. The WVTR was normalized with respect to the water vapour pressure and the film thickness to yield the water vapour permeability (WVP). Before insertion. was measured for pure water by first putting 0. M is the molar mass of water and t is the film thickness.2827 cm2) and ∆p is the water vapour pressure difference across the film. The film sample was tightly fastened onto the upper part of the steel ampoule by a stainless steel ring that was anchored using a screw. Qo. The measurement principle is based on the assumption that there is an equilibrium exchange of water molecules between the liquid. After 26 . the film and the upper compartment at steady-state. The calorimetric response at steady-state measured by the microcalorimeter is the heat flow due to evaporation of water from the liquid. 2. As explained by Gennadios et al (1990) [23]. Before the measurements. Specimens were tightly sandwiched between two pieces of aluminium foil leaving a 5 cm2 exposure area for the WVTR measurements (see Figure 13). The heat of water evaporation from the liquid. The RH perfusion ampoule was designed to humidify a gas to a specified relative humidity by mixing a flow of dry gas with a flow of gas of 100% relative humidity. the lower compartment. this low RH was used in order to avoid swelling and non-Fickian behaviour of the films. 1: The water vapour transmission rate at steady-state Where Pe is the heat flow rate. The oxygen transmission rate was normalized with respect to the oxygen pressure and the film thickness to yield the oxygen permeability (OP). is given by Eq. It is also assumed that the heat of condensation at the lower film surface is the same as the heat of evaporation at the upper film surface and that the heat of solution and desolution of water in the film are included in these terms. MN) according to ASTM F 1249-90. Sweden) of the heat conduction type equipped with a 4 ml stainless steel RH perfusion ampoule.2 Microcalorimetry A microcalorimeter system (2277 Thermal Activity Monitoring. 1: Qο = Pe ⋅ M ⋅ t ∆H v ⋅ Α ⋅ ∆p Eq. the water contained in the steel ampoule was weighed. was used for the measurements. referred to as TAM. A closed and empty 4-ml stainless steel ampoule was used on the reference side. Thermometric AB. Water vapour permeabilities were evaluated for the films by applying a small RH gradient (11%) across film samples.4 g of water into a steel ampoule with an inner diameter of 6 mm.Experimental Measurements were interrupted when steady-state was attained. In the present case water was added to the lower compartment (inside the steel ampoule). the specimens were conditioned in isolated diffusion cells with one side in contact with an atmosphere of a saturated LiCl-solution (giving 11% RH) and the other side facing dry nitrogen gas. ∆Hv.

The heat of evaporation of water in this case was obtained by a procedure similar to that for pure water. The WG films were tensile tested at 50% RH and 23°C. The tensile strength was defined as the maximum stress in the nominal stress-strain curve. the perfusion ampoule was withdrawn and weighed and the heat of evaporation calculated.4 Content of volatile mass The loss of volatile mass of gluten films was measured as described in ASTM D 644-94. The specimens were subsequently cooled in desiccators at 0% RH and 23°C and then weighed to determine the loss of volatile mass. The WG films were hanged inside the recipient in order to expose the entire surface to the sublimed iodine. The clamped length of the specimen was 40mm.3. 10 replicates of each sample were measured. Sweden). 2.3.0±0.1® computer program supplied from Zwick GmbH & Co. using a JEOL JSM-5400 scanning electron microscope. Optical Microscopy Starch turns deep blue (almost black) in the presence of a small amount of iodine vapour.3. In order to achieve a relative humidity of 11% in the lower compartment. The strain rate was 100 mm/min and. Dumpbell shaped specimens were punched out from the films with a length and width of the narrow section of 16. The thickness of each specimen was taken as the average of five readings. The samples were kept in iodine vapour for 24 hours. Iodine powder was dispersed in the bottom of a hermetically closed recipient. whey and gluten composite specimens. 27 . a solution saturated with LiCl crystals was used instead of pure water. The test pieces were weighed and then dried for 24 h at 105°C in a Nûve FN400 oven (LabRum Klimat AB. Three replicates from each sample were measured.0±1.0 mm and 4.Experimental approximately 15 h. Three samples with a length of 50 mm and a width of 15 mm were tested for each blend. Prior to testing the samples were conditioned in a room at 50%RH and 23C. 2.3 Tensile test The stress-strain properties of chitosan and whey films were obtained at 23°C (50% RH) according to SCAN-P 38:80 (34) using an Alwetron TCT 10 tensile tester.5 Microscopy Scanning Electron Microscopy Scanning electron microscopy was performed on fractured cross-sections and surface of gold/palladium (60%/40%) sputtered chitosan. This reaction serves as a test to check the presence of starch in the surface of the gluten films. using a ZwickZ010 tensile tester controlled by testXpert 7. The measurements were performed as described in ASTM 882-01 with a crosshead speed of 100 mm/min and a clamp distance of 40 mm. Germany.1 mm. respectively (ISO 37:1994(E)). 2.

Experimental

The structure of the surfaces was analysed with a Leitz Ortholux POL BK II optical microscope.

2.3.6 Water uptake
Small whey and chitosan test specimens were cut and put on aluminium foils. The specimens were dried in a vacuum oven for two days and then weighed. After that they were stored in a chamber at 11%RH and 23°C for two days and then they were weighed again. The water uptake was obtained as the percentage of gained weight.

2.3.7 Spectroscopy
UV/VIS spectroscopy

A Hewlett-Packard 8415A Diode Array spectrometer was used to measure the light absorption in whey and chitosan films.
IR spectroscopy

Fourier transform infrared spectra (12 scans) of gluten films were recorded on a Perkin Elmer Spectrum 2000 FTIR spectrometer equipped with a normal single reflection ATR accessory (Gloden Gate) from Specac Ltd. (Kent, England).

2.3.8 Protein solubility
A biochemical control parameter of the changes in the gluten polymer network is the percentage of protein, which is insoluble in non-ionic detergents such as sodium dodecyl sulphate (SDS). The percentage of SDS-insoluble protein is a well established quantity, which reflects the covalent cross-linking degree of the protein network [165, 166] Amount and size distribution of proteins extractable with sodium dodecyl sulfate (SDS) and SDS+sonication of cast films were obtained using size-exclusion high performance liquid chromatography (SE-HPLC), together with a three-step extraction procedure according to Gällstedt et al [63]. Proteins soluble in dilute SDS were extracted in the first step and additional proteins were extracted with repeated sonications [63]. The new films were analyzed approximately 6 days after being prepared. Aged films were at least 120 days old. In the first step, 16.5 mg of each film were suspended in 1.5 ml 0.5% SDSphosphate buffer (pH 6.9) and vortexed for 10 s. The suspension was then stirred for 5 min at 2000 rpm and centrifuged for 30 min at 10000 rpm to obtain the supernatant protein. In the second step, the pellet was re-suspended in the SDS buffer and sonicated in an ultrasonic disintegrator (Soniprep 150, Tamro, Mölndal, Sweden) for 30 s, amplitude 5, fitted with a 3 mm exponential microtip. The samples were then centrifuged (30 min, 10000 rpm) to obtain a supernatant of proteins. For the third step, the pellet was again re-suspended in the SDS buffer and sonicated as above for 30+60+60s. The extracts were filtered through 0.45 mm filters (Millipore, Durapore Membrane Filters) before the SE-HPLC operation. SE-HPLC analyses were performed on a Waters HPLC system using a BIOSEP SEC-4000 Phenomenex column. Separation was

28

Experimental

obtained after 30 min by loading 20 µl of sample into an eluant of 50% (v/v) acetonitrile and water containing 0.1% (v/v) trifluoroacetic acid at a flow rate of 0.2 ml min-1. Proteins were detected by UV absorption at 210 nm. The amount of extracted proteins after each of the extraction steps were normalized the protein solubility of unprocessed WG powder exposed to the third extraction step [63]. The SE-HPLC chromatograms were divided into polymeric and monomeric fractions (Johansson et al 2001) and the protein polymer/monomer ratio was calculated [63].

2.3.9 Characterization of Polymer-Clay Nanocomposites
X-ray Diffraction (XRD)

X-ray diffractograms of films were obtained in a Siemens D5000 diffractometer with Cu radiation (50 kV, 40 mA). The scanning speed and the step size were 0.15°/min and 0.02°. The specimen rotation speed was 15 rpm.
Transmission Electron Microscopy (TEM)

Specimen preparation constitutes a major difficulty in transmission electron microscopy analysis. TEM specimens need to be sufficient thin in order to optimise the so-called mass thickness contrast. One way to prepare TEM specimen from polymer samples is ultramicrotomy. Samples were embedded in epoxy and curated at 60 degrees during 24 hours. Then, using an RMC ultramicrotome, 50 nm thick sections were obtained at cryogenic temperature (-80 or -120°C) (Figure 14). After heating to room temperature the sections were removed from the knife using a grid with a water droplet.

Figure 14: Ultramicrotomy - the sample is moved across a knife-edge (diamond). [154]

The grids were then transferred to the transmission electron microscope (TEM) Philips Tecnai 10 operating at 80 kV for observation of the clay-polymer structure.

2.3.10
Printability

Packaging related tests

A red ethanol-based dye was applied as a single straight line on the surface of the films. After intervals of 2s and 30s, a dried poly(methyl methacrylate) plate was drawn over the dye line to smear the line out. The distance of the smearing was measured. The
29

Experimental

shape and amount of dye left on the dye line after this ‘draw-down’ procedure were analysed qualitatively.
Seal strength Measurements

Films (thickness = 0.1-0.3mm) were welded in a Multivac A300 during 10 consecutive 0.2 s heat pulses. The seal strength of the welded films was determined in an Alwetron TCT10 tensile tester at 23±2°C (50% relative humidity) and 100 mm/min strain rate. The clamped length of the specimen was 40 mm.
Transparency

The transparency of the films was assessed by measuring the distance (L) between the specimen (polymer film) and a letter, at which the letter became invisible when viewed through the specimen. The letter had a thickness of 300 µm and was printed with black ink on white paper. The distance between the polymer film and the observer was 40 cm.
Flexural Stiffness

The stiffness of the blends was obtained by the standard test according to SCAN-P 29:95 using a Lorentzen and Wettre stiffness tester. The force to bend the specimen through an angle of 7.5°C was monitored. The specimen had the dimensions 55mm x 40 mm (clamp width) and the distance between the clamp and the point where the force was applied was 10mm. The samples were conditioned at 23°C and 50% RH for 3 h before the measurements. The stiffness index (Nm2/g) was obtained from the force (F) and the density (w) of the specimen: Si = F/w3. The stiffness index was calculated as the average of 3-4 specimens.
Folding Endurance Test

The folding test was performed according to SCAN-P 17:77 using a Lorentzen and Wettre folding endurance tester. Specimens preconditioned at 23°C for 24h at 50%RH (chitosan blends) and 25%RH (WPI blends) were subjected to cyclic folding through 180° until fracture occurred. Specimens were fixed in vibrating clamps at one end and the vibrational amplitude caused imposed folding of the specimen. The 10-logarithm of the number of cycles until fracture (log(Nf)) was calculated and averaged over 5 samples.

30

95%RH 23°C.05 0. but the conditions in these studies were not specified. The values were in reasonable agreement 31 .04 ± 0.3±0.04 0.11 0. Film Chitosan Chitosan PCL5% Chitosan PCL10% Chitosan PCL15% WPI Chitosan Chitosan (plasticized) Chitosan (unplasticised) WPI: glycerol (5.33 3. 50%RH 23°C. 0%RH unspecified 25°C. Film permeabilities were much lower than values reported by Muzzarelli in 1974 (0. 0%RH 25°C.10 ± 0.708 0.46±0. and similar to that of dry ethylene vinyl alcohol copolymers. 3. 0%RH 25°C.27 ± 0. 50%RH 23°C.1 Oxygen permeability The oxygen permeability was measured in pure WPI and chitosan films and in some blends of chitosan. 50%RH OP (cc µm/m2 day kPa) 0.1 18.5 76. 0%RH 25°C. 50%RH 23°C.1. Table 2: Comparison between oxygen permeability values in this study and earlier studies.28 ± 0. this study this study this study this study this study [167] [55] [60] [41] [41] [30] [30] [30] [30] [30] It was proved that chitosan and whey (at low relative humidity) had a lower oxygen permeability than that of poly(vinylidiene chloride). Attempts to find a procedure to blend PCL with chitosan and whey are reported and mechanical and barrier properties of these films are discussed. 50%RH 23°C.3 cc µm/m2 day kPa) [60].1 BIOPOLYMER/PCL BLENDS In this section the results of the study of chitosan and whey protein films blended with polycaprolactone are discussed.325 ± 0. 0%RH 23°C.Results and discussion 3 RESULTS AND DISCUSSION 3.15 0.1 1865 427 0.7:1) WPI: glycerol (2.708 cc µm/m2 day kPa) [167] and Wong in 1992 (3.3:1) LDPE HDPE EVOH (70%VOH) EVOH (70%VOH) PVDC based films Conditions 25°C. which are commercial polymer films commonly used as high oxygen barriers.1 12 0.14 0. 0%RH 25°C.4-5 Ref. 0%RH unspecified 23°C.

13 24.8° C 0-11%RH 26.2 Water vapour permeability Pure chitosan and WPI water vapour permeability values were compared and found in agreement with former research studies. PCL lowered the values of oxygen permeability even if there was no relation found between the percentage of PCL and the permeability decrease.34 0. 24.723 4. which could be an explanation to the higher permeability to oxygen.76 813. it was found that the gas barrier properties of these materials were very good in dry conditions.8° C. This took into account that PH2O [11%RH.1. When adding PCL to the films a substantial decrease of the water vapour permeability was obtained.135 0. (To compare the data a unit conversion was done.8 °C.82 110. The oxygen barrier properties of these edible polymer films indicate good potential as food packaging supplements.8°C] = 0.33 12. In this case it should be noted that the chitosan was plasticized. 0-11 %RH 25° C.46 32 .6:1) 0.315 0.70 Permeability (g. as shown in Table 4. Chitosan films WVP Composition (g/m day atm) Chitosan Chitosan plasticized Chitosan WPI: glycerol (2:1) WPI: glycerol (15:1) WPI: glycerol (2. PCL (%) 0 5 10 15 469. Table 4: Water vapour permeability for blends with different percentage of PCL and at 11%RH and 24.3:1) WPI: glycerol (1.3°C 0-88%RH 25°C 0-77%RH 25° C 0-65.86 1774. 0-50%RH 24. Table 3: Comparison between WVP-values for pure chitosan and WPI films in this study and earlier studies.day) Chitosan films WPI films 2513.00346 atm. 3.44 160. Thus.59 9. where the values are higher but of the same magnitude.1%RH this study [55] [60] this study [71] [48] [77] Conditions Reference The values confirmed the influence of the plasticizer. temperature and relative humidity.µm/m2.40 46.18-0.58 2065. 11-50%RH 23° C.Results and discussion with the data obtained by Butler [55].

µm/m2.069 (g. The PCL particles were small (in the order of 1-10 µm) indicating good miscibility (compatibility) between the two components. Considering the physical similarity between solute permeability and electrical conductivity.3 vol% (15 wt%) of PCL particles exhibited a 90% decrease in water vapour permeability compared with the pure chitosan films. The assumption in this derivation was that the content of spheres is low enough to ensure that no direct contact exist between neighbouring spheres. WPI blends with 16. ⎡ ⎤ ⎢ 3(1 − υ m ) ⎥ ⎥ Q = Qm ⎢1 + q+2 ⎢ − (1 − υ m )⎥ ⎢ ⎥ q −1 ⎣ ⎦ Eq. νm is the relative amount of permeable matrix (chitosan or WPI) in the blend. Maxwell‘s equation was used to predict data that describe the impedance of a heterogeneous system of dispersed low permeable spheres (PCL) in a permeable matrix.Results and discussion Chitosan blends with a volume fraction of 18. hence very small compared to the values of chitosan and WPI.8 vol% (15 wt%) PCL showed a 70% decrease in water vapour permeability compared to pure WPI. Maxwell derived a relationship for predicting the electrical conductivity of a system of low conducting spheres in a high-conducting matrix [168]. for this study q can be neglected. it was possible to apply the Maxwell equation to this study. Thus. 2: Maxwell’s equation Here q is the ratio of the water vapour transmission rate of PCL to that of chitosan/WPI.day) at 100% RH. The water transmission rate of PCL is 0. SEM pictures (Figure 15 and Figure 16) showed that a great amount of the PCL particles have ellipsoidal shape in chitosan and long shape in WPI films instead of spherical shape as supposed in the Maxwell equation. Figure 15: Chitosan SEM micrograph showing the morphology of the cross section surface of specimen containing 15wt% PCL particles in the chitosan matrix Figure 16: Whey SEM micrograph showing the morphology of the surface of specimen containing 5-wt% PCL particles in the WPI matrix 33 .

Ellipsoidal particles. Figure 18: Schematic diagram of the PCL particle Thus. In this case the ratio of water saturation concentration in Eq. especially if they are oriented parallel to the film surface.Results and discussion As showed in Figure 19 and Figure 21 the resulting Maxwell curves don’t fit the data obtained in this study. the model of Fricke (see Eq. Again. are more effective than spherical particles in decreasing the permeability. 3 is close to zero and it simplifies to: 34 .m are the water saturation concentrations in the dispersed and matrix components. 3) was applied to the same system. C∞.d and C∞. the similarity between electrical conductivity and solute permeability was taken into account. making it possible to use the model here. 3: Frickes equation The parameter k1 and k2 are described as: and where w is the thickness and t is the width of the less permeable particles (PCL). It was considered that the PCL particles were the low-conducting ellipsoids and that the chitosan and the WPI particles were the conducting or permeable matrix: Eq. t w Figure 17: Tortuosity of the permeant particle across the film with hydrophobic PCL particles. This is because the solute diffusion path (tortuosity) is increasing with the width-to-thickness ratio of the particles. because in this model the low conducting particles can also have ellipsoidal shapes [169].

Results and discussion ⎛ ν m ⋅ k1 ⎞ Q = Qm ⎜ ⎜ 1 -ν + k ⎟ ⎟ m 1 ⎠ ⎝ Eq. 35 . limiting its ability to swell freely by absorbing moisture. 4: Simplified Fricke equation. 600 500 day)) Q (g µm/(m 2 400 300 200 100 0 0 20 40 60 80 100 PCL content (vol%) Figure 19: Water vapour permeability Q of PCL/Chitosan. In the SEM picture (Figure 15). Figure 20 indicates that the equilibrium degree of swelling and hence the water uptake decreased drastically with increasing PCL content. In the case of chitosan/PCL blends this equation fit the obtained data for a width-tothickness ratio of the dispersed PCL phase of 150 (see Figure 19). That means that the pronounced water vapour permeability decrease was not only due to the increase in the water diffusion path. the geometrical blocking. ellipsoidal PCL particles could be observed but the aspect ratio was far less than 150. i. It seemed that the PCL particles constrained the chitosan matrix phase. (•) The solid line is from the Maxwell equation and the broken line is the fitted curve according to the Fricke model using a w/t ratio =150. It was suggested that the large decrease in water vapour permeability with increasing PCL content was due to both geometrical blocking of the diffusing water molecules and to a reduction in the swelling of the matrix.e.

0 0 0 20 40 60 80 100 PCL content (vol%) Figure 20: Equilibrium water uptake at 11% RH as a function of the PCL content in PCL/chitosan films.0 Water uptake (wt%) 6. Density measures and SEM pictures revealed good adhesion between PCL and the 36 . 4000 3500 day)) Q (g µm/(m 2 3000 2500 2000 1500 1000 500 0 0 20 40 60 80 100 PCL content (vol%) Figure 21: Water vapour permeability Q of PCL/WPI (•). The densities of PCL/chitosan and PCL/WPI systems were basically a linear function of the content of PCL. The SEM picture in Figure 16 showed that PCL particles were longshaped and small.0 2. The water vapour improvement rate for PCL/WPI was lower than for chitosan.0 4. It was believed that free swelling induced by moisture was reduced by the constraining action of the PCL particles in the matrix phase. which again seemed to be too high. even if it was still large. The solid line is from the Maxwell equation and the broken line is the fitted curve according to the Fricke model with a w/t ratio of 29 for PCL. These values do not include strongly bound water in chitosan.Results and discussion 8. indicating a good miscibility. But it was found that the w/t ratio that best fitted Fricke’s model for ellipsoidal particles was 29. which indicated that the systems were essentially free of pores.

When comparing the two materials it can be said that WPI is tougher but weaker than chitosan. 80 70 Nominal stress (MPa) 60 50 40 30 20 10 0 0 20 40 60 80 100 PCL content (vol%) Figure 22: Yield stress (•) and fracture stress (o) for PCL/ chitosan blends.6 4 1657.271 67.6 31. as shown in Figure 22. even though the values of this study were a bit lower. 64. This was also confirmed by the strain values that decreased only mildly with increasing PCL content (Figure 23).8% vol% PCL particles lowered the water vapour permeability of WPI by 70%. Yield stress Yield strain (MPa) (%) Young’s modulus (MPa) Fracture stress (MPa) Fracture strain (%) Chitosan WPI 59. The line is drawn to show the linearity. In PCL/chitosan blends. Blends with a volume fraction of 18.165 12. 3. 16.703 1. 37 .196 3.3% PCL particles lowered the water vapour transmission rate of chitosan by 90%.3 Mechanical properties The tensile test that was performed on pure materials lead to the following results: Table 5: Mechanical properties of pure chitosan and WPI films. which can be due to different drying or testing conditions.Results and discussion matrices.4 2.1.5 100 WPI data seemed to be in agreement with earlier studies [42. the fracture stress decreased linearly with increasing PCL content. 74].

38 . 4 3.05 0 0 20 40 60 80 100 PCL content (vol%) Figure 23: Yield strain (•) and fracture strain (o) for PCL/ chitosan blends. displayed that the toughness of WPI was not greatly altered by the addition of PCL.5 Nominal stress (MPa) 3 2. The fracture stress was low for WPI.1 0. as shown in Figure 24.15 0. The line is drawn to show the linearity.5 0 0 20 40 60 80 100 PCL content (vol%) Figure 24: Yield stress (•) and fracture stress (o) for PCL/WPI blends.2 Nominal strain 0. Figure 25 shows that the fracture strain decreased with increasing PCL content while the yield strain remained constant.5 2 1.Results and discussion 0.5 1 0.25 0. The absence of any large decrease in fracture stress at intermediate PCL content and the very smooth increase on yield stress. Hence all blends were considered tough even though toughness decreased as a result of the addition of PCL.

2 0 0 20 40 60 80 100 PCL content (vol%) Figure 25: Yield strain (•) and fracture strain (o) for PCL/WPI blends.2 1 Nominal strain 0. hence it is also a measure of the intrinsic stiffness of the film. as displayed in Figure 26 The data lay within the series-parallel upper and lower boundary estimates and were adequately predicted by the Halpin-Tsai relationship (Eq.Results and discussion 1.6 0.8 0. The modulus of elasticity is a measure of the force that is required to deform the film by a given amount of tension. 5: Halpin-Tsai relationship The parameters η and ζ are defined as: η= ((E d ((E d E c ) − 1) Ec ) + ζ ) and ζ = 3 0. In this study the incorporation of PCL led to a rapid decrease in stiffness for chitosan and a relatively slow increase in stiffness for WPI. 5): E = Ec 1 + ζ ⋅η ⋅ Φ 1−η ⋅ Φ Eq.5 ⋅ log⎜ ⎟ ⎛ w⎞ ⎝l⎠ The rest of the parameters in the equation: w and l are the width and thickness of the dispersed component φ and Ed are respectively the volume fraction and modulus of the dispersed component Ec is the modulus of the continuous component 39 .4 0.

The solid line represents best fits of the Halpin Tsaimodel for a width-to-thickness ratio of 1. The modified model yielded a fibre length-to-diameter ratio of 4-6. in agreement with the Young’s modulus values that were obtained.5 for the dispersed PCL phase. Thus. The stiffness index appeared to be much higher for chitosan than for WPI.1. The stiffness can be considered as the film’s resistance to distortion and. The best fit of the PCL/chitosan data yielded an average width-to-thickness ratio of 1. but the seals were brittle. to bending. clearly inconsistent with the moduli data. The width-to-thickness ratio of the PCL domains in PCL/WPI was found to be approximately 50.4 Other packaging related properties The sealability of a packaging is one of the most important properties when considering its use on wrapping or bag making equipment and. 3. For packaging materials it is also essential to know the bending stiffness of the material. The addition of PCL to chitosan reduced the bending stiffness. sealing chitosan requires alternative techniques. in particular. this is done by measuring the stiffness index. as was expected considering the tensile modulus data.5 (PCL/ chitosan) and 50 (PCL/WPI). It was found that it was impossible to seal chitosan and its PCL blends with standard heat sealing equipment.Results and discussion 2000 200 Young's modulus (MPa) Young's modulus (MPa) 1500 150 1000 100 500 50 0 0 20 40 60 80 100 0 PCL content (vol%) Figure 26: Modulus for PCL/chitosan blends (•) and PCL/WPI blends (o). but it may be explained by the fact that the PCL domains are long (Figure 16). This value seems to be rather high. 40 . when the tensile tests were performed. The fractures. the integrity of the seal is also important to the ultimate package. The Halpin-Tsai model predicts a value of unity for ζ. WPI and the WPI blends were readily sealable. Lewis and Nielsen modified the Halpin-Tsai model and using their model it was possible to obtain a perfect fit for PCL/WPI as shown in Figure 26. Young’s modulus is one measure of the inherent stiffness of a film. The dashed lines are upper and lower boundary estimates. The seal strength of the PCL/WPI blends decreased with increasing PCL content. occurred at the seal lines. of course. This is in agreement with the SEM picture (Figure 15).

the log(Nf) in the folding test was not lowered. 3. Transparency is an often-desired property for food or medical packaging materials. whereas the strain data (Figure 23 and Figure 25) showed that WPI was the tougher material. were inferior to those of chitosan. suffer from aging.1 General film characteristics The film and film forming properties are strongly dependent on the pH of the dispersion and is normally inferior close to the isoelectric point. mechanical and permeation properties were obtained as a function of aging time. Interestingly. 3. WPI contained globular particles and a ‘wavy’ surface that decreased the optical clarity. The uniform colour suggested a homogeneous dispersion of the PCL phase.2. between 220 nm and 1100 nm for pure chitosan. The physicochemical. Regarding the printing properties. including WG films. which for WG is on the order of 7. Even if the addition of PCL reduced the bending stiffness of chitosan. The homogeneous colour suggested that the PCL particles were uniformly distributed in the blends. the resistance to repeated folding was lower for WPI than for chitosan. 41 .5 [102]. In order to limit aging.Results and discussion The resistance to repeated flexure or creasing is important for the use of the films. which were measured indirectly through the folding test.[37] WG homogeneous films are obtained at pH 2-4 and pH 9-13 whereas films are of poor quality at pH 5-6 and do not form at all at pH 7-8. the bending stiffness increased and log(Nf) decreased with the addition of PCL. These results therefore suggested that PCL was not present on the surface of the WPI blends. It seems that the fatigue properties of WPI. UV-VIS spectroscopy revealed that light transmission was high. at a given pace. The optical clarity of the chitosan blends was not affected below 5% PCL. This study aims towards the understanding of the mechanisms responsible for the aging of films of plasticized vital/commercial wheat gluten cast from water-ethanol solutions at pH=4 and pH=11. In accordance to Gennadios et al. The number of cycles until failure was recorded as the flex or folding resistance. which is promising for future packaging applications. However. transparency was acceptable even for films with 15 wt% PCL.2 AGING OF WHEAT GLUTEN FILMS Biopolymers. the resistance to flexing was measured by repeated folding of the films backwards and forward. In essence. it was proved that a red dye on pure chitosan and on pure WPI yielded a solid thick line. The optical clarity of chitosan was higher than that of WPI. The impact of PCL on the printability of WPI was negligible. In agreement with data for modulus and fracture strain presented earlier. the transparency decreased and the blends became more opaque. it is important to reveal and understand the mechanisms and reasons for the time-dependent physical and chemical changes. The red dye was absorbed more rapidly in WPI than in chitosan and the general quality of the print was better on WPI. Above 5%. The effects of adding PCL to WPI were different. and between 320 and 1100 nm for pure WPI. As regards WPI the transparency also decreased gradually with increasing PCL content and here the samples turned from beige to white.

Results and discussion The choice of casting pH=4 and pH=11 WG solutions was based on the idea of having acidic and basic solutions with approximately the same net intermolecular electrostatic repulsion. According to Morel et al. the ductility dropped and the stiffness and strength increased monotonically with time and the film became brittle (see Figure 29-Figure 32). i.e. associated with the C-C-O stretch or CH2 twist of glycerol [170]. the surface that faced the Teflon-coated petri dish during casting. However.2 Mechanical properties of unaged and aged WG films The unaged b-film had a higher modulus.[79]. the ductility and stiffness of the b-film changed only marginally with time. Both the a. IR. 2000. IR spectroscopy on dehydrated samples showed that the a-film surface was richer in glycerol than the corresponding bfilm. [90] at high pH disulfide bonds are cleaved and the released thiol groups would become available for formation of new disulfide linkages. The stiffness of the a-film increased 32 times during 120 days 42 . [37] and Kayserilioglu et al. i. as indicated by a more pronounced 850 cm-1 peak. The films prepared from basic solution (hereafter referred to as b-films) appeared more homogeneous and smooth than the films prepared from acidic solutions (a-films). also indicated that there was more glycerol on the lower side than the upper side of the cast films. Figure 27: SEM micrograph of the upper faces of a. SEM revealed a undulating a-film upper surface and an uneven film thickness (Figure 27 and Figure 28).film Figure 28: SEM micrograph of upper face of b-film 3. This was in accordance with the results of Gennadios et al. Most probably the gravitational force led to that a limited amount of glycerol migrated downwards through the solidifying film during the casting process. The lower surface. The unaged a-films were very ductile. in the former IR spectrum.and b-films contained particles at the surface but also in the bulk. which covers a depth of a few microns.2. In contrast to this. Iodine staining indicated that these were starch granules.e. the same pH distance from the isoelectric point. who reported stronger films at alkaline conditions. tensile strength and lower elongation at break than the unaged a-film (see Figure 29-Figure 32). was always smooth for both films. which gives strength to the film.

The b-film always yielded and showed a post-yield strain-hardening as indicated by the increase of the stress-strain curve after yielding. 900 800 E modulus (MPa) 700 600 500 400 300 200 100 0 0 20 40 60 80 100 120 140 Time (days) Figure 30: Young’s modulus of the a-film ( ) and the b-film ( ) as a function of time.Results and discussion of storage whereas it increased only 2 times for the b-film. 43 . The corresponding values for the tensile strength were 6. 250 200 Strain at break (%) 150 100 50 0 0 20 40 60 80 100 120 140 Time (days) Figure 29: Strain at break of the a-film ( ) and b-film ( ) as a function of time. As observed in Figure 31 this occurred also for the a-film but only until 30 days of storage.5 and 2.

The changes in mechanical properties were accompanied by microstructural changes. SEM revealed that the structure of the a-film became coarser/more heterogeneous during aging (see Figure 33 and Figure 34) whereas the structure of the b-film was unchanged (see Figure 35 and Figure 36). 44 . Figure 32: Stress-strain curves of the b-film during storage time.Results and discussion Figure 31: Stress-strain curves of the a-film during storage time. We have observed the same type of time-induced coarsening for cast whey films plasticized with either glycerol or diethylene glycol (unpublished data).

3 Permeability The permeability of water and oxygen provide additional information on the differences in the structure of the different films. It is expected that if the film looses plasticizing molecules during aging that the oxygen permeability would decrease.Results and discussion Figure 33: SEM micrograph of cross-section of unaged tensile fractures a-film Figure 34: SEM micrograph of cross-section of aged tensile fractures a-film Figure 35: SEM micrograph of cross-section of unaged tensile fractures b-film Figure 36: SEM micrograph of cross-section of aged tensile fractures b-film 3.5 (1. This suggested that the loss and agglomeration of plasticizer yielded a less dense and more 45 .5 (0.1) 2.5 (0.2) 0.6) This was however not the case. The OP increased with aging (see Table 6).2. Table 6: Oxygen permeability of a-films and b-films Days of storage 0 90 Oxygen permeability (cm3 mm/(m2 day atm)) a-film 1.0) b-film 0.9 (0.

which is considered to break primarily disulphide bonds. The protein solubility of the a-film was high already after the SDS-treatment. This would explain the 46 .and b-films. Interestingly. However.g. the b-film proteins would then show a more extensive aggregation and polymerisation than those in the a-film. simply because the calliper might not have reached the bottom of every valley. increased the protein solubility significantly. the water vapour permeability increased with aging. the formation of lysinoalanine. more than one third of the dissolved proteins were “polymeric” proteins. occurs more readily at high pH [79].and b-films were small. Table 7: Water vapour permeability of a-films and b-films Days of storage 17 60 120 Water vapor permeability (g mm/(m2 day atm)) a-film 240 (28) 920 (500) 690 (520) b-film 310 (140) 1150 (530) 670 (300) The small difference in WVP is probably due to that WVP is less sensitive to the presence of voids than OP [171]. cavities and voids. and consequently an overestimation of the OP. The more heterogeneous a-film probably contained a larger amount of voids/capillaries between starch. Figure 38 showed that a significant part of the dissolved matter of the a-film was large proteins. e. This is explained by that the high surface energy of water leads to that it adheres to the surfaces of capillaries. and that the conformation of the protein molecule is more stable in acidic conditions [172]. not involving thiol groups. it is likely that the b-film experienced a larger degree of denaturation during film formation than the a-film. Only a small part of the dissolved b-film proteins were large proteins. it could not explain the large OP differences between the a. indicating that the more heterogeneous a-film structure was also more permeable.4 Protein solubility of unaged and aged WG films The SE-HPLC data revealed that the protein solubility of the unaged a-film was considerably higher than the corresponding b-film (see Figure 37).Results and discussion permeable structure. the sonication had a larger impact on the a-film than on the bfilm. which dissolved protein molecules by breaking mainly secondary bonds. In fact. Even though the undulating surface of the a-film led to an overestimation of the thickness. Consequently the concentration of oxygen (and also the WVP) increases more rapidly than the concentration of water with increasing amounts of capillaries and voids. The OP of the a-film was approximately 3 times larger than that of the b-film. reflecting its large heterogeneity. 3. Also. Since it is known that basic conditions promote protein denaturation by reducing the number of intermolecular and intramolecular S-S bonds [35].2. the subsequent sonication. glycerol and protein particles than the b-film. Following the denaturation. other aminoacid crosslinking reactions. whereas oxygen is filling the same. after the last sonication extraction. In addition. In addition the scatter in OP data (standard deviation. Table 6) increased with aging and was largest for the a-film. However the differences between the a.

during the aging period.e. It became more difficult to dissolve proteins both by attempting to break secondary bonds (SDS) and by also attempting to break disulphide bonds (sonication). 1 Normalised protein extract 0. The high ductility of the unaged a-film was most likely a consequence of its low degree of denaturation.Results and discussion low protein solubility of the b-film.8 0. ( ) aged a-film. at least partly. as observed by the overall higher protein solubility of the a-film (see Figure 37). ( ) aged b-film 47 . Whereas the protein solubility was relatively constant during aging at basic conditions it decreased significantly with time in acidic conditions.2 0 Ext 1 Ext 2 Ext 3 Extraction process Figure 37: Normalized protein solubility. which was followed by protein aggregation and protein polymerisation. Also the amount of soluble a-film “polymeric” proteins was reduced during aging. SDS (EXT 1).6 0. the ratio of the protein solubility of the film to the protein solubility of the unprocessed vital wheat gluten powder in a solution of. a consequence of the changes of the protein structure.4 0. and SDS and 150s (30s and 60+60 s) sonication treatments (EXT 3): ( ) unaged a-film. i. However. SDS-solution and 30s sonication (EXT 2). the extent of protein aggregation for the a-film did not reach the level of the b-film. Thus the a-film experienced a timedependent additional denaturation. Based on the protein solubility results. it seemed as if the reduction of the a-film ductility during aging was. ( ) unaged b-film.

The reason for the mass loss could be loss of either water ethanol or glycerol. 3. ( ) aged a-film. ( ) unaged b-film.3 0.5 Mass loss The films lost mass continuously during the entire aging period (Figure 39). 102 100 98 Mass loss (%) 96 94 92 90 88 86 0 20 40 60 80 ( ) 100 120 140 Time (days) Figure 39: Mass loss of the a-film ( ) and b-film ( ) as a function of time.6 0. SDS-solution and 30 s sonication (EXT 2). Lines only serve as a guide for the interpretation.5 0. ( ) aged b-film.2 0.4 0. The afilm lost approximately twice as much (~12%) as the b-film (~6%). 48 .Results and discussion 0.1 0 Ext 1 Ext 2 Ext 3 Polymer/monomer ratio Extraction process Figure 38: The mass ratio of soluble protein polymers and soluble protein monomers in a solution of SDS (EXT 1).2. and SDS-solution and 150 s (30 s and 60+60s) (EXT 3) sonication: ( ) unaged afilm.

the loss of volatile mass (see Figure 40).2 (pH 4) of the initial mass of the WG film. In addition.Results and discussion In Figure 40 the contents of volatile mass left in the films after various times of aging are given. as compared to the bfilms (cf. must. 176] and polarized light microscopy revealed nonswollen. the evaporation of water and ethanol. The mass increase of the paper was therefore measured as a function of time. The loss in water/ethanol was approximately 2% and 9. Figure 39 and 40). i. to a large extent. 22 days) and ~4% (pH 4.7%±0. After 22 days of storage the mass that had migrated to the paper was 1. Again.g. probably due to a more complete protein network (aggregated structure).5% for the b. 22 20 Volatile Content (wt. its evaporation rate at room temperature was negligible. 49 . The concentration of glycerol at the b-film surfaces was stable or increased slightly with time. The decrease in volatile-mass during aging. The higher ductility of the new a-films and the more brittle behaviour of the aged films. high ethanol concentration is known to retard or inhibit gelatinisation [175. Consequently the b-film retained volatile mass better than the a-film.e. Note that. The decrease at the lower side was simply a consequence of migration to the paper and the loss from the upper side was probably due to an ongoing migration of glycerol downwards into and through the film.%) 18 16 14 12 10 8 0 20 40 60 80 100 120 140 Time (days) Figure 40: Loss of volatile mass of the a-film ( ) and b-film ( ) as a function of time. The mass loss which was not attributed to volatile mass. respectively. besides water.and b-films. IR spectroscopy revealed that the glycerol concentration at the upper and lower a-film surfaces decreased with aging. It could also be that the enhanced moisture holding capacity was due to full or partial gelatinisation of the starch granules in the film caused by high pH [173. it must be concluded that these values were close enough to explain the discrepancy between the loss of mass and loss of volatile mass. e. birefringent granules in both the a. 174].2 (pH 11) and 2. have been due to differences in their residual glycerol content and volatile mass. It may however have migrated into any surrounding material. the more uniform protein network in the b-film seemed to reduce also the migration to the paper support. However. any residual ethanol left in the films were included in the volatile mass. Lines only serve as a guide for the interpretation. Considering the scatter in data. in this case the paper support.and a-films. By using the volatile mass experiment on glycerol. was lower than the actual total loss of material from the films.8%±0. it was revealed that it did not evaporate to any measurable amounts. ~2% (pH 11. 20 days) must have been due to loss of glycerol.

increase in stiffness and strength) were observed for films produced from an acidic solution (a-films) whereas films from basic solutions (b-films) were more stable. 50 . which even without clay was not aging dramatically.3.1 Mechanical properties Figure 41 and Figure 42 show that the effects of adding clay to the WG had no effect on the kinetics of aging.Results and discussion 3. In this study blending of a gluten matrix with montmorillonite was performed in order to try to reduce aging by prolonging the path for the volatile mass and migrating plasticizers.3 GLUTEN /MONTMORILLONITE FILMS As reported in the previous section.5 wt% natural clay and ( ) WG film with 4. Figure 41: Maximum stress versus aging time for: ( ) WG film. Figure 42: Strain at break (%) versus aging time for: ( ) WG film.5 wt% modified clay. ( ) WG film with 4.5 wt% natural clay and ( ) WG film with 4. ( ) WG film with 4. i. As shown in Table 8 the effects of clay on the properties of relatively new films (9 days) was modest with the b-mod-film being the strongest. The largest mechanical changes (decrease in ductility. The a-film experienced the same rate of increase in stiffness and strength and decrease in ductility as observed without clay. 3. stiffest and most brittle of them all.5 wt% modified clay. by increasing the tortuosity. was negligible.e. The effect of clay on the b-film’s aging. wheat gluten films experience a change in properties during aging.

Values within parenthesis are standard deviations.616 − X X +3 51 .72) 111.27 (10. 177].53 (8.48) 5. τ is the tortuosity factor which increases with increasing blocking efficiency of the filler.52 (0.13 (0.5 4. water permeation data was obtained.01) 67. Provided that the montmorillonite layers were completely delaminated and that the reduction in permeability was a function solely of the content of filler and its width (w) and thickness (l).97) 7. The choice of the content of clay was based on the idea of allowing the individual clay layers to orient independently and thereby to allow for a more complete exfoliation.18) 5.5 Maximum strength (MPa) 3. 6.35 (0.45 (7.63) 97.77) Young’s Modulus (MPa) 94.86) 81.05) 64.02 (0.99) 134.33 (0.51 (8.14 (8. 7.Results and discussion Table 8: Effects of clay added to WG films.01) 148. The permeability of the clay-WG film (Pc) is: Eq.5 (14. the tortuosity factor could be estimated using the following relationship: Eq.55 (8. 3.09) 2.82 (0.08) 75.5 0 4.07 (15.15) 2.60) 92. 9 days a a-nat a-mod b b-mod b-nat Clay Content (%) 0 4.77) 62. w = l 1 0. Figure 6 shows that the small amount of clay greatly effected the water barrier properties. X= 1− vm τ −1 where X is obtained from Eq. The permeability of the composite film was modelled by first considering the sheets as oblate randomly oriented exfoliated spheroids uniformly distributed within the matrix [169.84 (12.41 (0.5 4.5wt%=~1-2 vol%). 8.785 − 0.2 Permeability In order to see if the small content of clay chosen was the reason why there were no effects on the aging kinetics. Pc = Pm vm τ where Pm is the permeability of the polymer matrix and vm is the volume fraction of glycerol/protein in the composite.37) The modest change was due to the small amount of clay in the films (4.26 (9.3.97 (14.24) Fracture strain (%) 112.

Results and discussion The drop in water permeability was. since the film collapses during drying. (6) and (9)) using an aspect ratio of 100 (upper curve) and 600 (lower curve). Consequently the simple relationship derived by Nielsen [178] was used. that the sheets become oriented with the long axis parallel to the film long axis. 9. During casting it is probable. a-mod ( ) and a-nat ( ) films. (6) and (9) with an aspect ratio of 100 and 600. The tortuosity is: Eq. 7 WVP (g mm/(m2 day kPa)) 6 5 4 3 2 1 0 0 1 2 3 4 5 100 600 Clay content (vol%) Figure 43: Water vapor permeability as a function of clay content for b-mod ( ). Consequently. it appears as that the casting process is beneficial in the sense that clay sheets orient with their long axis parallel to the film long axis. (6)-(8)) using from an aspect ratio of 100 (upper curve) and 600 (lower curve). b-nat ( ). τ = 1+ w v clay 2l Figure 43 shows that most experimental water vapor permeabilities lie within the curves corresponding to eqs. In addition even though tactoids can be observed in some systems it appears that exfoliated clay structure is a common feature in all systems. most clay sheet widths lie within 100-600 nm. larger than predicted even with a clay sheet aspect ratio as high as 600 (Figure 43). The decrease in the permeability of the b-mod-film (in bold) was the only significant trend within a 95% confidence limit (Students-t test). Solid lines represent the Fricke model (eqs. The dashed lines correspond to the model of Nielsen (eq. 52 . the corresponding aspect ratios are consequently 100-600 nm. in general. As observed in Figure 44 and Figure 45. and provided the sheets are exfoliated. which is based on that all clay sheets are oriented with the long axis parallel to the film long axis. Indeed Figure 44 and Figure 45 indicate that the orientation of individual sheets is not completely random.

were present for all but the b-nat-film. 53 . which explains why the b-nat-film had the highest stiffness and strength. Nevertheless water vapor permeation indicated that also this film contained exfoliated structures. (d) b-nat (4.3 X-ray and TEM TEM revealed that all films contained exfoliated clay (Figure 44 and Figure 45).5 wt% clay). indicative of the presence of clay tactoids.3.5 wt% clay). Figure 46: XRD spectra of (a). 2θ~5° pure modified clay). The tactoids were swollen as shown by the shift of the d-001 reflection to smaller angles. TEM and XRD indicated that the b-nat-film was almost fully exfoliated.Results and discussion 3. (b) wheat gluten pH=11. (c) wheat gluten pH=4.5%) and (e) a-nat (4. Evidently the pH 11 solution yielded the most efficient delamination of clay. In contrast TEM showed large tactoids in the a-nat-film and a large sharp d-001 reflex in the XRD spectrum. Figure 44: Transmission electron micrograph of the b-nat-film (4. Figure 45: Transmission electron micrograph of the a-mod-film (4.5%). However XRD (Figures 46 and 47) showed that the d-001 reflections (2θ~8° pure natural clay. natural clay.

Results and discussion Figure 47: XRD spectra of (a). The fact that the kinetics of aging was unaffected. i. 54 .e. aggregation and protein polymerization during storage. (b) wheat gluten pH=11. modified clay. (c) b-mod (4. Indeed the content of volatile mass at short times (9 days) was higher in the films with natural clay and the films with modified clay had a smaller volatile content than the clayfree film. observed for the a-film in the previous study determined the rate of loss of volatile mass and glycerol migration or phase separation. Instead it seemed that the changes of the protein structure. time-dependent denaturation.5%) It may be speculated that the excess of hydroxyl groups interact with the sodium-rich clay surface causing the sheets to delaminate.5%) and (d) a-mod (4. This was certainly due to a smaller affinity to moisture than experienced by the natural clays. indicated that the aging was primarily not diffusion-controlled. despite that the clay sheets did impose a large geometrical impedance in the film. Nevertheless the largest decrease in water vapor permeability was observed for the b-film with modified clay (the b-mod-film).

The addition of PCL to whey protein isolate (WPI) and chitosan reduced the water vapour and oxygen permeabilities. which was not altered by the addition of PCL. PCL reduced the Young’s modulus in chitosan but increased it in WPI. • 55 . which could be promising for future packaging applications. thickness and surface smoothness were higher for the alkaline films. Chitosan films were found to be difficult to heat sealable. The addition of PCL had different effects on the mechanical properties of the two materials. than WPI films. Surprisingly WPI. however. The composites were. in turn. • • • • Aging of wheat gluten films • The aging properties of the wheat gluten cast films depended strongly on the pH of the solution. too water sensitive to be used in direct contact with water. Chitosan films had a high folding endurance. but they had a low sealing strength. The WPI films were sealable.Conclusions 4 CONCLUSIONS Films of chitosan or whey blended with PCL • Poly (ε-caprolactone) (PCL) was ‘compatible’ with both whey and chitosan and it was possible to produce films of these polymer blends. was due to a lower degree of protein aggregation/polymerisation as compared to the basic film. The homogeneity. The increased film homogeneity of the alkaline films yielded enhanced oxygen barrier properties. The gluten films made from acidic solution experienced a larger loss of volatiles and migration of glycerol to the paper that supported the films during the storage. Chitosan showed lower water vapour permeability than WPI. while alkaline films remained more stable during the storage time. The more aggregated protein in the alkaline films seemed to be more effective in retaining the plasticizing molecules within the film. even if it had higher fracture strain than chitosan. Chitosan films showed higher strength and stiffness. The time-induced decrease of fracture strain of the acidic film was due to the loss of plasticizing molecules (water. which. High folding endurance indicated good fatigue properties for this material. ethanol and glycerol). but lower elongation at break. had much lower resistance to folding. Gluten films made with acidic solutions experienced a significant increase in fracture stress and Young’s modulus. using casting technique.

This can serve as a base for further research.Conclusions Composite films of wheat gluten and montmorillonite • It was possible to produce films of wheat gluten with montmorillonite clay by solution casting technique. which should be directed towards maintaining the oxygen barrier properties while improving water sensitivity. The water vapour permeability was lower for films with the less hygroscopic modified montmorillonite. The water vapour permeabilities indicated that the clay particles effectively acted as geometrical obstacles in the wheat gluten matrix. The montmorillonite clay showed a higher degree of exfoliation in films cast from alkaline solution than from acidic solution. The clay did not affect the aging rate of the films made from acidic and basic solution. water vapour barrier and mechanical properties. 56 . • • This study shows the potential of these biopolymers as blending materials for producing films by casting method.

57 . Prof... generosity.. his encouragement. Maria E. for their encouragement at the beginning of my polymer career and for being the origin of this long journey. optimism. Annika L. Roland K. Henrik U. Anders Hult. Prof. Sigbritt Karlsson are thanked for their leadership and for making this department scientifically stimulating. especially Maggan.. Anders B. Kamyar (R&R at Beckers) and Eugenia (Photoshop Queen and promising artist) for being great friends and a second family to me. Gedde for his kindness and for accepting me in his group. Bengt Stengberg and Prof. Jose Ramon Sarasua. Anders. Past and present members of Ulf’s Group: Mikael Krook. Richard O. Linda F. I am grateful to Mari Jose Suarez.. I wish also thank all my former colleges at Packforsk. Gunnar K.. kindness and the good moments we spent together (and more to come). I also thank Professor Ulf W. the fika-rum people. Associate Professor Mikael Hedenqvist. remember that we could have been a promising artistic ‘magdans’ couple (it’s never too late). John. especially Prof. for taking care of me and being a beautiful person and Fran for having the guts to take over Kamyar’s place as the only male in our Friday’s girl lunches. thank you for making the department a nice and enjoyable place to work. in Basque Country. Natalia (honeeeeeeey!) for her funny craziness.. Jinglan. Associate Prof.. Prof. Maria and Viktoria for their help with all administrative matters and their kindness. former room mates as Margaretha. encouragement and interesting talks about this work. for his support... Ann-Christine Albertsson. Ana for the nice times in the gym and in the dancing courses.”. Alessandro M.. his help and his sense of humour. Mats Johansson and Prof. Västsvenska Lantmännens Research Foundation (VL-stiftelsen) is acknowledged for financial support. Martin Svensson and Kurt Villwock at Lantmännen are thanked for all the valuable discussions during ‘gluten meetings’. I will always remember the good times with the rest of Polymer’s girl power as Clara S. and Sung-Woo. Johan. among others. I would like to mention: the ‘Spanish group’ for giving me the support and courage I needed in every moment. Eva Malmström are thanked for always being helpful and so nice and the administrative personnel of the department. David N. Bruska A. Jonas (sorry for leaving the MOCON nightmare to you) and to all of those of you I have not forgotten to mention but I didn’t have the space to do it. now part of STFI-Packforsk.Acknowledgements 5 ACKNOWLEDGEMENTS First I would like to express my very sincere gratitude to my supervisor. Peter Baeling. Carol... for his guidance. I would like to show my gratitude to all the people who made the work at the Polymer department a great experience for me. I thank people from the Engineering Faculty in Bilbao (University of Basque Country) for their kindness. Bereket N. Inma Angulo and my very good friend Laura Dominguez (hejsan!) at the Research Institute Gaiker. Viktoria B. I am indebted to Mikael Gällstedt for helping me so much. laugh and late chats. Janis R. especially Marie for her optimisms. for their kindness and support in both work and personal matters. Stefanie R. for his help. always finding time to answer and discuss my questions about “gluten et al.. I also thank Inger for her valuable assistance in every kind of matters and Ove for being an authentic help with all the technical problems I had.

for being so sweet. This work is dedicated to Pär for his love and dedication.Acknowledgements My friends outside the department are also thanked. for being the best that have ever happened to me. 58 . and for always being ready to help me. My friends in Bilbao are thanked for being there. maite zaituztet! Also warm thanks to Karin and Rolf and the rest of the family Weström for taking so well care of me and for being to me a real family. no matter the distance. for never remember the word biopolymer and for making me enjoy the trips home. My warmest gratitude goes to my family for their support and their love. unconditional friend and my moral support here in Stockholm. especially Natalia H.

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