DEVELOPMENT AND CHARACTERIZATION OF FLEXIBLE FILMS MADE OF SUGAR BEET LIGNOCELLULOSE By Zhu Shen A DISSERTATION Submitted to Michigan State University i n partial fulfillment of the requirements for the degree of Packaging - Doctor of Philosophy 2015 ABSTRACT DEVELOPMENT AND CHARACTERIZATION OF FLEXIBLE FILMS MADE OF SUGAR BEET LIGNOCELLULOSE By Zhu Shen The abundance of biological low value sugar beet lignocellulose (SBL) after sugar extraction makes it a potential raw material for biodegradable flexible packaging films. This study attempts to develop a sustainable film from SBL with potential novel appli cations in packaging. Firstly, the dissertation investigated the effects of chemical pretreatments on the structure and properties of SBL. The study then is focused on improving SBL properties and demonstrating additional applications by developing flexibl e films with antimicrobial activity . The f irst objective involved a two - step pretreatment technology to improve the tensile, barrier, and thermal properties of SBL based films. Chemical analyses were used to identify and characterize sugar beet lignocellul ose (SBL). Cellulose content of SBL was increased by sulfuric acid pretr eatment and bleaching from 22.2% to 80.4 %. This was attributed to removal of some lignin, pectin, and hemicelluloses as evidenced by non - destructive spectroscopic analysis. FT - IR spect roscopic analysis of fibers confirmed that the acid pretreatment led to partial removal of hemicelluloses and lignin from the structure of SBL. XRD results revealed that this acid pretreatment resulted in increased crystallinity of the SBL fibers. The ther mal gravimetric analysis (TGA) was used to demonstrate the increased thermal stability after acid pretreatment due to the increased contribution of stable cellulose crystal s . TGA curves after sulfuric acid pretreatment demonstrated a two - stage thermal degr adation behavior due to the introduction of sulfated groups during the sulfuric acid hydrolysis process. These improvements after pretreatments are promising for the use of acid pretreated SBL as a source of bio - based lignocellulose to reinforce polymer co mposites and high value products from agricultural residues. In the second objective, composite films of pretreated SBL and polyvinyl alcohol (PVOH) plasticized with sorbitol were successfully developed. Film - forming dispersion s of different ratios of SBL to PVOH (100/0, 75/25, 50/50, 25/75) were cast at room temperature. Films were evaluated for physical, tensile, water barrier, and thermal properties. The addition of PVOH gave significantly ( P 0.05) higher elongation at break ( 12.45 % ) and lower water vapor permeability (1.55 g s - 1 m - 1 Pa - 1 ) than that of control . The ESEM results showed that the compatibility of SBL 50/PVOH 50 was better than those of other composite films. These results suggest that when taking all the st udied variables into account, composite films formulated with 50% PV OH are most suitable for various packaging applications. In the third objective, SBL films were developed with cedarwood oil (CWO) and t ung oil to improve the water barrier properties and antimicrobial activity. The m icrostructure of the composite films was characterized through Fourier transform infrared spectroscopy (FTIR). The results showed that c edar and t ung oils can be used to decrease water vapor permeability of SBL films by more than 25 %; the contact angle to water of SBL film was increased by 134% when incorporated with 15 % w / w of t ung oil. These results showed the hydrophobicity of the SBL films was increased by adding oils. Antimicrobial properties of the films were im pr oved by the introduction of 5 % w/w CWO in the film. Results from antimicrobial tests revealed that the Inhibition Index of cellulosic films increased to 20 % by incorporating 20 % w / w of CWO. The introduc tion of oils showed no obvious change i n thermal properties. iv ACKNOWLEDGEMENTS Great thanks go to Professor Donatien Pascal Kamdem , mentor and friend, who introduced me to science and research. Thank you for giving me this great opportunity to become your student and enjoy your endless guidance, advice, and encouragement through my entire graduate studies. I hope this dissertation is an acceptable return on your investment in me as a scholar. I extend my thanks to my dissertation committee, Professor Karen Chou, Professor Laurent M. Matuana , and Professor Susan Selke for their comments, questions, and guidance throughout this project and my graduate school career. Thank you also for showing me, by example, how to be a good scientist. My accomplishments would not have been s o without the help of Dr. Xing Cheng, Dr. Yining Xia, Dr. Zhenglun Li, and Mehran Ghasemlou . I also owe a depth of gratitude to my colleagues Muyang Li, Hamoud Abdulaziz Alnughaymishi , Lei Wang, and Peng Gao for their friendship and assistance in lab. I w ould like to thank Dr. Tom M. Johnson and Mrs. Jane S. Johnson for offering me free housing and helping me adapt living in the United State s . I thank my wife Mingwei Yan for without her editing, insight, encouragement, love and support, I would be empty, w ithout form, and void. Finally, I dedicate this project to my grandfather, Zhenguang Shen; my parents, Guozh eng Shen and Yaping Zhou; my pa rents in law, Xiping Yan and Shanhua Wei; my uncle and aunt , Xiaoping Shen and Mingxia Ding, who provided a support s ystem that I could not do without. v TABLE OF CONTENTS LIST OF TABLES ................................ ................................ ................................ ................. viii LIST OF FIGURES ................................ ................................ ................................ ................. ix Chapter 1 Introduction ................................ ................................ ................................ ............... 1 1.1. I ntroduction ................................ ................................ ................................ .......................... 1 Chapter 2 Literature Review ................................ ................................ ................................ ...... 5 2.1. Sugar beet lignocellulose (SBL) ................................ ................................ ........................... 5 2.2. Composition and structure of SBL cell wall ................................ ................................ .......... 5 2.2.1 Chemical composition of SBL cell wall ................................ ................................ .......... 5 2.2.2. Structure of cell wall of SBL ................................ ................................ ......................... 7 2.3. Isolation and characterization of components from SBL ................................ ....................... 7 2.3.1. Isolation and characterization of pectin from SBL ................................ .......................... 9 2.3.2. Isolation and characterization of hemicellulose from SBL ................................ ............ 10 2.3.3. Isolation and characterizat ion of cellulose from SBL ................................ ................... 10 2.4. Potential application of SBL ................................ ................................ ............................... 13 2.4.1. Food ingredients ................................ ................................ ................................ .......... 13 2.4. 2. Sources as biofuel ................................ ................................ ................................ ........ 14 2.4.3. Sources for polymers and composites ................................ ................................ .......... 14 2.5. Lignocellulosic flexible film in packaging ................................ ................................ .......... 17 2.5.1. Tensile properties of film for packaging ................................ ................................ ....... 17 2.5.2. Factors affecting tensile properties of a film ................................ ................................ . 19 2.5.2.1. Effect of pulping process ................................ ................................ ....................... 19 2.5.2.2. Effect of moisture content ................................ ................................ ..................... 20 2.5.2.3. Effect of particle size ................................ ................................ ............................ 21 2.5.2.4. Effect of additives ................................ ................................ ................................ . 21 2.5.2.5. Effect of blending with other polymers ................................ ................................ . 22 2.5.3. Moisture barrier property of lignocellulosic film ................................ .......................... 22 2.5.4. Factors affecting water permeability of lignocellulosic film ................................ ......... 24 2.5.4.1. Effects of chemical structure and morphology ................................ ....................... 24 2.5.4.2. Effects of temperature and relative humidity ................................ ......................... 26 2.6. Flexible film based on biomass ................................ ................................ ........................... 27 2.6.1. Mechanical properties of polymers based on biomass ................................ .................. 27 2.6.2. Antimicrobial activity of flexible film and coating ................................ ....................... 30 2.6.2.1. Bacteriocins ................................ ................................ ................................ .......... 31 2.6.2.2. Enzyme ................................ ................................ ................................ ................. 32 2.6.2.3. Plant extracts ................................ ................................ ................................ ......... 33 Chapter 3 Isolation and Characterization of Sugar Beet Lignocellulose (SBL) ......................... 35 vi 3.1. Introduction ................................ ................................ ................................ ........................ 35 3.2. Materials and methods ................................ ................................ ................................ ........ 37 3.2.1. Materials ................................ ................................ ................................ ..................... 37 3.2.2. Preparation of SBL ................................ ................................ ................................ ...... 38 3. 2.3. Gravimetric method to assess chemical composition of SBL ................................ ........ 38 3.2.3.2 . Holocellulose content ................................ ................................ ............................ 39 - cellulose content ................................ ................................ ................................ 39 3.2.3.4. Hemicellulose content ................................ ................................ ........................... 39 3.2.4. Fourier transform infrared spectroscopy (FTIR) ................................ ........................... 40 3.2.5. X - ray diffraction (XRD) ................................ ................................ .............................. 40 3.2.6. T hermogravimetric analysis (TGA) ................................ ................................ ............. 41 3.3. Result and Discussion ................................ ................................ ................................ ......... 41 3.3.1. Characterization of SBL ................................ ................................ .............................. 41 3.3.2. Crystal Aggregation of SBL ................................ ................................ ......................... 42 3.3.3. FTIR spectroscopy analysis ................................ ................................ ......................... 44 3.3.4. Thermal Degradation Behaviour ................................ ................................ .................. 47 3.4. Conclusion ................................ ................................ ................................ ......................... 49 Chapter 4 Development and Characterization of Sugar Beet lignocellulose/Poly (vi nyl alcohol) Composite Film via Simple Casting Method ................................ ................................ ............. 51 4.1. Introduction ................................ ................................ ................................ ........................ 51 4.2. Materials and methods ................................ ................................ ................................ ........ 53 4.2.1. Materials ................................ ................................ ................................ ..................... 53 4.2.2. Prepa ration of SBL ................................ ................................ ................................ ...... 53 4.2.3. Chemical composition of SBL ................................ ................................ ..................... 54 4.2.4. Preparation of films ................................ ................................ ................................ ..... 5 4 4.2.5. Film characterization ................................ ................................ ................................ ... 55 4.2.5.1. Film thickness ................................ ................................ ................................ ....... 55 4.2.5.2. Film density ................................ ................................ ................................ .......... 56 4.2.5.3. Water vapor permeability ................................ ................................ ...................... 56 4.2.5.4. Mechanical properties ................................ ................................ ........................... 57 4.2.5.5. Thermogravimetric (TGA) analysis ................................ ................................ ....... 58 4.2.5.6. X - ray diffraction (XRD) ................................ ................................ ....................... 58 4.2.5.7. Film microstructure ................................ ................................ ............................... 58 4.2.5.8. Statistical analysis ................................ ................................ ................................ . 59 4.3. Results and discussion ................................ ................................ ................................ ........ 59 4.3.1. Chemical composition of SBL ................................ ................................ ..................... 59 4.3.2. Appearance and physical properties of the film ................................ ............................ 59 4.3.3. Water vapor permeability (WVP) ................................ ................................ ................ 60 4.3.4. Mechanical properties ................................ ................................ ................................ .. 61 4.3.5. Thermal stability assessment by TGA ................................ ................................ .......... 65 4.3.6. Assessment of compatibility of blend films by XRD ................................ .................... 66 4.3.7. Surface morphology of blend films ................................ ................................ .............. 68 4.4. Conclusion ................................ ................................ ................................ ......................... 68 vii Chapter 5 Antimicrobial Activity of Sugar Beet Lignocellulose films containing Tung and Cedarwood essential oils ................................ ................................ ................................ ........... 71 5.1. Introduction ................................ ................................ ................................ ........................ 71 5.2. Mater ials and methods ................................ ................................ ................................ ........ 73 5.2.1. Materials ................................ ................................ ................................ ..................... 73 5.2.2. Oil screening for antimicrobial activity ................................ ................................ ........ 74 5.2.3. Films preparation ................................ ................................ ................................ ......... 74 5.2. 4. Film characterization ................................ ................................ ................................ ... 75 5.2.4.1. Film solubility in water ................................ ................................ ......................... 75 5.2.4.2. Film thickness ................................ ................................ ................................ ....... 75 5.2.4.3. Moisture content and density ................................ ................................ ................. 75 5.2.4.4 Water vapor permeability (WVP) ................................ ................................ ............... 76 5.2.4.5. Tensile properties of films ................................ ................................ ........................ 77 5.2.4.6. FTIR spectroscopy ................................ ................................ ................................ .... 78 5.2.4.7. Thermogravimetric Analysis (TGA) ................................ ................................ ......... 78 5.2.4.8. Differential scanning calorimetry (DSC) ................................ ................................ ... 79 5.2.4.9. Contact angle measurement ................................ ................................ ...................... 79 5.2.4.10 . Antimicrobial activity of films ................................ ................................ ................ 79 5.2.4.11. Statistical analysis ................................ ................................ ................................ ... 81 5.3. Results and Discussion ................................ ................................ ................................ ....... 81 5.3.1. Physical properties of the films ................................ ................................ .................... 81 5.3.2. Water vapor permeability and Wettability properties ................................ ................... 82 5.3.3. Mechanical properties of the films ................................ ................................ ............... 83 5.3.4. Structural properties ................................ ................................ ................................ .... 86 5.3.5. Thermal properties of the films ................................ ................................ .................... 88 5.3.6. Antibacterial activity ................................ ................................ ................................ ... 92 5.4. Conclusion ................................ ................................ ................................ ......................... 95 Chapter 6 General Conclusions and Future Work ................................ ................................ ..... 97 6.1 General conclusions ................................ ................................ ................................ ............ 97 6.2 Future work ................................ ................................ ................................ ......................... 98 APPENDIX ................................ ................................ ................................ .............................. 99 REFERENCES ................................ ................................ ................................ ...................... 103 viii LIST OF TABLES Table 1 DP of native wood and non - woody celluloses after nitration using the viscometric method. ................................ ................................ ................................ ................................ ..... 12 Table 2 Mechanical properties of film from biomass at 50 % RH and 25 o C ............................. 23 Table 3 Water vapor permeability (WVP) of biomass based film at 25 o C ................................ 25 Table 4 Chemical composition of SBL powders at different stages ................................ ........... 42 Table 5 Main functio nal groups ................................ ................................ ................................ 45 Table 6 Effect of oil concentration on the physical and tensile properties of SBL films ............. 85 Table 7 TGA and DTG Curve Parameters of the Films ................................ ............................. 91 Table 8 Antimicrobial activity of SBL films incorporated with CWO ................................ ....... 94 ix LIST OF FIGURES Figure 1 Molecular structure of cellulose ................................ ................................ .................... 6 Figure 2 Structure of SBL from plant to the fiber (Pos tek et al., 2011; Xiao and Anderson, 2013) ................................ ................................ ................................ ................................ ................... 8 Figure 3 Strain - stress curve ................................ ................................ ................................ ...... 19 Figure 4 X - ray diffraction patterns of RSBL, ASBL, and DSBL fibers. Cellulose whisker was running as control ................................ ................................ ................................ ..................... 43 Figure 5 FT - IR spectra of RSBL, ASBL, and DSBL fibers. Cellulose whisker and lignin powder were running as control ................................ ................................ ................................ ............. 46 Figure 6 Correlation between the lignin content determined by gravimetric method and FTIR peak intensity ratio (1) I 1550/I1315 , (2) I 1550/1157 ................................ ................................ ............. 47 Figure 7 TG and DTG curves of RSBL, ASBL, and DSBL fibers. Cellulose whisker, xylan, and lignin powder were running as control ................................ ................................ ....................... 48 Figure 8 Water vapor permeability (WVP) of the different composite films made of sugar beet lignocellulose (SBL) and poly (vinyl alcohol)(PVOH) a, b and c are different letters represent ........................ 60 Figure 9 Tensile strength (A), elongation at break (B) and Elastic modulus (C) of the different composite films made of sugar beet lignocellulose (SBL) and poly (vinyl alcohol)(PVOH) Note: a, b and c are different letters represent significant differences (p < 0.0 5) between the means ................................ ................................ ................................ ......... 63 Figure 10 TGA (a) and DTG (b) curves for the sugar beet lignocellulose (SBL) a nd poly (vinyl alcohol)(PVOH) and different composite films made of SBL and PVOH. ................................ . 64 Figure 11 X - ray diffractograms of SBL/PVOH c omposite films (a) SBL/PVOH ratio of 100/0 (v/v), (b) SBL/PVOH ratio of 75/25 (v/v), (c) SBL/PVOH ratio of 50/50 (v/v), (d) SBL/PVOH ratio of 25/75 (v/v) and (e) SBL/PVOH ratio of 0/100. ................................ .............................. 67 x Figure 12 Typical scanning electron micrographs of SBL/PVOH composite films (a) SBL/PVOH ratio of 100/0 (v/v), (b) SBL/PVOH ratio of 75/25 (v/v), (c) SBL/PVOH ratio of 50/50 (v/v), and (d) SBL/PVO H ratio of 25/75 (v/v). ................................ ................................ . 69 Figure 13 Water vapor permeability (WVP) of the SBL films including different concentration of cedarwood oil (CWO) and Tung oil. ................................ ................................ ..................... 86 Figure 14 FTIR spectra of the films incorporated with different concentration of (a) CWO and (b) Tung oil. ................................ ................................ ................................ ................................ ... 87 Figure 15 Typical results of TGA and DTG curves of SBL films including different concentration of CWO ................................ ................................ ................................ .............. 89 Figure 16 Typical results of TGA and DTG curves of SBL films including different concentration of Tung oil ................................ ................................ ................................ .......... 90 Figure 17 Inhibition Index of SBL films incorporated with various concentration of CWO ...... 93 Figure 18 Typical DSC thermograms of SBL films i ncorporated with different concentration of (a) CWO and (b) Tung oil ................................ ................................ ................................ ....... 100 Figure 19 Volume kinetics of water droplets deposited on surface of SBL films with different levels of CWO and Tung oil ................................ ................................ ................................ .... 101 Figure 20 Petri dishes circular disks of films incorporated with SBL films incorporated with different contents of CWO showing the inhibitory zone against three types of bacteria ........... 102 1 Chapter 1 Introduction 1.1. Introduction Owing to their aesthetic appearance, light weight, low cost, and stable properties, petroleum - based packaging materials are widely used in the packaging industry. It is reported that 30 % of packaging materials are made of plastics ( Pearson , 2009 ) . However , with the volatility in oil prices and environmental concerns on the use of petroleum - based materials, consumer s will likely prefer to switch to eco - friendly, green, sustainable, and biodegradable relatively low cost materials. Attributes of eco - friendly sustainable products include the use of (1) raw materials from biological and other sustainable sources not in competition with the food supply, (2) low energy consumption during processing, (3) low to neutral carbon footprint, (4) low density, (5) low to negligible release of pollutants with negative impacts on h uman and environment during production, (6) efficient utilization, recyclable, compostable, and biodegradable. For decades, numerous investigations and studies have been made on using wood as a bi o - refinery platform to extract cellulose and hemicellulose for the production of flexible packaging alternative s (Björkman, 1956; Epstein et al., 1976; Lee et al., 2009) . Today, competition from different sectors, suc h as the construction industry , furnitu re manufacturing, solid wood packaging, and the pulp and paper industry, has made this option less cost competitive. Several other potential bio - products from agricultur al residues and industries are available including starch from rice and potatoes (Piyada et al., 2013) , corn (Ghasemlou et al., 2013) ; proteins from peas and peanut s (Sun et al., 2013) , straws (Ruiz et al., 2013) , sugarcane (Sun et al., 2004a) , sugar beet residue (Dinand et al., 1996) , banana leaf (Reddy and Yang, 2005) , coconut, and j ute (Phan et al., 2006) . Although some of the above mentioned raw materials provide advantages 2 from a carbon footprint perspective, some are not cost competitive due to their primary uses and importance in the human food chain. Readily available low value agricultural and forestry residues with a high content of cellulose, hemicellulose, protein and pectins are good candidates for the extraction and production of raw materials to be used in the manufacturing of sustainable packaging materials. One of this is sugar beet ( Beta vulgaris ) residue , which is also known as sugar beet lignocellulose (SB L ). SB L is a by - product generated from the production process, which involves grinding, refining, and washing off the sugar extract. It is reported that about 32.7 million tons of sugar beet was produced in the USA in 2012 (Magana et al., 2011) . Most of the SB L generated after the extraction of sugar is used for low value animal food and energy production through combustion or fermentation to generate alcohol. SB L co ntains about one - third cellulose, one third hemicellulose s , one third pectin in primary cell walls , and a low amount of lignin (Dinand et al., 1996) . The fractionation of lignin, hemicellulose, and cellulose from SB L tissues may likely require less energy and fewer chemicals compared to woody material , providing a potential material for the manufacture of sustainable, bio - renewable and biodegradable flexible packaging as well as extra income to beet growers and the sugar beet industry. Overall if successfu l, this will also reduce the production cost of sugar. Flexible packaging is made of flexible materials such as paper or other easily yielding materials that when filled and closed, can readily change shape. The flexible film industry is one of largest sec tors of the business in US and in the world. Some efforts have been reported on the use of SB L for the formulation of flexible film . Dufresne et al. ( 1997) noted that cellulose extracted 3 from SB L can be used to make film with tensile strength below 10 MPa. This mechanical property was lower in comparison to most conventional packaging materials, such as polyethylene (PE) or polypropylene (PP). The low tensile strength did not promote the use of SB L film in packaging. Leitner et al. ( 2007) investigated a thin film with tensile strength higher than 100 MPa made from SB L nano - cellulose. A serial of chemical and high pressure pretreatment was used to extract less than 10% nano - cellulose from SB L resulting in relatively high cost, which is a we ll - known limit ing factor for the development of new materials in packaging (Auras et al., 2004) . The use of SBL in the manufacture of flexible film requires a technology that will result in a cost competitive and environmental ly acceptable film from the pr ocessing perspective. As a bio - based polymer, films made from SBL are like ly to be sensitive to environmental conditions. The physical and mechanical properties of these films are not adequate for many applications (Shi and Dumont, 2014) . Their hydrophilic character also promotes the growth of several microorganisms which can be a menace to human health and food safety. As a result, several studies have been carried out to increase the hydrophobicity and antimicrobial activity of lignocel lulosic based material s . One simple and economic al way to improve the properties of biopolymer s is introduce a synthetic polymer or antimicrobial agent into it (Espitia et al., 2014) . For example, polyvinyl alcohol (PVOH), as a non - toxic and water - soluble synthetic polymer with excellent film - forming, chemical resistance, and good biodegradability, has been widely utilized for the preparation of blends and composites with several natural, renewable polymers (Chiellini et al., 2003) . Besides this, various or ganic lipids that naturally occur in wood and plants contain saturated and unsaturated aromatic compounds, such as terpenes, monoterpenes, thujone, polyphenols, tannins, alkene s , flavonoid s , cedrol, and phenolic acids (F ernánde z Pan 4 et al. 2013; T r nç and Meier 2013) . Most of t hese compounds are typical ly hydrophobic with a wide range of antimicrobial properties (Seydim and Sarikus 2006) . This dissertation study was intended to investigate the possibility of producing workable and antimicrobial films from SBL with the following steps : (1) Develop an economical and environmentally sound pretreatment method for the manufacturing of flexible packaging film from SBL with acceptable properties. (2) Evaluate the effec t of pretreatment on the chemical composition of SB L including lignin, hemicellulose, pectin, and cellulose content. (3) Introduce PVOH into the SBL film matrix and evaluate the effects of the addition. (4) Incorporate cedarwood oil (CWO) and t ung oil into SBL films to improve the hydrophobicity and antimicrobial activity. 5 Chapter 2 Literature R eview 2.1. Sugar beet lignocellulose (SB L ) Sugar beet ( Beta vulgaris) , is used to extract sugar and the byproduct after the removal of sugar is known as sugar beet lignocellulose (SB L ). SB L produced by the sugar beet refinery is mainly used for animal feeding and energy cogeneration (Warren et al., 2008) . The USA is one of the largest producers of sugar beet. It is estimated that about 31.9 trillion tons of sugar beet was produced in the USA in 2012 and will continue to grow due to high sugar demand (Kracher et al., 2014) . Once harvested, sugar beets are water washed to remove soil and dirt, and then sliced into very thin strips to facilitate the sugar extraction with water, heat and compression. The remaining pulp must be dried in order to store for later animal feeding (Gurbuz and Coskun, 2011) . In the p ast decade, the chemical composition of SB L has been well documented. SB L contains relatively low lignin content ra nging from 2 to 6 %, which may facilitate the isolation of carbohydrates (Dinand et al., 1999; Sun and Hughes, 1998) . The carbohydrates in the cell walls are roughly one - third cellulose, one - third hemicelluloses and one - third pectin (Dinand et al., 1996; Dinand et al., 1999) . 2.2. Composition and structure of SB L cell wall 2.2.1 Chemical composition of SB L cell wall The major components of the SB L cell wall are several carbohydrates including cellulose, hemicellulose, and pectin. The composition and percentages of these three major polymers vary with age, cultivar, stage of growth, soils, climate, ways of harvest and conditions of SBL processin g (Mojtahedi and Mesgaran, 2009) . 6 It is reported t hat cellulose makes up about 30 % of the dry weight of the SB L cell (Dinand et al., 1996; Dinand et al., 1999) . This line carbohydrate consist s of a linear chain of - D - glucopyranose units linked by glucoside bond s between their C - 1 and C - 4 hydroxyl groups. Fig ure 1 shows the structure of cellobiose molecule, consisting of long chain glucose linked together by hydrogen bonds and Van der Waals forces (Pérez et al., 2002) . These long chains of cellobios e constitute the microfibrils, which are group ed together as cellulose fiber. Within cellulose macromolecules, there are a number of intra - and intermolecular hydrogen bonds. These hydrogen bonds result in various ordered crystalline region s , called as crystalline cellulose (Park et al., 2010) . Besides these ordered celluloses, there are also a small percentage of cellulose chains known as amorphous/non - order, as thus there are bundles of disordered region s besides the crystalline regions, wh ich is often referred to as amorphous (O'S ullivan , 1997) . In t hese area s , the cellulose chains are randomly oriented in a spaghetti - like arrangement leading to lower properties, such as density and strength, in these domains (Li et al., 2009) . Fig ure 1 Molecular structure of cellulose In most biomass, such as SB L , the cellulose microfibrils are lin k ed to hemicelluloses, pectin, and lignin. Hemicelluloses are a series of complex carbohydrate polyols which make up 25 - 30 % of the t otal dry weight. Compared to cellulose, hemicellulose is a carbohydrate with lower molecular weight. The major hemicellulose s type in SB L is reported to be arabinan, a branched polymer - 1,5 - linked L - - 1,3 - linked L - arabinose (Sun and Hughes, 7 1998) , along with galactose, xylose and rhamnose (Kobayashi et al., 1993) . Pectin is a family of oligosaccharides and polysaccharides that makes up about one third of the cell wall dry weight of SB L (Phatak et al., 1988) . The h ighest concentration of pectin is found in the middle lamella between adjoining cells , with gradual decrease in the primary and secondary walls (Willats et al., 2001) . 2.2.2. Structure of cell wall of SB L The c ell wall of SBL has both a primary cell wall, which a ccommodates the cell as it grows; and a secondary cell wall, which develop s inside the primary wall after the cell is fully grown (Ralet et al., 1994) . The thickness, composition, and organization of cell walls are varied significantly. The primary cell wall of SB L is normally thinner than 100 nm, in sharp contrast with the secondary cell wall, which can reach several micro meter s (Xiao and Anderson, 2013 ) . The main chemical components of the primary cell wall include cellulose and two groups of carbohydrates, the pectins and hemicelluloses. However, besides these carbohydrates, the secondary cell wall of SB L has additional substances, such as lignin (Van Soest and Wine, 1967) . In the primary cell wall, cellulose microfibrils are organized in a loose network embedded in an abundant matrix consisting of hemicelluloses and pectin, in contrast to the secondary wall where the cellulose microfibrils are packed i n a tight network (Dinand et al., 1999) . The structure of the S BL cell wall is shown in Fig ure 2 . 2.3. Isolation and characterization of components from SB L After harvesting of sugar beet, SB L is produced through several steps: (1) the cleaned and washed sugar beets are sliced into long, small strips, which are called cossettes; (2) the cossettes are placed in a tower diffuser with hot water, in which the sucrose is extracted; (3) the wet pulp 8 residues are pressed, collected, and then dried; (4) the resulting product is usually pelletized, and is known as sugar beet lignocellulose (SB L ) (Lapp and Shrager, 1996) . Fig ure 2 Structure of SB L from plant to the fiber (Postek et al., 2011; Xiao and Anderson, 2013) It was reported total approximately 67 % of SB L dry weight consists of carbohydrates and it is therefore a potentially source of cellulose, hemicellulose, and pectin (Phatak et al., 1988; Wen et al., 1988) . Sev eral pre - treatments are applied before the extraction in order to eliminate the factors that affect the separation of these carbohydrates. Milling is a conventional mechanical pretreatment of the biomass before extraction. The objective of this procedure i s to reduce the 9 particle size of the biomass and therefore increase the available surface to the chemical s (Palmowski and Mller, 2000) . Then, extractives and protein can be easily removed from SB L before isolation. 2.3.1. Isolation and characterization of pectin from SB L Pectin can be extracted from biomass by hot water, weak acids or chelating agents like e thylenediaminetetraacetic acid (EDTA) or ethylenediaminetetraacetic acid (CDTA) (Ebringerova et al., 2005) . Lin et al. ( 1978) investigated an acidic met hod to isolate pectin from sunflower head s . This method was modified by Phatak et al. ( 1988) and they were able to isolate pectin from SB L . In detail, SB L free of extractives and protein was first poured into a hot acidic solution using hydrogen chloride with pH below 3.0. The objective of this procedure was to dissolve pectin into solution. After filtration, the solution was poured into 95 % ethanol to precipitate the pectin from acidic solution. Then, the resulting material was centrifuge d, filtered, and washed with 45 % ethanol. The resulting solid was pectin. It was also reported that pectin in SB L can be isolated by chelating agents (Furda, 1981) . In this method, e thylenediamine tetraacetic acid (EDTA) and d isodium hydrogen phosphate were used to dissol ve pectin into chelating agents and then precipitated and isolated using ethanol. The pectins from SB L obtained by these two methods were characterized by Phatak et al. ( 1988) . They indicated that the main sugar components linked to pectin were arabinose, galactose, glucose, and rhamnose. Moreover, the author s also investigated the molecular we ight distribution of the isolated pectin (Furda, 1981) . Pectin isolated using the EDT A method had higher average molecular weight (44,700 Daltons) than that with the acidic method (35,500 Daltons). However, pectin from the acidic methods had a wider molecular weight range than that from the EDTA method. This report clearly indicated that a cidic treatment is more included to modify/degrade pectin from SBL. 10 2.3.2. Isolation and characterization of hemicellulose from SB L Many attempts have been made to isolate hemicelluloses from a large number of biomass sources such as wood and plant tissues (Ebringerova and Heinze, 2000) . Among these procedures, the alkali method has been well documented as effective to be used to fract ionate and isolate hemicelluloses from wood and SB L (Sun and Hughes, 1998; Wen et al., 1988) . In general, KOH and NaOH are used to dissolve hemicellulose from pectin - free SB L . After filtration, the hemice lluloses are precipitated in 95 % ethanol solution. S un and Hughes ( 1998 , 1999) reported on the properties of SB L hemicelluloses isolated using this method. They indicated that the main sugar components of isolated hemicellulose s from SB L are arabinose, glucose, galactose, xylose, and minor quantities of rhamnose. Some scientists compared the effect of delignification raw SB L on the range of molecular weight of hemicelluloses . Hemicelluloses isolated from delignificated SB L have a much lower molecular weight (21,620 to 21,990) compared to those from SB L wi thout delignification. This confirmed that the delignification triggered degradation of hemicelluloses in SB L . 2.3.3. Isolation and characterization of cellulose from SB L After the removal of extractives, lignin , and hemicellulose s from SB L , the remain der is assumed to be cellulose and minerals. After alkaline treatments to dissolve hemicellulose s, the insoluble products were immersed in a sodium chlorite solution for delignification. Then, the mineral s were removed by washing with abundant running water u sing a nylon sieve filter. Physical, mechanical and chemical properties of cellulose are strongly related to the d egree of p olymerization (DP) and the c rystalline i ndex (CI) which depends on the process method used to isolate the cellulose - Char got et al., 2011) . 11 The DP of cellulose is defined as the number of repeating glucose units that make up one cellulose molecule or the chain length of cellulose. The two most common methods used to estimate the DP of cellulose are viscometry and gel - permeat ion chromatography (GPC). The v iscometry method is more popular for the characterization of lignocellulosic biomass (Kumar et al., 2009) . Dinand et al. ( 1999) reported that the DP of cellulose isolated from SB L is around 1000, which is lower than that of cellulose obtained from wood (Hallac and Ragauskas, 2011) . Table 1 shows the DP of wood and non - woody celluloses after nitration using the viscometry method. CI is the crystallinity i ndex , defined as the relative amount of crystalline material in cellulos e (Park et al., 2010) . The two most common methods used to measure the CI of cellulose are XRD and solid - state 13 C NMR (Park et al., 2010) . The e quation below is used to calculate the CI of cellulose from the XRD spectra using the peak intensity method (Se gal et al., 1959) . w here I 002 is the intensity of the = 22.5 o and I am is the intensity corresponding to the = 18 o after subtraction of the background signal obtained from XRD without cellulose. Another method using solid - state 13 C NMR can be used to calculate the CI of cellulose. Using to the method from Hall et al. ( 2010) , solid - state 13 C NMR method was performed o n a Bruker Avance/DSX - 400 spectrometer (Bruker Instruments, Inc., Billerica , MA, USA) operating at frequencies of 100.55 MHz for 13 C. Air dried cellulose was packed in 4 mm zirconium dioxide 12 Table 1 DP of native wood and non - woody celluloses after nitration using the viscometric method. Species DP Reference Balsam fir 4400 Snyder and Timell, 1955 White spruce 5500 Timell, 1955 Beech 4050 Ivanov, 1957 Wheat straw 2660 Alemdar and Sain, 2008b Cotton liners 3170 Puri, 1984 Bagasse 925 Puri, 1984 Sugar beet 1000 Dinand et al., 1999 rotors and then spun at 10 kHz. Acquisition was carried out with a CP pulse sequence using a 5 pulse and a 2.0 ms contact pulse over 4 h. The CI was calculated according to the equation described below (Bommarius et al., 2008) : where is the area of the crystalline peak (79 to 86 ppm) and the total area (crystalline and am orphous) assigned to the C 4 peak (79 - 92 ppm). Heux et al. ( 1999) investigated the CI of cellulose from SB L before and after the removal of pectin and hemicelluloses by the NMR method. They indicated that the CI of cellulose in SB L before pectin and hemicelluloses removal was around 40 %. The CI increase d to 51 % after pectin and 13 hemicelluloses removal. The authors concluded that the removal of hemicelluloses and pectin linked to the cellulose led to the increase of CI. 2.4. Potential application of SBL Once the sugar is extracted from sugar beet tissues, the leftover residues known as sugar beet residues or byproducts are used in several applications. The residues are dehydrated before storage to control the biological degradation fro m mold, mildew, fungi and bacteria and also to reduce their weight for efficient transportation. Currently, the dehydrated residues are used as fodder for cows, horses or energy cogeneration (Teimouri Yansari, 2014) . However, these low value - added utilizat ions of sugar beet residues bring little economic benefit to farmers (Finkenstadt , 2013 ) . Several attempts have been made to create high value - added products from competi tiveness while protecting our natural resources and environment. Uses of SB L will contribute to the above objective. 2.4.1. Food ingredients It is reported that su gar beet fibers contain about 8% protein and 67 % carbohydrate including hemicellulose, cellul ose and pectin (Michel et al., 1988) . Sugar beet fiber showed a wide range of beneficial effects on human health (Ralet et al., 2009) . Leontowicz et al. ( 2001) reported the levels. Protein isolated from SB L was evaluated as a food component in comparison to other leafy green matter and the outcome is positive (Jwanny et al., 1993) . Moreover, SB L can be used as the carbohydrate sources to produce x anthan, which is used as a food thickener (Moosavi and Karbassi, 2010) . Pectin from sugar beet has shown excellent properties for gel formation that 14 may be used in the food industries (Norsker et al., 2000) . Sugar beet contains some extractable colored phe nolic materials, which have been used as antioxidants in food (Mohdaly et al., 2010) . 2.4.2. Sources as biofuel Several researchers have used SBL as a raw material for the production of ethanol (Finkenstadt; Kawa - Rygielska et al., 2013; Sutton and Peterson , 2001; Zheng et al., 2012) . Tian et al. ( 2013) investigated the formation of methane based on SB L . ( 2012) reported producing biogases from SB L after a variety of pre - treatments. 2.4.3. Sources for polymer s and composites A great deal of research efforts have been focused on the use of SBL as raw materials for biopolymers. SBL w as chemically and/or physically pretreated before uses to improve some properties. Rouilly et al. ( 2006) reported on the mechanical properties of co mposite made with sugar beet residues using injection - molding after twin - screw extrusion modification. In this paper, using an injection - molding method with a nose temperature at 130 o C and an injection pressure of 1500kg · cm - 2 , they successfully produced S B L based thermoplastic. This SB L composite was brittle with a tensile strain around 1 % for a tensile modulus of 2 GPa. A further reported made by Rouilly et al. ( 2009) investigated the improvement of mechanical properties of SB L film made through extrusion methods at 100 o C by adding plasticizers or cross - l inkers at a concentration of 30 % w/w. They reported that the addition of xylitol increases the elo ngation at break from 1 to 11.3 %. The authors attributed this phenomenon to th e strong hydrogen bonding interactions between the cellulose microfibrils and the xylitols. 15 Liu et al. ( 2011b) use d SB L without pretreatment using various co ncentrations of glycerol (20 - 50 % w/w ) as a plasticizer in a common twin - screw compounding extruder to make thermoplastic sheet. This study showed that with 20 % w/w glycerol, SB L can be used to make sheet with tensile strength of 9.3 MPa. It was further found that lower strength and m odulus of elastic occurred at a higher concentration of glycerol. In a ddition, SB L were shown to be useful as an additive to reinforce other materials. al. ( 2007) studied the properties of paper made by mixing various amounts of sugar beet residues and some pulp fibers from a semi chemical process. The paper prop erties such as water retention value, internal bond strength, tensile energy absorption (TEA) and resistance to air penetration were increased with the addition of SBL . The only explanation was the high cellulose content and low lignin content of SB L . Chen et al. ( 2008) reported using a twin screw extruder to make poly (lactic acid) (PLA) and SB L composites at various concentrations. They found that the tensile strength of the combination of SB L and polylactic acid (PLA) at a ratio of 30/70 (w/w) approached that of neat P LA with an addition of 2 % of p olymeric diphenylmethane diisocyanate (pMDI). They attributed this phenomenon to the penetration of PLA into the SB L particles and the improved interfacial adhesion produced by pMDI. Liu et al. ( 2011a) demonstrated SB L can make thermoplastic sheets with poly butylene adipate - co - terep h thalate (PBAT) by extrusion. However, these resulting plastics have relatively low tensile s trength (8.4 MPa). They added 3 % w/w of pMDI as a compatibilizer in sheets to improve the tensile properties from 8.4 MPa to 17.1 MPa. They demonstrated the addition of 16 pMDI work ed as an interfacial modifier and improved the adhesion and the dispersion of SB L phase in the blends, which resulted in increas ed tensile strength. On the other hand, it has been shown that with some chemical treatments, SB L can be purified to get plastics with higher properties. Dufresne et al. ( 1997) first address ed the mechanical properties of film based on SB L before and after alkali extraction. In this paper, the author s using a casting method produced SB L film with maximum tensile strength around 8 MPa. Moreover, the author s indicated that at a high relative humidity, the tensile modulus of the resulting film with purified SB L significantly increases. They attributed this phenomenon to the strongly hydrophilic behavior of pectin, which absorbed water in the materials and impaired interaction of water with cellulose. Further investigation by Leitner et al. ( 2007) reported on f ilm made with nano - cellulose from SB L by the cast method. T he author purified cellulose using alkali extraction followed by sodium chlorite bleaching. Then, nano - cellulose was obtained through a high - pressure homogenization. The film produced had tensile s trength higher than 100 MPa. Such high tensile strength was attributed to the good distribution of fibers after high - pressure homogenization. Unfortunately, these methods were very time consuming and energy wasting, which made them impractical for industri al use. In addition to making plastics and composites, a few attempts have been made to a ss ess the effect of SB L coatings on actual foods for human consumption. Most of the work has centered on the barrier properties of SB L film. ( 2004) evaluated the capacity of c arboxymethyl cellulose (CMC) from SB L to extend the shelf - life of peaches and pears at 25 o C and 75 % relative humidity. The study showed 17 that the coated samples delayed the losses on soluble solids, titratable acidity and ascorb ic acid in comparison to the uncoated peaches and pears. Shelf - life of the coated sample had been extended to 12 and 16 days for peach es and pear s , respectively. Extensive investigation Arslan, 2004) on mandarin orange s and apple s (Togrul and Arslan, 2005) also showed similar result, extend ing the shelf - life of mandarin orange s and apple s up to 27 and 34 days, respectively, without significantly (P < 0.05) loss of soluble solids, titratable acidity and ascorbic acid. 2.5. Lignocellulosic flexib le film in packaging Bio - based polymers used in film production can be from various sources. Among them, lignocellulosic based products from agro - forestry resources are one of the most promising candidates due to their abundance, renewability, strength properties, low density, biodegradability, and relatively low competition with the food chain (Azizi Samir et al., 2005) . 2.5. 1 . Tensile properties of film for packaging The tensile strength of a material is one of the strength characteristics used to comp are rigid and flexible materials. It is defined as a material resistance to be pulled apart by a load or stress applied at a certain rate of speed. Tensile tests provide information on the load applied and the deformation of the material. The tensile stren gth ( of a material is defined as the ratio of load/force applied (F) divided by the material cross sectional are (A). A for a film is the width multipl ied by the thickness. The values are expressed in pound - force per square inch ( p si) in US standard and in N/m 2 in SI. It is also known as stress. 18 The change in length (L) of a film under tensile stress is the strain ( ) calculated as the percent change in length with the units in %. . L is the stretch or change in the ini tial length from L o to L under tensile stress. A typical stress - strain curve is shown in Fig ure 3 . The tensile modulus is obtained from the ratio of the stress divided by the strain in the linear zone of the curve of stress versus strain and this value is an indication of the stiffness and the resistance to elongation or deformation or how extensible a film is under tensile stress. A higher modulus value of material suggests that it has a higher stiffness and better resistance to deformati on. E is the an index of the rigidity of the film. The total area under the stress - strain curve is the total energy absorbed per unit volume of the film during stretch. Tensile test s are used to determine the ultimate tensile strength ( TS ), the maximum stress a material can withstand before failure; the percent elongation at break ( EB ), the strain of a film before failu also known as toughness, the total energy absorbed per unit volume of the film at rupture ( ASTM D882, 2010 ). 19 Figure 3 Strain - stress curve 2.5. 2 . Factors affecting tensile properties of a film 2.5. 2 .1. Effect of pulping p rocess There are three main categories of pulping processes: mechanical, chemi - mechanical, and chemical pulping. The tensile properties of resulting films can be affected by the pulping process (Gierer, 1980) . Mechanical pulp is produced using only mechanical means to reduce raw materials into discrete fiber s . This pulp type has hi gh yield , up to 95 %, which preserves most of extractives and lignin (Sundholm et al., 1999) . Pressure, steam and water are used during the pulping process to soften the lignin and release fibers with lignin on surface. The retained lignin will interfere wi th the hydrogen bonding between fibers , yielding a relatively low strength film as in the case of newsprint (Biermann, 1996) . E 20 The c hemi - mechanical pulping process consists of a particularly mild chemical treatment followed by mechanical refin ing to liberat e fibers. Some lignin, extractives and hemicellulose are released in the pulping effluents (Biermann, 1996) . The action of the mechanical process is less intense compare d to mechanical pulping, long er fibers and less lign in are present on fiber surface . Yi eld of c hemi - mechanical pulping range from 70 to 85% , which is due to loss of lignin and other extractives , but result in higher tensile strength papers (Zhang et al., 1994) . The c hemical pulping process is defined as combining biomass chips with chemicals to break down the lignin (to 3 - 5 %) without considerable cellulose fiber degradation. The reduced lignin offsets the interferes with hydrogen bonding between cellulose fiber, which leads to stronger tensile strength of the resulting pulp compare d with that of other pulping process. The c hemical pulping process produces longer fiber s due to the absence of grinding, which also lead s to a higher tensile strength (Gullichsen and Fogelholm, 1999) . 2.5. 2 .2. Effect of moisture content Moisture content al so affects the tensile properties of lignocellulosic materials. This is attributed to both the binding and lubricant function s by increasing the area of contact between particles (Grover and Mishra, 1996) . Several studies showed that the tensile properties of lignocellulosic based materials improved with increasing moisture content until an optimal level. Chang et al. ( 2000) reported the water acted as a binder in the bio - based film at low moisture content and then as a plasticizer at high moisture content. Gennadios et al. ( 1993a) also found that the tensile strength of cellulose based film was reduced as the moisture conten t increased above a specific point. Stamboulis et al. ( 2001) reported that 21 the water in flax fibers helped hydrogen bond formation between hemicellulose and cellulose molecule s 2.5. 2 .3. Effe ct of particle size Particle size of fiber also affects the tensile properties of the lignocellulosic based film. In general, the smaller the particle size, the higher the tensile properties. This is attributed to the presence of uniform small particles in the film matrix which may result in large surface area per gram of materials and thus better bonding (Arzt, 1998) . Dikobe and Luyt ( 2007) recommended vinyl acetat e that will have higher tensile properties than that produced with larger particles. They also demonstrated that small er size composites had better filler dispersion and filler - matrix interaction than composites made from large r particles. 2.5. 2 .4. Effect of additives Additives are used in a small amount to improve the properties of polymers. These additives include cross - linkers, plasticizers, and reinforcing agents. Cross - linkers are used to promote covalent bonds or ionic bonds between molecules includ ing polymers like cellulose. Cross - linkers can be solid, liquid or gas; their role is to create link or bridge between molecules. Samal and Ray ( 1997) investigated mixing formaldehyde and p - phenylenediamine with pineapple leaf fiber. They indicated that th ese two additives acted as cross - linker and improved the mechanical properties of the product considerably. Plasticizers are molecules that improve the plasticity or fluidity. Chiellini et al. ( 2001) 22 agro - waste and poly (vinyl alcohol). They indicated that the plasticizer improves the elongation of the films but reduces the value of the tensile strength and the modulus. Reinforcing agents are agents used to strengthen certain specific properties of polymer. Bilbao - Sainz et al. ( 2011) reported on hydroxypropyl methylcellulose (HPMC) film reinforced with cellulose nano - particles. In this report, the authors fo und that the mechanical properties of HPMC film were significantly improved by the addition of nano - cellulose whiskers. The authors attributed these phenomena to the high surface area of nano - cellulose promoting hydrogen bond formation with HPMC, leading t o a higher efficiency of the stress transfe r from the matrix to the fiber. 2.5. 2 .5. Effect of blending with other polymers Mixing lignocellulosic based materials with other polymers to improve the mechanical properties has been reported in several studies. Colom et al. (2003) investigated a composite film made of aspen fiber and HDPE (high - density polyethylene). They found that the adhesion mechani sm between cellulose molecules ethylene results in higher tensile properties. Mikkonen et al. (2008) investigated blended film based on hemicellulose from spruce and PVOH . These authors indicated that increasing the amount of PVOH in the hemicellulose base d film improve s the tensile properties significantly. 2.5. 3 . Moisture barrier property of lignocellulosic film The moisture barrier property is a fundamental property for fiber - based packaging materials to control the shelf life of packaged items and to during the lifetime of the packaging. The water vapor transmission rate (WVTR) is defined as the steady water vapor flow that is transmitted through a material per unit time and unit area, 23 Table 2 Mechanical properties of film from biomass at 50 % RH and 25 o C Sample TS (MPa) E B (%) (GPa) Reference Gluten/xylan pH 4 2.3 130 0.04 Gluten/xylan pH11 7.1 26 0.14 2003 Starch 6.3 67 Ghanbarzadeh et al., 2011 Starch/CMC 16 60 Ghanbarzadeh et al., 2011 Chitosan 18 26 Mi et al., 2006 Chitosan/GA 27 17 Mi et al., 2006 Chitosan/aGSA 29 17 Mi et al., 2006 Whey protein 3.0 13 0.09 Ghanbarzadeh and Oromiehi, 2008 Whey protein/zein 6.7 7.3 0.2 Ghanbarzadeh and Oromiehi, 2008 Gluten 14 0.50 2.6 Cho et al., 2010 Gluten/PLA 34 2.6 2.0 Cho et al., 2010 Chitosan 25 33 Xu et al., 2005 Chitosan/Starch 40 54 Xu et al., 2005 Chitosan/KGM 51 10 Jia et al., 2009 Starch 4.5 110 Huang et al., 2006 Starch/MMT 25 100 Huang et al., 2006 Chitosan 47 7.9 1.3 Azeredo et al., 2010 Chitosan/nanocellulose 57 7.6 1.6 Azeredo et al., 2010 24 under specified temperature and humidity conditions. According to the thermodynamic s of irreversible process, the water vapor potential difference is the driving force of the water transfer through a film. The most commonly used technique to measure the WVTR of bio - based film is the cup method. This method can be divided into wet cup and dry cup methods as described in ASTM E96 - 96 . For the wet cup method, the procedure involves using a dish filled with dist illed water and covered with a film. The mass of water lost from the dish is mo nitored as a function of time. For the dry cup method, the procedure involves using a dish filled with desiccant and covered with a film. The mass of water gain in the dish is monitored as a function of time. For the wet cup method, the procedure involves using a dish filled with distilled water and covered with a film. The mass of water lost from the dish is monitored as a function of time. For the dry cup method, the procedure involves using a dish filled with desiccant and covered with a film. The mass of water gain in the dish is monitored as a function of time. 2.5. 4 . Factors affecting water permeability of lignocellulosic film Water per meability of lignocellulosic based films is affected by many factors, depending on the chemical structure and morphology (crystallinity, cross linking, and fiber size), and thermodynamics such as temperature and vapor pressure. 2.5. 4 .1. Effects of chemical structure and morphology Water vapor barrier properties of lignocellulosic film are affected by a series of factors. Spence et al. (2010) investigated the water vapor barrier properties of cellulose based film with and 25 Table 3 Water vapor permeability (WVP) of biomass based film at 25 o C Sample WVP ( g m - 1 s - 1 Pa - 1 10 10 ) Reference Whey protein 38 Anker et al., 2002 Whey protein/acetylated monoglycerides 19 Anker et al., 2002 Chitosan 8.3 Bonilla et al., 2012 Chitosan/Basil oil 4.3 Bonilla et al., 2012 Starch 3.3 Bertuzzi et al., 2007 Soy bean 20 Pol et al., 2002 Soy bean/Corn - zein 9.0 Pol et al., 2002 Cassava Starch 2.2 Müller et al., 2011 Cassava Starch/MMT 0.8 Müller et al., 2011 Basil seed gum 1.7 Khazaei et al., 2014 Xylan 2.1 Alekhina et al., 2014 Corn Starch 0.9 Ghasemlou et al., 2013 Starch/PVOH/nano - SiO 2 1.2 Tang et al., 2008 without lignin. Although lignin is reported to be less hydrophilic than cellulose, the report found that the cellulose film without lignin had lower water vapor permeability than that with lignin. The author attributed this phenomenon to the larger density of the film due to the lignin removal. Spence et al. ( 2010) also compared water vapor permeability of cellulose films made with different particle sizes. The report showed that the micronized cellulose based film had much lower water vapor permeability than that of macro - cellulose based film. The author indicated that 26 the reduced pore size matrix increase d the possibility of intermolecular inte ractions of cellulose, which le d to less mobility and more tortuosity of the produced film. Adding a crosslinking agent into the film matrix also affects the moisture barrier properties of lignocellulosic based film. Coma et al. ( 2003) reported using polycarboxylic acid as a crosslinking agent to improve moisture barrier properties of the cellulosic bas ed film. These authors found that by forming ester bond s with cellulose, the polycarboxylic acid acted as a cross - linker, which decrease d cellulose chain mobility and increase d the resistance to water vapor transport (Kester and Fennema, 1986) . Moisture ba rrier properties are also affected by the crystallinity of materials. Saxena and Ragauskas ( 2009) investigated adding cellulosic whiskers into xylan/sorbitol films. Ten percent of cellulosic whisker addition resulted in a 74 % reduction in water transmissio n properties of the resulting film. The authors mentioned that the high degree of crystallinity of cellulosic whiskers and their rigid hydrogen - bonded network increase tortuosity of the film matrix. This kind of integrated matrix contributed to the improve ment of moisture barrier properties of the films. 2.5. 4 .2. Effects of temperature and relative humidity Many attempts have been made to investigate the effects of temperature and relative humidity on moisture barrier properties of lignocellulosic based film. Generally, higher relative humidity will result in higher water vapor permeability. Müller et al. ( 2009) investigated the water vapor permeability of cellulose/starch based film. The authors indicated that the water vapor permeability increase d 2 - 3 times when relative h umidity increased from 33 to 64 %. Coma et al., ( 2003 ) and Minelli et al. ( 2010) also showed similar trends in cellulose based film. These authors 27 attributed this phenomenon to the plasticization by water , which also increased the hydrophilic character of films (Kester and Fennema, 1986) . 2. 6 . Flexible film based on biomass To date, many attempts have been made to investigate flexible films based on biomass such as the polymers from agro - resources, includi ng polysaccharides, protein, and lipids. For development and application s in packaging , mechanical and barrier properties are very important concerns during their service life. The hydrophilic character and relatively low strength of these raw materials st ill make them difficult to make it with acceptable tensile and water vapor barrier properties. Improvements of mechanical and barrier properties of biomass based film have been an area of intensive investigation during past few years. 2. 6 .1. Mechanical properties of polymers based on biomass Improvement of mechanical properties of polymers based on biomass can be classified into chemical modification and physical modification. ( 2003) investigated the mechanical properties of xylan - wheat gluten based film at various pH. The authors found that a higher pH in the cast ing solvent result ed in a higher d the flexibility of the film. A similar result was found by Gennadios et al. ( 1993b) on wheat gluten - soy protein films. Kim et al. ( 2006) investigated the mechanical properties of chitosan films made at vario us pH using different acids . This report indicated that chitosan with acetic acid had the highest tensile strength , which was due to interactions between the chitosan and the acid solution. 28 Adding salt and a crosslinking agent to improve mechanical properties of bio - based films has been well documented by many researchers. Ghanbarzadeh et al. ( 2011) investigated the mechanical propert ies of corn starch base edible film in the effect of citric acid as a crosslinking agent. The report showed that the mechanical properties of the starch films were improved by increasing citric acid content to 10 %, w/w. Moreover, the report also indicated that the addition of citric acid result in the transition of starch film from ductile to plastic materials. Mi et al. ( 2006) investigated chitosan film cross linked with two kinds of crosslinking agents. The authors found that both glutaraldehyde and aglycone geniposidic acid (aGSA) had the ability to improve the mechanical properties of the film. For physical modification, there have been several attempts, such as lamination, formation of composites or addition of reinforcing particles (Johansson et al., 2012) . Ghanbarzadeh and Oromiehi ( 2008) reported mechanical properties of whey protein film laminated and unlaminated films made with zein protein . The author indicated that the lam inated film had higher TS and E B than that of unlaminated film. Cho et al. ( 2010) also found the laminated wheat gluten/ poly lactic acid had higher TS and E B than neat wheat gluten film. By using intermolecular force s between different polymers to form composites, the mechanical properties of the film can be improved. Xu e t al. ( 2005) investigated the chitosan - starch composite film at various ratios. They showed that the formation of inter - molecular hydrogen bonds between NH 3 + of the chitosan backbone and OH - of the starch as well as the cross - linking between chitosan and starch increased the TS of resulting film. Jia et al. ( 2009) demonstrated preparation and characterization of konjac glucomannan (KGM) - chitosan - soy protein films. The report found that the in termolecular force between KGM and chitosan is higher than that between soy protein and chitosan. Another research direction to improve mechanical properties 29 of bio - based film is the addition of reinforcing particles into films. Haq et al. ( 2008) determine d the mechanical properties of film based on polyester/ soybean and reinforced with nanoclay . The author indicated that 1.5 % addition of nano - clay can improve stiffness and toughness of the film. Huang et al. ( 2006) investigated mechanical properties of st arch film improved by using activated montmorillonite (MMT). This paper indicated that a low concentration of MMT addition (up to 8 %) improved the mechani cal properties, including TS, E of the starch film. To date, oil addition, one of the most popular strategies, ha s been selected to reduce the water vapor transmission rate (WVTR). Anker et al. ( 2002) investigated the ability of lipid addition to improve the water vapor barrier property of whey protein films. T he acetylated monoglyce ride (AMG) addition reduced water vapor permeability of whey protein films by 50 %. Bertan et al. ( 2005) also investigated the effect of fatty acid s on water vapor barrier property improvement of gelatin/triacetin film was positive result s . Another possibi lity to improve the moisture barrier of bio - based film is the modification of the polymer structure by a crosslinking reaction. McHugh et al. ( 1993) investigated the WVP of caseinate - based edible films as affected by pH and calcium crosslinking. This report found that the calcium crosslinking resulted in the decrease of film WVP. They also found an acidic environment (pH=4.6) was likely to promote the protein - protein crosslinking. Le Tien et al. ( 2000) investigated the moisture barrier property of films based on whey proteins and cellulose; they found that introducing cellulose into whey proteins film resulted in improvement of WVP. They attributed this phenomenon to the covalent cross linking and hydrogen bonds associations of cellulose - cellulose, cellulose - protein as well as protein - protein. Rhim et al. ( 2004) determinate d that polyvinyl alcohol ( PVOH ) was a good crosslinking agent for bio - based film. Many studies have been carried on lamination and coating methods to 30 improve the barrier properties of the bio - based film. Pol et al. ( 2002) investigated soy protein film laminated with corn - zein. They showed that laminated film presented higher barrier properties than the un - laminated one. The author attributed this improvement to the hydrophobic nature of the corn - zein. Mondal and Hu ( 2007) investigated the water vapor permeability of shape memory polyurethane (SMPU) coated cotton fabrics. The author found that for the coated film, there w as no abrupt change of WVP under 35 o C. However, the WVP of uncoated film increased significantly from 25 o C to 35 o C. They related this phenomenon to the crystal phase of SMPU under 35 o C. A dding reinforc ing agents such as nano - clay has also been investigated (Hussain et al., 2006; Vertuccio et al., 2009) . Müller et al. ( 2011) investigated the influence of MMT incorporation procedure on the water vapor barrier p roperty of starch/MMT composite films . They noted that addition of MMT into starc h films improved the water vapor barr ier property of the film, which was attributed to the better dispersion of MMT in the starch based film. Aulin et al. ( 2012) investigated the influence of barrier properties of bio - hybrid films at various relative humid it ies based on nano - cellulose and vermiculite nano - platelets through high - pressure homogenization in various ratios. The report found that hybrid film containing 80 % nano - cellulose and 20 % nano - vermiculite presented the best water va por barrier property at both 50% and 80 % RH. This phenomenon was attributed to two factors: (1) the good dispersion of two types of nanoclay in the film matrix; (2) the interaction between these two clays increased the tortuosity of the film . 2.6.2. Antimicrobial activity of fle xible film and coating One of the most important applications of flexible film and coating is active packaging material s . The main purpose of active packaging is to help prolong the shelf life and improve the safety of foods and pharmaceuticals (Bari et al ., 2007; Sen et al., 2012) . Among them, antimicrobial 31 packaging is one of the most promising version s (Suppakul et al., 2003) . Many studies have already been done in developing flexible film and coating with antimicrobial activit y, most of which focused on incorporating a known antimicrobial compound into the packaging (Appendini and Hotchkiss, 2002; Cha and Chinnan, 2004; Jideani and Vogt, 2014; McMillin, 2008) , such as bacteriocins, enzymes, and various plant extracts. 2.6.2.1. Bacteriocins Bacteriocins refer to protein - based toxins produced by bacteria that exert a lethal effect on closely and similar bacteria (Deegan et al., 2006) . They have often been used as promising valuable biological additive s to prevent packed product from foodborne illnesses and extend the shelf life. To date, one of the predominantly produced bacteriocins is nisin, which is a polypeptide produced by some strains of the lactic acid bacterium Lactoc oc cus lactis (Y ousef , 1999) . It has been widely suggested that nisin could be incor porated into flexible films as an antimicrobial agent to maintain the activity of the packaging system during food storage. For example, Pranoto et al. ( 2005) found incorporation of nisin into chitosan film at 51,000 IU/g can effectively improve the antimi crobial activity of the film against Gram positive bacteria, such as S. aureus , L. monocytogenes , and B. cereus . Sivarooban et al. ( 2008) studied the antimicrobial activity of protein film containing grape seed extract, nisin, and EDTA. They suggested tha t by combining with EDTA, nisin (10,000 IU/g) showed inhibit ability to several Gram - negative bacteria, such as E. coli and S. typhimurium . They found that as a chelating agent, EDTA can help to destroy the protective cell wall by sequestering divalent cations (notably Ca 2+ and Mg 2+ ) and therefore improve the antimicrobial activity of nisin. 32 However, as a natural antimicrobial peptide, the widespread application of b acteriocins in food packaging is limited due to their narrowly screen spectrum and the pr operties of products. For example, Delves - Broughton et al. ( 1996) suggested nisin will lose antimicrobial activity above pH 7, while another lactococcal lantibiotic, lacticin 3147, was found to retain activity at neutral pH (McAuliffe et al., 1999) . The ac tivity of nisin is not as effectively in meat as it is in dairy products. This is attributed to the phospholipids in meat components, which are thought to weaken the antimicrobial ability of nisin (Deegan et al., 2006) . 2.6.2.2. Enzyme Enzymes are protein - based macromolecular biological catalysts. Lysozyme is one of the popular and most frequently used enzyme s examined as an antimicrobial agent . This agent possesses enzymatic ability against the - 1 - 4 glycosidic linkages between N - acetylmuramic acid and N - acetylglucosamine on peptidoglycan, which is the main component of the cell wall of bacteria (Cha and Chinnan, 2004) . Lysozyme has been used in many antimicrobial flexible film s because of its sta bility over a wide spectrum of temperature and pH (Proctor et al., 1988) . It is effective against Gram - positive bacteria, but is not particularly effective against Gram - negative bacteria, such as E. coli and S. typhimurium , which restricts its application in the food industry (Zhong et al., 2011) . Several studies have been done to enhance the antimicrobial activity of lysozyme by introducing other substances. For example, Valenta et al. ( 1998) suggested that a combination of lysozyme and caffeic acid was ef fective against E. coli . Padgett et al. ( 1998) found the addition of EDTA and lysozyme increases the inhibitory effect of protein based film against selected Gram - negative bacteria. They attributed these phenomena to the weakened cell wall of bacteria due to the introducing of a chelating agent. 33 2.6.2.3. Plant extract s Plant extract s are becoming more and more widespread as additive s to replace synthetic chemical product s in flexible films and coating due to the consumer demand for more eco - friendly ingredients and their antimicrobial capabilities. The antimicrobial activities of plant extract s are possibly related to the functions of their constituents such as phenolic compounds, terpenoids, essential oils, and weak acids (Hammer et al., 1999) . The m echanism of their antimicrobial activity are reviewed and well documented in several references. Helander et al. ( 1998 ) and Juven et al. ( 1994) suggested the hydrophobicity character of the plant oils help them partition the lipids of the bacterial cell me mbrane and result s in the degradation of the cell wall and damage s the membrane proteins. Denyer and Hugo ( 1991 ) and Sikkema et al. ( 1995) claimed the phenolic compounds in plant oil, such as carvacrol, eugenol, and thymol contribute to the antimicrobial a ctivity by damaging the cytoplasmic membrane and disturb ing the proton motive force (PMF). Burt ( 2004) suggested that antimicrobial activity of plant extracts is likely the combination of the above mechanisms rather than specific one. Seydim and Sarikus ( 2006) investigated protein based film containing oregano, rosemary, and garlic essential oils. Through the agar diffusion method, they found the oregano essential oil was more effective against several selected bacteria than garlic and rosemary. Delaquis et al. ( 2002) examined the antimicrobial activity of essential oil from dill, coriander, cilantro, and eucalyptus. By determining the minimum inhibitory concentrations (MICs) of the essential oils, they found cilantro essential oil is particularly effectiv e against Listeria monocytogenes ( L. monocytogenes ). Several stud ies focused on the antimicrobial activity of purified components. Ramos et al. ( 2012) produced polypropylene film incorporated with carvacrol and thymol by extrusion. They found 34 these additi ves can effectively inhibit the selected bacteria without loss of mechanical and thermal properties of the film. 35 Chapter 3 Isolation and Characterization of Sugar Beet Lignocellulose (SBL) 3.1. Introduction Today, with the increased pressure from the general public and government and non - government regulatory organizations, bio - based products are becoming more and more used to replace synthetic products made of derivatives of petroleum and natural gas . Decade s ago, the main goal of using bio - based p roducts was to reduce western dependenc e on imported oil. Nowadays, t he driving forces behind the use of bio - based products in clude the control of greenhouse gases, the sustainability of our planet , and the current climate variation. It is estimated that more than 280 million ton s of plastic were produced worldwide in 2012 (McCabe and Block, 2014) . The packaging sector uses about 39% , following by building construction w ith 20% and automotive with 8% (Fuentes, 2014) . Of the total volume of plastic s , about 29 million tons will be used to manufacture flexible packaging films in 2018 (Agrawal, 2013) . Agricultural and forestry residues contain considerable amount of lignocellulose, starch, pectin and extr actives known as good raw materials candidates for the fabrication of ecological sustainable , bio - renewable , biodegradable packaging with relatively low carbon footprint , and mild impact on climate variation. Among the agriculture residues, sugar beet ( Beta vulgaris ) lignocellulose (SBL) available after the extraction of sugar is available in US and in other region s of the world where farmers grow sugar beet for the sugar industry (Salman et al., 2008) . About 32.7 million tons of sugar beet was produced in USA in 2012 (Magana et al., 2011) . To date, SBL is usually marketed and sold as cattle feed with relatively low value, generat ing only a low income to farmers. SB L has been investigated as a raw material for the production of ethanol after acid and bio - fermentation (Dufresne et al., 1997) . Further investigations were launched using high - pressure homogenization to produce nano - cellulose from SB L and to mix with food to increase 36 fiber content (Leitner et al., 2007) . Today, limited information is available in the open literature describing commercial high value products from SB L . C ommercial uses of SB L in the manufacturing of high value products such as flexible packaging will help alleviate the burden on the environment by replacing slow to no n - degradable plastic based material s with degradable and renewable mater ials . I t will also help generate additional income for sugar beet farmers and promote rural econom ics . However, to achieve this goal, several issues have to be resolved due to the complex nature of the sugar beet vegetal tissues. For example, the high hemicelluloses content in SBL resulted in large amorphous regime in the cell wall matrix and limit s the stiffness of resulting packaging. The lignin in the matrix result ed in a large pore of cell wall matrix, which cause low barrier properties (Spence et al., 2010) . Pretreatment has been used for the removal of lignin and hemicelluloses from woody and non - woody lignocellulose biomass structure s for centuries (Moon et al., 2011) . It can be carried out i n different ways: physical, chemical, biological, and a combination of the above treatments . Among these pretreatments, sulfuric acid at high temperatures is reported to affect the structure of the cell wall by altering the hemicelluloses and lignin structure s (Esteghlalian et al., 1997) . Several delignification processes , also known as bleaching , are selected to remove residual lignin from lignocellulosic materials (Villaverde et al., 2009) . Peracetic acid, persulfate and percarbonate processes invo lving the use of oxygen and /or carbonate/sulfate are effective methods due to their abili ty to produce important oxygen and peroxoacid oxidizing agent s in solution (Jääskeläinen et al., 2003) . One of the advantages of peroxoacid is the use of biodegradable and low er toxicity acetic acid and hydrogen peroxide known than chlorine based bleaching agents (Pan and Sano, 2005) . Few references are available in the open literature on the use of acid and peracetic acid treatments to release lignin from SBL (Shafie e t al., 2009; Zhao et 37 al., 2007) . It is postulated that the use of lignin free SB L will result in improvement properties of films such as tensile properties due to the increased hydrogen bonding interactions between fibers , similar to the increase of tensil e strength from mechanical to chemical pulp fibers. The use of acid pretreatment will also help increase the crystallinity of the resulting lignocellulose by hydrolyzing some amorphous cellulose zones and hemicellulose with some effects on the water absorp tion properties. The specific objective of this paper is to evaluate the modifications generated by the pretreatment o f the sugar beet res idue based lignocellulose by a combined acid and peroxoacid treatment. Gravimetric analysis using established TAPPI methods w as used to monitor and evaluate the lignin, hemicellulose, and cellulose content before and after pretreatment. Nondestructive solid state techniques incl uding Fourier Transform Infrar ed (FT IR) and X - ray diffraction (XRD) were used to identify and tentatively quantify the modifications o f the SB L, such as lignin and cellulose crystallinity after pretreatment . 3.2. Materials and methods 3.2.1. Materials Sugar beet pellets were donated from th e Michigan Sugar Company (Bay City, USA). The pellets were dried and ground into powder to go through a 60 - mesh sieve ( 230 µm) using a high speed Laboratory Wiley Mill. The ground SB L powder was defined as RSB L . The moisture content of RSB L was measured by the oven dr ying method to be 7 1% according to ASTM D442 - 07 (ASTM, 2007) . Anhydrous c alcium s ulfate , c alcium nitrate and p otassium s ulfate (used to equilibrate films at 0% RH, 50% RH and 97 % RH, respectively) , sorbitol (99 %), glycerol, 38 sulphuric acid, sodium chlorite (80 %), acetic acid and hydrogen pero xide (30 %) were purchased from Sigma Aldrich (St. Louis, MO, US) and used as received without further modifications. 3.2.2. Preparation of SBL RSB L was Soxhlet extracted by following ASTM D1105 (ASTM, 1996) to remove the non - structural component s including waxes and oils . The extractive free sample was soaked in 10 % w/w sulphuric acid solution at 75 o C for 45 min, and then washed with distilled water until pH neutral and labelled as ASBL . The ASB L was then delignificated during a 24 h treatment with aqueo us solution containing 40 % v/v acetic acid, and 3 % v/v of hydrogen peroxide at 75 o C. The delignifi ed pulp was washed with distilled water until a neutral pH and labelled as DSBL. All the s amples were air dried and ground to less than 250 m for further use. 3.2.3. Gravimetric method to assess chemical composition of SBL Chemical analysis follow ed Technical Association of Pulp and Paper Industry (TAPPI) standards with some modifications. Briefly, ash, extractive, and Klason lignin content were determined as specified in the National Renewable Energy Laboratory (NREL) procedure (Sluiter et al., 2008) . 3.2.3.1. Lignin content Weight 0.1 g of defatted sample and placed in a n autoclave tube, and 1ml of 72 % sulphuric acid was added. The mixture was stirred frequently for 1 h at 30 o C water bath, and 28 mL of distilled water was added to the tube. Then the mixture was autoclaved for 1 hour at 121 o C. After cooling, the lignin was transferred to the crucible and washed with distilled water repeatedly. The collected lignin was dried at 105 o C for 24 h and cooled down in a desiccator and weighed. The different weight of crucible before and after lignin washing was obtained as lignin content. 39 3.2.3.2 . Holocellulose content One gram of air dried defatted sample was weighted and placed in an Erlenmeyer flask, and then 80 mL of distilled water, 0.5 mL of glacial acetic acid, and 1 g of sodium chlorite were added, successively. The flask was placed in a water bath and heated up to 75 o C for an hour, and then an addition of acetic acid and sodium chlorite were repeated hourly, reaction was finished when sample turned to white and the solution turned into colorless. After cooled down into room temperature, the holocellulose was fil tered and washed in crucible with ethanol and water respectively. After the washing, the sample was dried in oven at 105 o C for 24 h before weighing. - cellulose content One gram of holocellulose was placed in a beaker, and 10 mL of 17.5 % sodium hydroxide solution was added. The fibres were stirred vigorously so that they could be soaked with sodium hydroxide solution. Then the sodium hydroxide solution was added to the mixture every five minutes, for 6 times. About 35 mL of distilled water was ad ded to the beaker and stirred for 1 h. The holocellulose residue was filtered and transferred to t he crucible and washed with 8.3 % of sodium hydroxid e, water and 10 - celluloses was dried and weighed. 3.2.3.4. Hemicellulo se content The hemicellulose content of SB L sample was determined by calculating the difference between - cellulose. 40 3.2.4. Fourier transform infrared spectroscopy (FTIR) Fourier transform infrared spectroscopy (FTIR) was conducted using a Shimadzu IR - Prestige 21 (Columbia, MD., USA) equipped with Pike Technologies horizontal attenuated total reflectance (HATR) (Madison, WI., USA) . Three types of samples (RSB L , ASB L , and DS B L ) , approximately 250 mg for each, were pressed uniformly against the diamond surface using a spring - loaded anvil to maximize the contact between the samples and the crystal . Sample s were scanned using an average of 64 scans over the range between 6 00 cm - 1 and 4000 cm - 1 with a spectral resolution of 8 cm - 1 . Each sample was run in triplicates. Spectra were displayed in absorbance and limited to the region of interest 800 - 2000 cm - 1 . Pure cellulose and lignin were collected and used as control reference . Prio r to data analysis, all the FTIR spectra were normalized by ratio ing the intensity of the peaks to the intensity of the highest peak in the region between 2000 and 8 00 cm - 1 . 3.2.5. X - ray diffraction (XRD) X - ray powder diffraction patterns of RSBL, ASBL, DSBL, and pure cellulose powder , approximately 500 mg for each, were obtained using a Bruker D8 advance X - ray diffractometer (Bruker AXS GmbH, Karlsruhe, Germany) operated at 40 kV and 40 mA, equipped with Cu - radiation source 0.154 nm ). Samples of particle size less than 250 were casted with double sided tape on a quartz sample holder and scanned at a speed of 2 o /min, range from 2 = 10 - 40º , at room temperature. Biomass crystallinity as expressed by the crystallinity index (CrI) was d etermined according to a method by Segal et al. ( 1959) : 41 i n which, I 002 is the intensity for the crystalline portion of biomass (cellulose) at about 2 = 22.4 , and I am is the peak for the amorphous portion (i.e., cellulose, hemicelluloses and lignin) at about 2 = 16.6 . 3.2.6. Thermogravimetric analysis (TGA) The t hermal stability of RSBL, ASBL and DSBL was determined using a thermogravimetric analyser with the Universal Analysis Software package V.3.9a (TA Instruments, DE, USA). Cellulose and xylan were running as control s . Five milligram for each sample was in sample cup made of aluminium and performed in a nitrogen environment and at a heati ng rate of 10 o C/min from 50 to 650 o C. All the measurements were conducted in duplicate. 3.3. Result and Discussion 3.3.1. Characterization of SBL Table 4 summarizes the chemical composition of SBL at different sta ges. The RSBL consists of 22.2 % - cellulose, 19.3 % hemicellulose , and 5.9 % lignin. This composition of ho l locellulose and lignin is similar to that found by Concha Olmos and Zúñiga Hansen ( 2012) in SB L from Chile. The differences between our data and theirs may be attributed to various sa mple sources and removal of some hemicellulose through the sodium chlorite and acetic acid method (Rowell, 1980) . After acid pretreatment, the - cellulose and hemicelluloses content s was increased by 115 and 43 %, respectively, while Klason lignin was redu ced to half of originals. The increased cellulose and hemicellulose content was attributed to the remov al of pectin from SB L after acid extraction (Phatak et al., 1988) . This result suggests that cellulose and hemicelluloses in SB L are more stable than other polysaccharides such as pectin. The reduction of lignin content suggests diluted sulfuric acid treatment could be used as delignification, which causes swelling of biomass, 42 destroying the links between lignin and cellulose. This destruction made the cellulose fraction more reactive and accessible to further treatment (Castañón - Rodríguez et al., 2013) . Compar ed with ASBL, the DSBL had more - cellulose, less hemicellulose and Klason lignin ; the contents of - cellulose, hemicellul ose s , and Klason lig nin were 80.4%, 10.5%, and 1.0 %, respectively , showing that the acetic acid and hydrogen peroxide pretreatment result ed in a significant level of delignification. The higher cellulose content and lower hemicellulose content in DSB L comp are d with that of ASB L revealed that bleaching remove d hemicellulose from ASB L . This result was similar to that of Kumar et al. ( 2013) , which indicated that the per acetic acid delignification procedure also result in hemicellulose removal. Table 4 Chemical composition of SBL powders at different stages RSB L ASB L DSB L Cellulose (%) 22.2 47.9 80.4 Hemicellulose (%) 19.3 27.5 10.5 Lignin (%) 5.9 3.3 1.0 Other (%) 52.6 21.3 8.1 Cr.I (%) 23.2 34.5 59.9 3.3.2. Crystal Aggregation of SBL Fig ure 4 shows the powder X - ray diffraction (XRD) patterns of the RSBL, ASBL, and DSBL. Cellulose powder was running as a control. The patterns show the typical form of cellulose I in the main peak at 2 = 22 o . From RSBL, ASBL to DSBL, the diffraction peak at 22 o for the cellulose I form became sharper and sharper, indicating an increase of crystallinity. The 43 crystallinities of each sample were calculated and listed in Table 4 . An increase of crystallini ty from 23.2% for the RSBL, 34.5% for the ASBL and 59.9 % for the DSBL was observed. These Figure 4 X - ray diffraction patterns of RSBL, ASBL, and DSBL fibers. Cellulose whisker was running as control results are in agreement with the work of Li et al. ( 2010) on dilute acid treatment o f switch grass. From the RSBL to the ASBL , the increase of crystallinity was due to the removal of hemicellulose, pectin, and lignin, which exist in the cellulose amorphous regions leading to more crystallites exposed (de Souza Lima and Borsali, 2004) . When the delignification was done (DSBL), the cr ystallinity increased from 34.5% to 59.9 %. This increase in crystallinity by means 10 15 20 25 30 35 40 Intensity Diffraction Angle 2 - theta, Degree RSBL ASBL DSBL Cellulose 44 of delignification treatment agreed with the results obtained by Roncero et al. ( 2005) , who found an increase in crystallinity after oxygen delignification. They attributed this phenomenon mainly to the degradation of the amorphous portion of cellulose by using oxygen, in addition to elimination of h emicellulose and lignin during delignification. This fact was confirmed by the chemical characterization of our samples. In addition to the peak at 22 o generally referenced in the literature for cellulose, another peak appeared in the diffractogram s of RSBL, ASBL, and DSBL at 2 values of 26.4 o . This may be attributed to the pectin in SBL (Combo et al., 2013) . 3.3.3. FTIR spectroscopy analysis Fig ure 5 shows the FTIR spectra of RSBL, ASBL, and DSBL. FTIR spectra in the region between 800 - 1800 cm - 1 represent the major chemical functional groups in lignocellulosic biomass (Bodirlau and Teaca, 2009; Olsson and Salmén, 2004; Shin and Rowell, 2005) and those regions are typically used to identify cellulose, hemicellulose, and lignin. Table 5 shows the m ain functional groups of lignocellulose. FTIR spectra showed obviously structural differences in SBL samples before and after pretreatment. The prominent peak at 1735 cm - 1 in the RSBL is attributed to the acetyl and uronic ester groups of the hemicellulose , lignin, and pectin (Monsoor, 2005; Sun et al., 2005) . The intensity of this peak decreased and disappeared completely in the ASBL and DSBL, which was attributed to the removal of most hemicellulose, pectin, and lignin from the SBL by the chemical pretrea tment. The peaks at 1550 and 1508 cm - 1 in RSBL represent the aromatic C=C stretch of aromatic rings of lignin (Alemdar and Sain, 2008b; Li et al., 2009) . The peaks were reduced and eliminated in the ASBL and DSBL because of the removal of lignin during pre treatment. The peaks at 1315, 1203, 1157, and 1103 cm - 1 result from C - O - C and CH reflection, deformation, and stretch on cellulose. Compared with those in RSBL, these peaks 45 Table 5 Main functional groups Wave number (cm - 1 ) Functional groups Reference 1735 Acetyl, uronic ester, ester linkage Sun et al., 2005 1643 Absorbed water Li et al., 2009 1550 C=C benzene stretching ring Li et al., 2009 1427 methoxyl - O - CH 3 Yang et al., 2007 1375 C - H cellulose, hemicellulose Bodirlau et al., 2009 1315 C - OH stretching cellulose Barry et al., 1989 1246 C - O - C stretching Yang et al., 2007 1157 C - O - C stretching vibration Barry et al., 1989 1103 - OH association alcohols Bodirlau et al., 2009 1053 C - O stretching and deformation Kacurakova et al., 2000 986 O - CH 3 coupled with C - O - C stretch Barry et al., 1989 975 C=C alkenes Bodirlau et al., 2009 895 Anomeric carbon group Barry et al., 1989 were sharper and sharper in ASBL and DSBL, which is due to the fact that the cellulose content in SBL was increased by removal of hemicellulose, pectin, and lignin during pretreatment. Th ese result s w ere consistent with the XRD pattern, which demonstrated the increase of cellulose crystallinity resulting from the removal of non - crystal chemicals such as lignin, hemicellulose and some amorphous cellulose during the pretreatment. 46 Figure 5 FT - IR spectra of RSBL, ASBL, and DSBL fibers. Cellulose whisker and lignin powder were running as control FTIR spectra as analysed are the sum of all functional groups in the SBL samples. The intensity at 1550 cm - 1 is sensitive to the amount of lignin, while the intensit ies of 1157 and 1315 cm - 1 are sensitive to the amount of carbohydrate. The intensity ratio s I 1550 /I 1157 and I 1550 /I 1315 were defined as an empirical lignin content (Pandey and Pitman, 2004; Zhou et al., 2011) . Many researchers have correlated lignin content measured by wet chemical method s and empirical lignin content measured by these intensity ratios (Scholze and Meier, 2001; Schultz et al., 1985) . Fig ure 6 shows typical correlation curves between lignin content of SBL measured by the gravimetric method and lignin content from the ratio of the height of the lignin peak at 1,550 with that at 1,157 or 1,315 ( I 1,550 / I 1,157 and I 1,550 / I 1,315) ) corresponding to carbohydrate peaks . The coefficient of 0.93 and 0.98 are shown for the correlation curve between I 1,550 /I 1,157 and I 1,550 /I 1,315 . 800 1000 1200 1400 1600 1800 Absorbance Wavenumber (cm - 1 ) RSBL ASBL DSBL Lignin Cellulose 1550 1315 1246 1203 1157 1103 1053 895 1735 1643 47 Fig ure 6 Correlation between the lignin content determined by gravimetric method and FTIR peak intensity ratio (1) I 1550/I1315 , (2) I 1550/1157 3.3.4. Thermal Degradation Behaviour Fig ure 7 shows the thermogravimetric analysis of RSBL, ASBL, and DSBL. Cellulose, xylan, and lignin were run as control s . The initial weight loss at 70 o C was attributed to the evaporation of the free w ater in the samples. The curve of RSBL shows a wide decomposition temperature between 210 and 400 o C, which was attributed to the pyrolysis of cellulose, hemicellulose, pectin, and lignin (Li et al., 2009; Tripathi et al., 2010) . This phenomenon is consistence with the chemical characterization of the sample. The DTG curve of RSBL shows a peak at 470 o C, which might be attributed to the degradation of charred residue or a small amount of lignin. y = 10.703x - 0.0384 R² = 0.9388 y = 9.9896x - 0.4617 R² = 0.9813 0 2 4 6 8 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Lignin Content (%) Intensity ratio Intensity Ratio 1 Intensity Ratio 2 48 Fig ure 7 TG and DTG curves of RSBL , ASB L , and DSB L fibers. Cellulose whisker, xylan, and lignin powder were running as control 0 20 40 60 80 100 50 150 250 350 450 550 650 Weight (%) Temperature ( o C) RSBL ASBL DSBL Cellulose Xylan Lignin 0 0.1 0.2 0.3 0.4 0.5 0.6 50 150 250 350 450 550 650 Deriv . Weight Change (%/ o C) Temperature ( o C) RSBL ASBL DSBL Cellulose Xylan Lignin 49 Compared with the curve of RSBL, the TG and DTG curve of ASBL shows a higher decomposition temperature which starts at 230 o C and reached a dominant peak at 324 o C. The increased decomposition temperature was attributed to the removal of pectin, some ligni n, and hemicellulose from SB L during the dilute acid pretreatment (Li et al., 2009) , which resulted in the increase of the cellulose content of the sample. On the other hand, the dominant decomposition peak of ASBL was significantly lower than that of the cellulose control. Similar changes in the degradation behaviour of cellulose fibre from wheat straw (Alemdar and Sain, 2008a) . The lower temperature state may be attributed to the introduction of sulphated groups into cellulose crystals during acid pretrea tment, therefore reduc ing the thermal stability of cellulose as a result of the dehydration reaction (Kim et al., 2001) . A nother decomposition peak was found at approximately 550 o C after acid pretreatment. This peak was ascribed to the breakdown of sulpha te groups that interacted with cellulose during the pretreatment (Nguyen et al., 2013) , which acted as flame retardants. When compared with that of ASBL, the curve of DSBL show ed a higher thermal stability at temperature lower that 250 o C, which may be caused by the further removal of hemicellulose and lignin during bleaching. However, the two decomposition peak of DSBL were earlier than those of ASBL. Similar results w ere found by Sun et al. ( 2004b) for straw fibre, which implied that the unbleached cel lulose had a higher thermal stability than the corresponding bleached cellulosic sample. 3.4. Conclusion In this work, sulfuric acid hydrolysis and peracetic acid delignification pretreatments at 75 o C and ambient pressures were applied to SBL. Chemical composition, structural, crystallinity, and thermal stability of the SBL before and after the pretreatments were characterized to investigate their usability in biocomposite applications. Chemical analysis and FTIR measurements of the 50 sample s revealed the partial removal of hemicelluloses and lignin. The crystallinity o f the SBL was increased by 48.7 % after sulfuric acid pretreatment and subsequ ently increased by another 73.6 % after peracetic acid delignification. The pretreated SBL exhibited enhanced thermal stability by up to 30%, which makes them promising candidates for use in thermoplastic composites. 51 Chapter 4 Development and Characterization of Sugar Beet lignocellulose /Poly (vinyl alcohol) Composite Film via Simple Casting Method 4.1. Introduction Over the past two decades, the use of plastic from synthetic polymers has increased extensively. These polymers, i.e., polyethylene (PE), polypropylene (PP), polyethylene terephthalate (PET), polystyrene (PS), and polycarbonate (PC), are usually petroleum based and are regarded as non - degradable (Yoon et al., 2012) . One of the current research trends is the replacement of synthetic polymers with biodegradable plastics made of renewable raw materials. This is closely connected with growing consumer demand for high - quality and long - shelf - life products and increased awareness of environmental problems (Gh asemlou et al., 2011b) . T he study of natural - polymer films has attracted much attention due to their excellent biodegradability, biocompatibility and the range of their potential applications. However, films based on these biopolymers are usually sensitive to environmental conditions and the physical and mechanical properties of these films are not adequate for many applications (Bonilla et al., 2014) . As a result, several studies have been carried out to develop films based on mixtures of biopolymers and s ynthetic polymers (Bahrami et al., 2003; Kanatt et al., 2012) . Sugar beet residue is a lignocellulosic byproduct from the sugar refining industry and is mainly used for animal feeding. About 26.7 million tons of sugar beet lignocellulose (SBL) in dry matte r equivalent was left over by the sugar industry in 2011 in the United States (Finkenstadt , 2013 ) . On a dry weight basis, SB L contains 75% 80% polysaccharides, consisting roughly of 22% 24% cellulose, 30% hemicelluloses ( mainly arabinans and (arabino) gala ctans ), and 25% pectin. Small amounts of fat, protein, ash and lignin contents are also present in SB L , 1.4%, 10.3%, 3.7% and 5.9%, respectively (Sun and Hughes, 1999) . Of the compounds mentioned above, SB L cellulose has 52 been shown to have a strong potential for number of packaging applications. Unlike most cellulose originating from secondary wall fibers , the cellulose obtained from SB L is typical primary wall cellulose (Sun and Hughes, 1999) . SB L has been traditionally e mployed as an excellent emulsifier, thickener or stabilizer, among other potential non - food industrial applications (Mishra et al., 2012) . Dufresne et al. ( 1997) have shown in their research that SB L can produce films with good appearance and satisfactory mechanical properties; it appears to have good potential as a film forming agent. However, to the best of our knowledge, limited studies have been carried out to evaluate the effectiveness of biodegradable films made from SB L for possible applications as p ackaging material s . Polyvinyl alcohol (PV OH ), as a non - toxic and water - soluble synthetic polymer with excellent film - forming ability and chemical resistance and good biodegradability, has been widely utilized for the preparation of blends and composites wi th several natural renewable polymers (Chiellini et al., 2003) . Many researchers have studied various biodegradable packaging composite films made from PV OH and other renewable biopolymers such as corn starch (Luo et al., 2012) , chitosan (Yang et al., 2010 ) , sodium alginate (Jegal et al., 2001) , and carboxymethyl cellulose (El - Sayed et al., 2011) . Nevertheless, to our knowledge this is the first study that would explain water vapor barrier and thermal propertie s of PVOH - SB L blend films. Based on the conside rations mentioned above and the motivation of the fundamental research and potential industrial applications of biodegradable films, the aim of this study was to develop new biocomposite biodegradable films by blending PVOH with SB L via a simple casting me thod, using sorbitol as a plasticizer and to evaluate some characteristics of these films, such as their mechanical, barrier, thermal stability, crystallinity and microstructural properties to examine their potential applications as packaging material s . 53 4. 2. Materials and methods 4.2.1. Materials Sugar beet pellets were donated by the Michigan Sugar Company (Bay City, USA). This SB L was dried and ground into powder to go through an 80 - mesh sieve using a high speed Laboratory Wiley Mill. The moisture content of the powders was measured around 7% (d.b . ) according to ASTM D442 - 07 . They were stored at room temperature (23 °C) until used. All chemical reagents used in this research were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA) and were of analytical grade. Millipore water (deionized and filtered) was used in the preparation of the film - forming dispersion (FFD). 4.2.2. Preparation of SBL The dried and ground SBL was d efatted by extraction with a Soxhlet apparatus for at least 24 h in accordance with ASTM E1690 (ASTM, 2008). The dewaxed samples were allowed to stand in a mild acid aqueous solution (1 M H 2 SO 4 ) adjusted to pH = 1 inside an Erlenmeyer flask with temperature set at 75 °C. Mild acid hydrolysis was chosen as the most appropriate system for the selective hydrolysis of hemicellulose in SB L (Harmsen et al., 2010) . The residual was then filtered and washed with distilled water several times until its pH was neutral. After acid treatment, the bleaching process was used to remove the lignin. Forty grams of the acid treated sample was heated in a water bath for 24 h at 70 80 °C together with 160 ml of water containing 40 g of hydrogen peroxide (30% solution) and 200 g of acetic acid. Then, the residue was hand - squeezed in a nylon cloth, and washed with a distilled water and boric acid (2%) solution . This was followed by adding distilled water to the residue to a volume of 800 mL and placed in a 1 - L aluminum vessel (Chicago Boiler Company, Chicago, USA) and homogenized using 250 g glass 54 beads and 50 g ceramic bead abrasives for 15 h at room temperature at a speed of 610 rpm. All purified SB L was refrigerated at 4 °C in bottles c overed with aluminum foil to prevent direct exposure to light , until further analysis. 4.2.3. Chemical composition of SB L The chemical composition of the SB L at the initial and final stage of treatment was determined according to the standards provided by Technical Association of Pulp and Paper Industry (TAPPI) taking into account the modification described by Silvério et al. ( 2013) . This method is based on the sequential extraction and separation of three fractions of lignocellulose. Briefly, lignin conten t was determined as specified in the TAPPI standard T13m - 54. This method is based on the isolation of lignin after hydrolysis of the polysaccharides (cellulose and hemicellulose) and dissolution with concentrated sulfuric acid (72%). The hol lo cellulose (hemicellulose + cellulose) content was estimated according to TAPPI T19m - 54 by selective degradation of the lignin by sodium hypochlorite at 70 °C. The cellulose content was determined by the removal of hemicellulose from the hol l ocellulose using sodium hydroxide (NaOH) at room temperature. The hemicellulose content was found by subtracting the cellulose content from the ho l locellulose content. The ash content was also determined by considering the percentage difference before and after calcination for 6 h at 550 °C. 4.2.4. Preparation of films SB L / PVOH composite films were manufactured by a casting and evaporation method as follows. PVOH solution was prepared by dissolving 5 g of PVOH in 50 ml distilled water under magnetic stirring at 40 °C for 1 h. SB L / PVOH composite films were prepared by mixing different levels of 1% (w/w) purified SB L solutions provided from 4.4.2 section with various levels of PVOH 55 solution (denoted as SB L 100, SB L 75/ PVOH 25, SB L 50/ PVOH 50 and SB L 25/ PVOH 75). To achieve complete dispersi on, the mixture was stirred constantly for 40 min usin g a magnetic stirrer at 500 rpm at 30 o C . The films prepared without plasticizer were brittle and cracked on the casting plates during drying. Thus, plasticizer was incorporat ed into the FFD to achieve more flexible films. Preliminary experiments were performed to compare the effectiveness of using sorbitol or glycerol as a pla sticizer. S orbitol gave significantly better results than glycerol with the latter producing wet films that were difficult to pee l. Accordingly, the dispersion was mixed with sorbitol as a plasticizer at a loading of 10% (w/w) of the total solid weight. Following the addition of plasticizer, stirring was continued for an additional 15 min. Following this process, the resulting dispersions were allowed to rest for several minutes to allow natural removal of most of the air bubbles incorp orated during stirring. The FFD were spread over polystyrene petri dishes (15 cm diameter, 30 g FF D per plate) placed on a leveled surface and allowed to dry for approximately 48 h at 30% RH and 22 °C. Dried films were peeled off the casting surface and maintained at 22 °C and 53% RH (produced with saturated Ca(NO 3 ) 2 solution) in a conditioning desicca tor until further evaluation. For each test, three different samples were prepared by taking 3 portions from each film at different positions (two at the edges and one at the center) with the exception of the water vapor permeability analysis, where the wh ole sample was used . R eplicates of each type of film were evaluated. 4.2.5. Film characterization 4.2.5.1. Film thickness Film thickness was determined using a hand - held digital micrometer (Mitutoyo No. 293 - 766, Tokyo, Japan) having a precision of 0.0001 mm. Measurements were carried out on at least five 56 random locations and the mean thickness value was used to calculate the permeability and mechanical properties of the films. 4.2.5.2. Film density For determining film density, samples of 1 × 1cm 2 were mai ntained in a desiccator with calcium sulphate desiccant (0% R H) for 20 days and weighed. Then , dry matter densities were calculated by Eq. (1). where A is the film area (1 cm 2 s is the dry matter density of the film (g/cm 3 ) (Jouki et al., 2013). The film density was expressed as the average of three determinations. 4.2.5.3. Water vapor permeability The WVP of films was determined gravimetrically in accordance with the ASTM E96/E96M (ASTM, 2012) with some modifications. Films without pinholes or defects were cut into discs with a diameter slightly larger than the diameter of the cup and then placed over a glass c up w ith a circular opening of 0.000324 m 2 . The inside of the cell was filled with calcium sulphate desiccant (0% RH), leaving an air gap of 1 cm between the film underside and the desiccant and then the whole system was placed in a desiccator containing a satu rated sodium chloride solution (75% RH). The RH inside the cell was lower than outside, and water - vapor transport was determined from the weight gain of the permeation cell at a steady state of transfer. The cups were weighed every 1 h to the nearest 0.000 1 g during the first nine hours and finally at 24 h intervals over the rest of 4 - day period. Changes in the weight of the cup were recorded and 57 plotted as a function of time. The slope of each line was calculated by linear regression using Microsoft ® Offic transmission rate (WVTR) was obtained by dividing the slope (g/h) by the effective film area (m 2 ). This was multiplied by the thickness of the film and divided by the pressure difference between the inner and outer surfaces to obtain the WVP. The WVP value expressed as [g m 1 s 1 Pa 1 ], was calculated according to : Where is the weight of moisture gain per unit of time (g/s), X is the average film thick ness (m), A is the area of the exposed film surface (m 2 ), and is the water vapor pressure difference between the two sides of the film (Pa). WVP w as measured for three replicate samples for each type of film. 4.2.5.4. Mechanical properties The mechanical properties of the composite films were determined at 22 °C and 30% RH with an Instron 5565 Universal Testing M achine (Instron, Canton, MA, USA) according to ASTM standard method D882. Films were cut in rectangular strips 50 mm long and 6.35 mm wide. The films were fixed with an initial grip separation of 25 mm and stretched at an extension speed of 0.8 mm/min. A microcomputer was used to record the stress strain curves. Tensile strength (TS), elongation at break ( EB ) and were cal culated. Four replicates of each test sample were run. 58 4.2.5.5. Thermogravimetric (TGA) analysis Thermogravimetric (TGA) analysis was performed to evaluate thermal stabilities of SB L 100, SB L 75/ PVOH 25, SB L 50/ PVOH 50 and SB L 25/ PVOH 75 using a TGA 2950 with U niversal Analysis Software package V.3.9a (TA Instruments, New Castle, DE, USA). Approximately 5 mg samples were heated from 50 °C to 500 °C at 15 °C/min heating rate under nitrogen flow of 70 mL/min. Weight losses of samples w as measured as a function of temperature. TGA (weight loss as a function of temperature) and derivative thermogravimetry (DTG) curves were recorded. All the measurements were conducted in duplicate. 4.2.5.6. X - ray diffraction (XRD) XRD patterns of the composite films were taken using a Bruker D8 advance d X - ray diffractometer (Bruker AXS GmbH, Karlsruhe, Germany) operated at 40 kV and 40 mA, equipped with Cu - = 1.5406 Å). Samples were scanned over a diffraction angle - t room temperature. The d - spacing was = - ray = 1.5406 measured diffraction angle. Data was collected in duplicate . 4.2.5.7. Film microstructure An environmental scanning electron microscope (ESEM, Phillips Electroscan 2020 equipped with a Lab6 filament) at 20 kV acceleration voltage was used to observe the surface characteristics of the composite films. All composite samples were fractured in liquid nitrogen and the fractured surfaces were sputter - coated with gold thin film using a Denton sputter coater to improve image quality. 59 4.2.5.8. Statistical analysis The data are presented as the mean ± standard deviation of each treatment. The experiments were factorial with a completely randomized design using analysis of variance (ANOVA), analyzed using SAS software (version 9.3; Statistical Analysis System Institute Inc., Cary, NC, USA) . s among the mean values for the film properties at the a level of 0.05. 4.3. Results and discussion 4.3.1. Chemical composition of SB L The chemical composition of SBL was describ ed in Section 3.3.1 . 4.3.2. Appearance and physical properties of the film The SB L films were flexible and resistant when handled. The composite films formed from SB L and PVOH were visually homogeneous, with no bubbles or cracks, as well as good handling charact eristics. This means that these films could be easily peeled from the casting plates without tearing. Those without PVOH were relatively whitish; however, with the inclusion of PVOH in the formulation, they became less whitish and translucent. Moreover, it was observed that the color intensified and the transparency increased as the content of PVOH increased. The film thicknesses were found to be similar with an average thickness between 48 ± 2 53 ± 2 PVOH did not significantly cha nge ( P > 0.05) the average thickness of the films. The thicknesses were controlled well because all FFDs were weighed to the same mass prior to casting. The film density increased upon PVOH addition; demonstrating that composite films were significantly ( P < 0.05) more dense than the SB L films. 60 4.3.3. Water vapor permeability (WVP) One of the main functions of food packaging is to avoid or minimize moisture transfer between the food and the surrounding atmosphere. Water vapor permeability (WVP) should therefore be as low as possible to optimize the food package environment and potent ially increase the shelf - life of the food product (Salarbashi et al., 2013) . Fig ure 8 shows the WVP for different composite films made with SB L and PVOH . The WVP was 1.78 × 10 10 g s 1 m 1 Pa 1 for the plasticized SB L film sample. In the present study, the W VP of the SB L / PVOH composite films was not Fig ure 8 Water vapor permeability (WVP) of the different composite films made of sugar beet lignocellulose (SBL) and poly (vinyl alcohol)(PVOH) a, b and c are different letters represent significantly ( P > 0.05) affected by the inclusion of 25% of PVOH compared to SB L films. The further addition of PVOH up to 50% to SB L resulted in a decreased WVP of the resulting composite films (1.61 × 10 10 g s 1 m 1 Pa 1 ), this was most likely associated with the interactions 1.4 1.5 1.6 1.7 1.8 1.9 SBL100 SBL75/PVA25 SBL50/PVA50 SBL25/PVA75 WVP (g S - 1 m - 1 Pa - 1 × 10 - 10 ) Film type) a a b b 61 between SB L and PVOH molecules which have the effect of preventing water molecules from diffusing through the f ilms, thus decreasing WVP values. However, when PVOH content of 75% was incorporated, the results did not significantly ( P > 0.05) affect the WVP of blend films. The same behaviors were shown in research by Limpan et al. ( 2010) , who found that increasing P VOH concentration decreased the WVP of myofibrillar protein/ PVOH composite films. The results presented in this study are more promising than those reported by Bonilla et al. ( 2014) who prepared biodegradable films based on PVOH and chitosan and reported r elatively high WVP values between 6.14 and 19 × 10 10 g s 1 m 1 Pa 1 . However, the WVP obtained in this work were high compared to those of high barrier synthetic polymers at 23 o C and 75 % RH : 0.0127 g s 1 m 1 Pa 1 × 10 10 for PVC, 0.0092 g s 1 m 1 Pa 1 × 10 10 for LDPE, and 0.0023 g s 1 m 1 Pa 1 × 10 10 for HDPE (Smith, 1986) . T he WVP of SB L / PVOH films were slightly higher than those of cellophane (0.84 × 10 10 g s 1 m 1 Pa - 1 ) (Tajik et al., 2013) and there is indeed some scope for use in some food packaging applic ations. 4.3.4. Mechanical properties Mechanical properties of films were characterized by measuring the tensile strength (TS) elongation at break ( EB ) and flexibility. Thus, determination of these properties is of great importance not only in scientific but also technological and practical application of these films. Results of the mechanical tests are shown in Fig ure 9 . Neat SB L films exhibited average TS and EB values of 50.24 ± 1.22 MPa and 4.10 ± 0.41%, respectively being in the same range as those reported by other authors (Liu et al., 2011b; Liu et al., 2005) . SB L / PVOH composites with PVOH content of 25% weight were less elastic and less resistant and had significantly ( P < 0.05) lower values of TS and EB than neat samples. Ghasemlou et al. ( 2011a) have discussed plasticization effectiveness of glycerol 62 and sorbitol in detail. They suggested that larger s orbitol molecules compared to glycerol molecules would make them less effective in trapping hydrophilic sites; this may be the explanation of this behavior. Nevertheless, the composite films in which the PVOH concentration was more than 50% (SB L 50/ PVOH 50) had significantly ( P < 0.05) higher TS values (59.68 ± 4.22 MPa). However with further increase in PVOH content, no further increase ( P > 0.05) in TS value (53.84 ± 0.45 MPa) was observed for composite films used in this study. The PVOH fraction thus contr ibuted to increase in TS in which a higher force was required to rupture those films but our study on the SB L / PVOH composite films, that is, replacing some fractions of SB L by PVOH , did not give such a strengthening effect on the composite film. This resul t might be attributed to such factors as the poor hydrogen bonding interaction between the two main components and plasticizer or the weak plasticizing effect of water absorbed in the films. This observation did not agree with the findings of Zhang et al. (2004), who investigated the mechanical properties of wheat protein/ PVOH blend films and indicated that the TS of the composite films were significantly improved as compared to those of neat films. These results were not also in accordance with the work of Bahrami et al. (2003) who reported that chitosan/ PVOH films showed higher TS and substantially reduced E B values. They suggested that the formation of intermolecular hydrogen bonds between - OH groups of PVOH with the - NH 2 groups of chitosan is able to imp rove the mechanical properties of the blend films. However, these are only assumptions and these authors did not display or measure these interactions. Although comparison of the TS of the composite films containing PVOH with those of SB L films did not sho w striking change, the EB of the resulting composite films was greatly affected by the addition of PVOH . In the film containing PVOH , there was a significant ( P < 0.05) increase in EB of the films especially in films in which the PVOH content was 75% (12.4 5 ± 63 Fig ure 9 Tensile strength (A), elongation at break (B) and Elastic modulus (C) of the different composite films made of sugar beet lignocellulose (SBL) and poly (vinyl alcohol)(PVOH) Note: a, b and c are different letters represent significant differences (p < 0.05) between the means b c a ab 0 10 20 30 40 50 60 70 SBL100 SBL75/PVA25 SBL50/PVA50 SBL25/PVA75 Tensile strength (MPa) Film type b d c a 0 2 4 6 8 10 12 14 16 SBL100 SBL75/PVA25 SBL50/PVA50 SBL25/PVA75 Elongation at break (%) Film type b a a b 0 0.5 1 1.5 2 2.5 3 SBL100 SBL75/PVA25 SBL50/PVA50 SBL25/PVA75 Elastic modulus (GPa) Film type ( A ) ( B ) ( C ) 64 Fig ure 10 TGA (a) and DTG (b) curves for the sugar beet lignocellulose (SBL) and poly (vinyl alcohol)(PVOH) and different composite films made of SBL and PVOH. 0 20 40 60 80 100 50 150 250 350 450 Weight (%) Temperature ( o C) SBL100 SBL75/PVA25 SBL50/PVA50 SBL25/PVA75 PVA 100 0 0.1 0.2 0.3 0.4 0.5 0.6 50 150 250 350 450 Deriv. Weight Change (%/ o C) Temperature ( o C) SBL100 SBL75/PVA25 SBL50/PVA50 SBL25/PVA75 PVA 100 ( a ) ( b ) 65 1.21%). The Young s modulus increased with increasing PVOH content up to 50% and then decreased at higher PVOH content. 4.3.5. Thermal stability assessment by TGA The thermal stability of SB L / PVOH films was evaluated by thermogravimetric analysis. Fig ure 10 shows the TGA weight loss and the derivative thermogravimetric (DTG) curves for the pure films and SB L /P VOH composites in the temperature range from 50 °C to 500 °C. Previous studies showed that the thermal degradation of SB L follows a two - step weight loss process . The first weight loss , which was observed at 50 150 °C , is generally due to the loss of free water adsorbed in the film. The weight loss in the second stage , which corresponds to the elimination of hydroxyl groups and decomposition and depolymerization of the carbon chains occurred at 170 270°C. PVOH had a similar trend of degrad ation because PVOH also con tains of hydroxyl groups. Our results indicated that 25% of PVOH did not influence the matrix thermal degradation. However, it was found that with increased addition of PVOH up to 50%, SB L / PVOH films start ed thermal degradation a t lower temperatures. This could be associated with the interaction between the SB L and PVOH matrix which might delay the thermal degradation of the composite films. It can be seen from Fig ure 10 that the onset degradation temperatures of the composites are found to be slightly higher with the addition of PVOH , and the major degradation peaks shifted to higher temperatures (~15 °C higher) ; however there was no definitive trend with increasing the loading content of PVOH . Correspondingly, there are two major peaks in the DTG curves: one is due to dehydration and the second is due to decom position and carbon burning. C omplete weight loss with a maximum at 333 and 278 °C for pure SB L and PVOH , respect ively, was detected. A similar behavior with SB L was observed in SB L 75/ PVOH 25, with a maximum at 331 °C, corresponding to the thermal decomposition of 66 the polymer. However, in the SB L 25/ PVOH 75 composite films, a shift to lower temperatures of about 40 °C w as detected, indicating that the thermal degradation process of SB L 25/ PVOH 75 happened at lower temperatures. It appeared that blending SB L with a synthetic polymer like PVOH could improve the thermal stability of the composite polymer. 4.3.6. Assessment of compatibility of blend films by XRD The films based on blends of SB L and PVOH were subjected to X - ray diffraction (XRD) analyses. Some typical examples of the results obtained from these analyses are shown in Fig ure 11 . As can be seen from this figure, a very broad peak could be recognized at around 2 =22.27° ( d = 0.395 nm), which is characteristic of the typical cellulose structure and agreeing well with the results obtained by Li et al. ( 2014) working with pure SB L fibers. PVOH showed an obvious diffraction peak at 2 =19.58° ( d = 0.453 nm). Similar XRD patterns can be observed in the studies of Xiao et al. ( 2000) for pure PVOH films. While the pattern of the composite films should be the superposition of those of the two compon ents, we expect ed that the composite films made from SB L and PVOH would be partially crystalline materials, because the films made with both pure SB L and pure PVOH , showed partially crystalline structures. The diffraction peaks at 2 = 22.27° of SB L crysta l and 2 = 19.58° of PVOH crystal were also obviously shown in the XRD of composite SB L / PVOH film as shown in Fig ure 11 . This shows that the blend of SB L and PVOH cannot effectively break the crystals of SB L and PVOH , suggesting that the addition of PVOH had no influence on the internal structure of the film, but the intensity of the diffraction peak decreased. With increase of PVOH content up to 75% in the films, the intensity of the diffraction peak of the blend film, compared with SB L and PVOH , became flatter and broader. It could be assumed that intermolecular interactions between SB L and PVOH existed which means that these two polymers have relatively good compatibility. 67 This conclusion was in agreement with previous work of Xiao et al. ( 2000) who rep orted that films made with blends of PVOH and konjac glucomannan showed partially crystalline structures. Fig ure 11 X - ray diffractograms of SBL/PVOH composite films (a) SBL/PVOH ratio of 100/0 (v/v), (b) SBL/PVOH ratio of 75/25 (v/v), (c) SBL/PVOH ratio of 50/50 (v/v), (d) SBL/PVOH ratio of 25/75 (v/v) and (e) SBL/PVOH ratio of 0/100. 5 10 15 20 25 30 35 40 45 Relative intensity 2 Theta (degrees) (a) ( b ) ( c ) ( d ) ( e ) 68 4.3.7. Surface morphology of blend films Fig ure 12 shows the representative electron scanning micrographs of the surfaces of SB L / PVOH composite films plasticized with sorbitol. As can be seen, the surface of the SB L film w as relatively smooth, homogeneous without any pores or cracks and with good structural integrity, which was similar to that reported by Li et al. ( 2012) . Even though macroscopically both pure SB L and composite films showed similar surface characteristics, ad dition of PVOH at higher content brought out notable difference in the fil nsoluble SB L blended with PVOH was visible in SB L 75/ PVOH 25 films, indicating that SB L and PVOH did not dissolve each other sufficiently. SB L 50/ PVOH 50 ( Fi g ure 12 (c) ) was comparatively smooth and the distribution was more uniform with some particles still existing, indicating that the compatibility of the PVOH and SB L was good. An apparent phase separation was observed in the SB L 25/ PVOH 75 composite films as shown in Fig ure 12 (d ). This is most likely due to the fact that when the content of PVOH in the blend was beyond a certain threshold, the s miscibility deteriorated . Despite this observation, all composite films generally had a compact matrix with good structural integrity, leading to acceptable mechanical properties, as confirmed by the mechanical test results. These results were similar to those of Chen et al. ( 2008) , who attributed phase deterioration in their work to the relatively poor compatib ility between starch and PVOH . 4.4. Conclusion This is the first report that demonstrates the feasibility to form biodegradable films made from SBL and PVOH via a casting and solvent - evaporation method . SBL could be a promising raw material for the prepara tion of biodegradable films and coatings . The mechanical properties, 69 Fig ure 12 Typical scanning electron micrographs of SBL/PVOH composite films (a) SBL/PVOH ratio of 100/0 (v/v), (b) SBL/PVOH ratio of 75/25 (v/v), (c) SBL/PVOH ratio of 50/50 (v/v), and (d) SBL/PVOH ratio of 25/75 (v/v). water resistance and thermal stability of the SBL/PVOH film improved compared to the neat SBL film . XRD results reveal ed that SBL and PVOH are compatible, and a ddition of PVOH reduce d the crystallinity of SBL / PVOH blends . The results generated in this study clearly indicated that there is a major requirement to understand how preparation and processing would affect biodegradable film stru cture. While successful films were produced as part of this study, it is clear that further studies are required to improve film formulation s , composition and their properties. Moreover, f urther studies need to be started using FT - IR s pectroscopy to provide evide nce for the presence of interaction between SBL and the PVOH matrix. Additionally, (a) ( b ) ( c ) ( d ) 70 thermal analysis using DSC needs to be performed to study the thermal properties of the resulting composite films . 71 Chapter 5 Antimicrobial Activity of Sugar Beet Lignocellulose films containing Tung and Cedarwood essential oils 5. 1. Introduction Conventional polymers from petroleum derivatives are commonly used in food packaging technology for a variety of applications over decades. General public perception o n the disposal of plastic packaging at the end of the service life is one of the drivers to replace plastic bags. Several alternatives with low greenhouse gases (GHG) emissions, renewable and biologically available have been proposed. It included abundant renewable raw materials, such as biopolymers from forest and agriculture residues. Cellulose, hemicellulose, and lignin are the most abundant nontoxic and biodegradable polymer. Sugar beet residue is a byproduct of the sugar industry available after the su gar extraction from sugar beet. It is usually and currently used as low value animal food and energy production with relatively low economic impact ( Fishman et al. 2011 ). Previous study suggested that cel lulosic flexible film made of sugar beet lignocellulose (SBL) exhibits tensile strength value of 50 MPa and water vapor permeability of 1.8 10 - 10 g·s - 1 ·m - 1 ·Pa - 1 (Shen et al. 2015) . Unfortunately, the poor moisture barrier property is one of the limitation s of its potential use in the manufacturing of flexible film for food packaging films where oxygen and water management are paramount in the prediction of shelf life. Microbial growth is known to request a certain level of water, oxygen, temperature and nu trients. Water and oxygen have been reported as difficult to control; the addition of substance capable of reducing the microbial growth is a well - established strategy in food packaging. Several compounds have been used to decrease the microbial growth in food packaging, including organic acids, enzymes, bacteriocins, chelating agents, radical scavengers, and antioxidants ( Salmieri et al. 2014 ; Cinelli et al. 2014; Shojaee - Aliabadi et al. 2013; Feng et al. 2014 ). 72 Plant essential oil contain saturated and unsaturated aromatic compounds, such as terpenes, monoterpenes, thujone, polyphenols, tannins, alkene, flavonoid, cedrol, and phenolic acids ( Ferná ndez Pan et al. 2013; Türünç and Meier 2013) . Most of these compounds are hydrophobic with wide range of antimicrobial properties (Seydim and Sarikus 2006) . Cedarwood essential oil (CWO), an essential oil obtained from cedar (thuja species) wood is reporte d to contain approximatively 9 to 12% widdrol, 10 to 11% thujone, 13 to 15% cedrol and 6 to 8% cedrene (Tunalier et al. 2004) . The efficacy of CWO to control insects including termites and mosquitoes is widely reported (Adams 1991; Regnault - Roger 1997) . So me studies mentioned the efficacy of CWO against E. coli O157: H7 (Hammer et al. 1999) , Bacillus subtilis , and Pseudomonas aeruginosa (Prabuseenivasan et al. 2006) . The mode of antimicrobial activity of CWO is not well documented but it is likely attribute - - cedrene (Johnston et al. 2001) . A probable antimicrobial mechanism is a combination of the antioxidant and chelating properties of phenol present in oil such as cedrol, and thujol and the probable disturbance of the bacteria cytoplasmic membrane due to the penetration of some oils components such as cedrene and other terpenes (Burt, 2004). Tung ( Vernicia fordii ) oil contains considerable amount of fatty acids such as oleic, eleostrearic , linoleic and palmitic acid which are capable of reacting with carbohydrates and then formation of hydrophobic esters (Sharma and Kundu 2006; Zhao and Baker 2013) . The highly unsaturated and conjugated fatty acids of tung oils esters contributed to the hy drophobic properties of the resulting esters (Li and Larock 2000) . Tung oil is reported to be applied on dry wood products to improve the water repellency (Brown and Keeler 2005; Mosiewicki and Aranguren 2013) . The purpose of this study is to incorporate tung oils containing fatty acids and CWO in hydrophilic lignocellulosic films to modify water interaction that will help control the microbial 73 growth using in vitro tests. Properties of the laboratory made films such as density; water vapor barrier, tensi le, microbial growth, thermal properties and chemical interactions between oil and SBL were monitored using TGA and FTIR. 5.2. Material s and method s 5.2.1. Materials Dried and roughly ground sugar beet chip was obtained from Michigan Sugar Company (Bay c ity, Michigan). Moisture content of the residues was 7%. Sugar beet chip was pretreated using a method previously described in chapter 3. The chips were milled to a particle size of 0.5 - 1 mm before further treatment. Twenty grams dry sugar beet powders w ere Soxhlet extracted for 24 h with ethanol and water. Extractives free powder was treated with a 1 M H 2 SO 4 solution at 75 o C temperature for one hour and then filtered and washed with distilled water until a pH of 6 ± 0.5. Subsequently, the slurry was treated with a solution containing 3% w/w of 30% hydrogen peroxide, 160 g of distilled water, and 200 g of acetic acid at 75 o C in water bath for 24 h. The resulting pulp was repeatedly washed with distilled water to pH 6 ± 0.5. The sample was placed in a 1 - L aluminum vessel (Chicago Boiler Company, Chicago) and homogenized using bead abrasives for 15 h at room temperature and 610 further processing. Pretreated sugar beet lignocellulosic (SBL) contained 78% cellulose, 12% hemicellulose, 1% lignin, and 2% of ash (Shen et al. 2015). Span 80, glycerol at 99% purity, and Tung oil with a densi ty of 0.94 g/cm 3 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Cedarwood essential oil (denoted as CWO) was purchased from Atomergic Chemicals Corp. (Farmingdale, NY, USA). Tung oil and CWO were stored in a closed dark glass containers at 22 °C until used. 74 The bacterial strains used in this study include one Gram - positive bacterium ( Listeria innocua ATCC33090), and two Gram - negative bacteria Escherichia coli ATCC25922, and Salmonella enterica ATCC29934. Bacteria were kept at 4 o C in freezer. Subculture was carried out each 14 days to maintain bacterial viability. Bacteria were grown in Brain - heart infusion (BHI) broth at 37 o C at incubation chamber (Sheldon Manufacturing Inc, Cornelius, OR, USA). The bacterial population in all the inoculated media was estimated to be more than 1 10 7 CFU/ml after 24 h incubation. 5.2.2. Oil screening for a ntimicrobial activity Antimicrobial activity of essential oil was screened before testing. Four types of oil were selected based on literature review and lab availability, including Juniper Berry oil, Argan oil, Neem oil, and CWO. CWO was then found best antimicrobial activity among these candidates. 5.2. 3 . Fil ms preparation Aqueous dispersion containing about 1.8 g of sugar beet lignocellulosic (SBL), 0.2 g of glycerol, 0.02 g of Span 80 and 198 g of DI water was stirred at room temperature on a magnetic plate. After 1 h stirring, various amounts of Tung oil or CWO was added to the slurry to achieve target oil concentrations of 0%, 5 %, 10 %, 15 %, and 20 % ( w / w ) basis on the weight of SBL . Take 5% w/w of oil adding as example, 0.1 g oil was added into slurry containing 1.8 g of sugar beet lignocellulosic (SBL), 0. 2 g of glycerol, 0.02 g of Span 80, and 198 g of DI water. The percentage of oil in film was 4.7% w/w and round up to 5% w/w. The slurry was emulsified using an Ultra - Turrax (IKA, Canada) at 2500 rpm for 4 min, and then degassed under vacuum for 5 min. Abo ut 25 ml emulsions were poured in a polystyrene Petri dish measuring 85 mm diameter and stored in a condition room set at 50% RH and 23 o C for 24 75 h until visual evidence of formation of a film. Dried films were peeled from the Petri dish and stored in a de siccator at 23 o C and 50 % RH until further evaluation. 5.2. 4 . Film characterization 5.2. 4 .1. Film solubility in water Specimens of each type of films measuring 1 3 cm 2 were cut , oven dried and weighed to the nearest 0.0001 g to determine the oven dry (OD) weight of the specimen before testing. Film specimens with known oven dry weight were immersed in 200 ml of DI water under constant agitation for 6 h at 23 o C. After 6 h, the undissolved specimens were dried at 105 o C for 24 h or until constant weight and weighed to obtain the OD weight of the undissolved specimen residual. Film solubility in water (%) was calculated by using the following equation : 5.2. 4 .2. Film thickness Film thickness was determined using a hand - held digital micrometer (Mitutoyo No. 293 - 766, Tokyo, Japan) with a precision of 0.0001 mm. Measurements were carried out on five random locations, and the mean thickness value was used to calculate the properties of the films. 5.2. 4 .3. Moisture content and d ensity Moisture content of the films was determined by measuring the weight loss of films upon drying in an oven at 105 o C for 24 hours. To determine film density, samples of 2 2 cm were maintained in a condition ed room at 50 % RH at 23 o C for 48 h and weighed. Densities were calcula ted using the following equation : 76 w here A is the film area (4 cm 2 ), is the film thickness (cm), and are the film mass (g) and density of the film (g/cm 3 ) at 50 % RH at 23 o C, respectively. The film moisture content and density were expressed as the average of three determinations. 5.2. 4 . 4 Water vapor permeability (WVP) The gravimetric Cup Method following standard method ASTM E96/E96M (ASTM, 2012) with some modifications was used to evaluate the WVP of films. Films conditioned a t 50% RH and 23 o C for 48 h were placed on a circular opening of 3.24 cm 2 surface area of a permeability capsule. Wax was used as sealant to ensure that humidity migration occurred only through the circular opening surface area of the specimen and not in t he cross section of the film edges. The permeability capsule was loaded with desiccant, the distance between the film underside and the desiccant was about 1 cm for air gap. Calcium sulfate also known commercially as Drierite was used to achieve a relative humidity of 0%. Capsule cups were placed in a Hotpack Stability Chamber (Thermo Electron Corp., Philadelphia, PA) with temperature and relative humidity set at 25 ± 1 o C and 75% RH, respectively. Each cup was weighted to the nearest 0.0001 g 6 times at 1 h intervals. Three replicates were tested for each type of films. Steady state of water vapor transmission rate was achieved within 4 h. The capsule humidity at the film underside dictated by the calcium sulfates and WVPs were calculated . The WVP of the films was calculated by multiplying the steady state water vapor transmission rate by the average film thickness and divide by the water vapor partial pressure difference across the films using the following equation : 77 where WVP is the water vapor permeability in g m 1 s 1 Pa 1 , WVTR is the water vapor transmission rate corresponding to the amount of water absorbed in grams per unit surface and per unit time thickness in meter, p i and p o the water vapor pressure insi de (i) and outside (o) the capsule. At 25 o C and 75% RH, the value of p o is 2369 Pa while the value of p i is 0 Pa for an absolute value of p i - p o of 2369 Pa. 5.2. 4 . 5. Tensile properties of films A modified standard method D882 - 12 (ASTM, 2012) was used to measure the tensile properties of films. The modifications consisted in using different sample size and different crosshead speed due to initial size of the film made in an 85 mm diameter Petri dish. Films were cut into strips of 50 mm long and 6.35 mm wi dth. The tensile properties were measured on specimens equilibrated at 23 o C and 50% RH with an Instron 5565 Universal Testing machine (Instron, Canton, MA). The initial gauge length was set at 25 mm and films were stretched using a crosshead speed of 1 mm/min. A microcomputer was used to record the stress - strain curves and used to calculate the t ensile strength (TS) , which is the ratio of the stress to the strain in compression and tensile testing and modulus of elasticity in bending as related to the equation below (4): where the Young modulus is expressed in force per unit area, stress in force per unit area and elongation in percentage of dimension change per initial dimension (Twede et al 2015). The 78 greater the stress or modulus, the greater the resistance to deformation or the rigidity of a material. Young modulus is a good indicator of the rigidity of a material while strain or elongation is more related to flexibility. The addition of oils is expected to increase the ur replicates were run for tensile testing of each type of films. 5.2. 4. 6. FTIR spectroscopy FTIR spectra of SB L films were recorded using a Shimadzu IR - Prestige 21 (Columbia, MD., USA) equipped with a Pike Technologies Miracle attenuated total reflectanc e (HATR) (Madison, WI., USA) accessory. Films were placed onto a zinc selenide crystal, and FTIR was performed within the spectral region of 600 - 4000 cm - 1 with 64 scans recorded at a 4 cm - 1 resolution. Prior to data analysis, FTIR spectra were normalized b y ratioing the intensity of peaks to the intensity of the highest peak in the fingerprint region between 2000 and 500 cm - 1 . FTIR spectra of films containing oils were compared to control film without oil to evaluate the effect of the addition of oil (Tung oil and CWO) on the intensity and shift of IR bands. 5.2. 4. 7. Thermogravimetric Analysis (TGA) The thermogravimetric curves were obtained in a TGA 2950 equipped with the Universal Analysis Software package V.3.9a (TA Instruments, New Castle, DE, USA). Samp les of approximately 5 mg were heated from 50 °C to 650 °C at 10 °C/min heating rate under a nitrogen flow of 70 mL/min. Weight losses of samples were recorded in function of temperature. All the measurements were conducted in duplicate. 79 5.2. 4. 8. Differe ntial scanning calorimetry (DSC) The electrical and mechanical properties of polymer s change significantly when the temperature is close to or exceeds the glass transition temperature (T g ). Therefore, the T g of SBL films provides important information about their properties. In this study, Differential scanning calorimetry (DSC) tests were conducted to determinate the T g of films using a TA DSC Q100 (TA Instrument, New Castle, DE, USA) under an inert nitrogen atmosphere with a flow rate of 70 mL/min. Fi ve milligram sample were placed in aluminum pans and heated up at a heating rate of 10 o C/min from 0 to 40 0 o C. All the measurements were conducted in duplicate. 5.2. 4. 9. Contact angle measurement Contact angle analysis was used to monitor changes on film wettability due to the addition of oil. A Video Contact Angle (VCA) 2000 instrument was used to record the contact angle between a A syri on the surface of a film specimen. Image processing and curve fitting of the contact angle from the drop profile was used to measure the contact angle between the baseline of the drop and the tangent at the drop boundary. Contact angles were recorded on 4 dif ferent specimens per each type of film. All measurements were taken at 50% RH and at a room temperature of 23 o C. 5.2. 4. 10. Antimicrobial activity of films Three bacteria namely two Gram - negative, including Escherichia coli ( E. coli ) and Salmonella enteric a ( S. enterica ), and one Gram - positive, Listeria inocua ( L. inocua ), were used for the antimicrobial assay of the films following a protocol reported in the literature ( Berndt et al. 2013 ) with some slight modifications as described below. 80 The agar dilution test was used to determine the density of the bacteria in broth. A serial ten - fold dilutions of broth from 1 to 1 10 - 9 g/mL were made by adding 1 mL of fresh broth into 9 mL of sterile 0.9 % sodium chloride solution. After shaking and completely mixing followed by serial dilutions, 1 mL of each solution was sub - cultured on agar plates and dispersed. The settled plates were sealed and placed in incubator for 24 h. The number of colony forming units (C FU) of bacteria that appear on the countable agar plate (between 30 and 300) was counted . For the agar diffusion methods, t he films were cut into 10 mm diameter discs with a scissor. Film cuts were placed on Brain - heart infusion (BHI) agar for L . innocua , Nutrient agar for S . enterica , and Tryptic Soy Agar (TSA) for E . coli . Films specimens measuring 10 mm diameter were then placed on the surface of inoculated agar petri dish plate, stored in an incubation chamber at 37 o C for 24 h. The area of the inhibiti on zone defined as zone without apparent visual growth of bacteria was measured with a caliper to the nearest 0.01 mm (A a ) The whole zone area that was pre - inoculated with bacteria was calculated and used as the potential surface of bacteria growth and cor rected by subtracting the film surface (A b ). The tests were carried out in triplicate for each type of film. Tetracycline was used as a positive control reference well known to inhibit bacteria growth and used to establish the antimicrobial growth. The ant imicrobial index was calculated as the percentage of the value of inhibited surface measured with a caliper by the total potential surface growth of inoculated petri dish. A 100% antimicrobial index corresponds to a complete surface inhibition of microbia l growth on the petri dish surface while a 0% surface inhibition index is a full microbial growth on the surface of petri dish or 0 cm 2 surface inhibition. The antimicrobial index growth for bacteria used in this work was computed by using the following equation (5): 81 The zero or minimum surface inhibition of bacteria wa s obtained by using film with no oil content while the maximum inhibition surface was estimated using films containing 2% tetracycline as reference antibacterial ( ). 5.2. 4. 11. Statistical a nalysis The results were presented as the mean ± standard deviation of each treatment. The experiments were factorial with a completely randomized design using analysis of variance (ANOVA), analyzed using SAS software (version 9.3; Statistical Analysis Sys tem Institute Inc., Cary, NC, g the mean values for the film properties at the level of 0.05. 5.3. Results and Discussion 5.3.1. Physical properties of the films The SBL films incorporated with oils up to 15% (w/w) of the slurry were flexible and easy to handle compare to that difficult to handle specimens containing 20% (w/w) oils. Samples with 20% oil also show uneven oil distribution color and air bubbles attributed to a poor dispersion of oils in the SBL. Further studies will explore potential increase of surfactant and pl asticizer to improve oil dispersion and control the foam formation. Strips of films containing 20% (w/w) oils were limited to microbial activity testing due to their small sizes. The thicknesses of all films laboratory made in this study were similar with an average of 38 Table 6 lists the value of the physical properties including density, solubility in water, and contact angle in function of the types and amount of oils added. The density of conditioned films containing oil was 1.0 ± 0.1 g/cm 3 , no t significantly different from that of control film with no oil added (P >0.05). The density of lignocellulosic cell wall without pores is close to 1.5 g/cm 3 (Özdemir et al. 2013) ; a 82 density of 1.0 ± 0.1 g/cm 3 will correspond to a porosity of about 33% following the equation below (Twede et all 2015): where Pores, % is the porosity or the percent volume of pores, SG cell wall the specific gravity of cell wall close to 1.5 for lignocellulose and SG film , the specific gravity of the film (Twede et al. 2015). The films solubility in water listed in percentage in Table 6 represents the percentage of films dissolved in water. A high percentage is an indication of high amount of film weight soluble in water. Contro l film without added oil shows the highest film solubility of 29.7%, while films containing 15% CWO exhibited significantly lower film solubility (p < 0.05) of 21.8% compared to control film, which in agreement with Ojagh et al. (2010), who reported simila r data on chitosan film containing essential oil. They attributed this phenomenon on the loss of free functional groups of chitosan after oil addition. The formation of a semi - interpenetrated network containing essential oils promotes hydrophobic interacti ons in film matrix that may contribute to the reduction of film solubility. However, the films containing tung oil have a film solubility of 22.0% with 10% (w/w) oil addition and then increases to 25.9% with 15% (w/w). This different behavior between tung oil and CWO may be due to their chemical composition. Fatty acid with numerous carboxylic groups in tung oils in excess may be a good source of water absorption to the contrary of essential oil with limited carboxylic groups. 5.3.2. Water vapor permeabilit y and Wettability properties The value of the water vapor permeability (WVP) of film without oil was 2.8 10 - 10 g m - 1 s - 1 Pa - 1 . WVP values decreased significantly (p < 0.05) with the addition of oil. This effect was more prominent with the addition of 15% tung oil than 15% CWO. The WVP of films made with 15% 83 tung oil exhibited a reduction of 29% from 2.8 to 2.0 10 - 10 g m - 1 s - 1 Pa - 1 , while film containing 15% cedar oil have their WVP reduced by only 18%. Similar reduction was observed by Hernandez (1994) ; he attributed these to the hydrophobic nature of oils. He indicated that the addition of oils into film matrix reduces the hydrophilic portion of the film and decreases the hydrophilic - hydrophobic ratio of the film components, therefore makes water vapor much harder to transfer through. The difference between films with tung oil and CWO may be attributed to the unique physicochemical properties of tung oil also known as drying oil due to its numerous polyunsaturated fatty acids or double bonds easily oxidi zed and polymerized. The water wettability of SBL film with and without oils was evaluated via the water contact angle on the film surface using the sessile drop method. The contact angles are listed in Table 1. Low contact angle of 39.0 o was obtained for control films indicat ing the hydrophilic nature of lignocellulosic film surface from the numerous hydroxyl groups of SBL polysaccharide s . The value of SBL film containing oils were significantly ( p < 0.05) higher than that of control film without oil, indicating that the addition of oils confer hydrophobicity to resulting films surface. Films containing tung oils have a higher contact angle in comparison to CWO. This was attributed to the presence of potential polymerizable polyunsaturated fatty ac ids of tung oils (Atkins and De Paula 2010) . 5.3.3. Mechanical properties of the films The value of average and standard deviations of the TS, EB, and of the laboratory made films were evaluated and listed in Table 6 as a function of the ty pe and percent of added oils. The values of the tensile strength of the film varied from 32.77 to 54.33 MPa. Film made of sugar beet residues without the addition of oils exhibited a tensile strength of 54.33 MPa, 84 1.3 GPa. The addition of 0.1 to 0.3% oils corresponds to a significant reduction of the tensile strength similarly to a study of apple puree edible film containing essential oil by Rojas - Graü et al. (2007) . The reduction of the tensile strength is attributed to weak bonds generated between the hydrophobic oils and the hydrophilic polysaccharides. Similarly to TS results, EB decreased with an increase of oils concentration but control was not affected by 0.3 % CWO whereas it was affected by Tung oil. This phenomenon was attributed to the longer carbon chains in Tung oil compared to CWO, which resulted in a small amount of oxygen containing groups and low level of free volume, therefore congruent with the great hydrophobicity of films containing Tung oi l. Films made with 0.1 % CWO experienced an elongation increase from 8.33% to 11.4%, representing an increase of almost 37 % compare d to control film with 0% CWO . The addition of CWO to 0.2 and 0.3 % led to a reduction in film elongation down to 9.9% and to 6.4%, respectively ( Table 6 ). The addition of CWO at 0.1% concentration may act like a plasticizer, facilitat ing the movement of the SBL polysaccharide chains and improv ing t he film flexibility. At a high er concentration level, the oil dispersion may be challenged, and contributing to the loss of flexibility and elongation . In the case of tung oil, reduction of elongation was observed from 8.33 to 2.38% and attributed to the presence of poly unsaturated fatty acids such as linoleic, stearic and palmitic acid in tung oil that are easy to oxidize and reduce the molecular mobility of the f ilm (Li and Larock, 2000) . The values of confirm the high rigidity or lower flexibility of the film s made with tung oil in comparison of CWO. 85 Table 6 Effect of oil concentration on the physical and tensile properties of SBL films Film type O il conc. (% w / w ) Den sity (g/cm 3 ) Moisture content (%) Solub le in Water (%) TS (MPa) E B (%) modulus ( G Pa ) Contact angle at 0 s ( o ) Control 0 1.00 0.03 a 12.35 0.46 a 29.66 0.97a 54.33 4.34 a 8.33 1.50 bc 1.22 0.13a 38.98 0.30a CWO 5 10.3 0.04 a 12.06 0.39 a 25.89 1.45b 47.41 5.01 ab 11.37 1.55 a 0.77 0.04b 57.73 0.43b 10 0.95 0.03 a 10.81 0.27 b 24.87 0.89b 36.08 5.13 cd 9.85 1.52 ab 0.81 0.03b 66.05 0.67c 15 1.00 0.08 a 9.85 0.60 c 21.75 1.22c 28.09 2.87 e 6.37 0.47 c 0.59 0.03c 75.35 0.28d Tung oil 5 1.05 0.08 a 11.41 0.42 ab 25.76 0.86b 45.72 6.91 bc 7.71 0.58 c 1.23 0.06a 67.58 0.47e 10 0.99 0.02 a 9.62 0.30 c 22.02 1.04c 34.25 2.98 de 3.21 0.54 d 1.40 0.10a 76.97 1.16f 15 0.99 0.04 a 8.62 0.21 d 25.94 0.81b 32.77 1.84 e 2.38 0.59 d 1.31 0.14a 89.36 0.29g Values represent the mean ± standard deviation of four replicates for TS, E and three replicates for other test Values in a column having different superscript letters are significantly different ( P < 0.05). 86 Fig ure 13 Water vapor permeability (WVP) of the SBL films including different concentration of cedarwood oil (CWO) and Tung oil. 5.3.4. Structural properties FTIR spectroscopy was used to monitor the functional groups and structural changes. FTIR spectra of SB L , SB L - span 80, and SB L containing 5 , 10 , and 15 % CWO or t ung oil are shown in Fig. 14 . The broad band at 3305 cm - 1 in the SB L film spectrum is from hydrogen bonds to the hydroxyl groups of the cellulose , pectin, hemicelluloses , and lignin (Olsson and Salmén, 2004) . The peak at 2900 cm - 1 is associated with CH stretching from polysaccharides in SB L (Yang et al., 2007) . The peaks at 1440, 1365, 1311, and 1157 cm - 1 are attributed to O - CH 3 , CH stretching, CH 2 stretching, and C - O - C stretching, respectively, which are typical peaks of polysaccharides (Oh et al., 2005) . The intensity of peaks at 3305 and 2900 cm - 1 increased after the addition of Span 80. These phenomena might be attributed to more hydrogen bondi ng forming from the hydroxyl groups of Span 80. 1.9 2.1 2.3 2.5 2.7 2.9 0 5 10 15 WVP (g.s - 1 m - 1 Pa × 10 - 10 ) Oil Conc. (% w/w) CWO Tung oil 87 Figure 14 FTIR spectra of the films incorporated with different concentration of (a) CWO and (b) Tung oil. 800 1300 1800 2300 2800 3300 3800 Absorbance Wavenumber (cm - 1 ) SBL SBL Span 80 CWO 5% w/w CWO 10% w/w CWO 15% w/w CWO 800 1300 1800 2300 2800 3300 3800 Absorbance Wavenumber (cm - 1 ) SBL SBL Span 80 Tung oil 5% w/w Tung oil 10% w/w Tung oil 15% w/w Tung oil 2927 2866 1741 1246 1463 a b 2927 2866 1741 1463 1236 88 The addition of CWO or t ung oil into SB L film resulted in the appearance of two new bands at 1741 and 1643 cm - 1 from the carbonyl (C O) and unsaturat ed bond from oil s, respectively. The y are associated with esters groups, carbonyl groups, acid groups, and double bonds from poly unsaturated palmitic, stearic acid, linoleic chains of t ung oil (Pereda et al., 2010; Trumbo and Mote, 2001) ; and from the sesquiterpene alcohol s , including cedrol, widdrol, sesquiterpenes , such as cedrene, thujopsene from CWO (Kamatou et al., 2010; Panten et al., 2004) . A n increase in intensity of four bands at 2927, 2866, 1463, and 1236 cm - 1 corresponding to methylene and methyl groups was observed with the addition of oils and with an in crease on the amount of added oils. 5.3.5. Thermal properties of the films TGA was performed to evaluate the thermal stability of the SB L film with and without oil s. TGA curves and their derivatives (DTG) are shown in Fig ure 15 and 16 . The maximum decomposition temperature (T d max ) and the percentage of residues are listed in Table 7 . The (T d max ) values were determined from the maximum temperatures of the peaks in the TGA curve derivatives. Five main stages of weight loss were observed in films con taining Tung oil at levels of 5, 10, and 15% w/w , and only four main stages in films containing CWO and films without oil . T he first stage of thermal degradation as revealed by TGA data in Table 2 indicated a weight loss of about 3 ± 2% in the region of 5 0 to 120 o C with T d max of about 82 ± 1 o C . At this temperature range, degradation of glycerol, SBL, Span and water is unlikely, the presence of some impurities may have promoted the evaporation and or degradation in the film reference without added oil (2.8%). 89 Fig ure 15 Typical results of TGA and DTG curves of SBL films including different concentration of CWO 20 40 60 80 100 50 150 250 350 450 550 650 Weight (%) Temperature ( o C) SBL SBL Span 80 CWO 5% w/w CWO 10% w/w CWO 15% w/w 0 0.05 0.1 0.15 0.2 0.25 50 150 250 350 450 550 650 Deriv. Weight Change (%/ o C) Temperature ( o C) SBL SBL Span 80 CWO 5% w/w CWO 10% w/w CWO 15% w/w 90 Fig ure 16 Typical results of TGA and DTG curves of SB L films including different concentration of Tung oil 20 40 60 80 100 50 150 250 350 450 550 650 Weight (%) Temperature ( o C) SBL SBL Span 80 Tung oil 5% w/w Tung oil 10% w/w Tung oil 15% w/w 0 0.05 0.1 0.15 0.2 0.25 50 150 250 350 450 550 650 Deriv. Weight Change (%/ o C) Temperature ( o C) SBL SBL Span 80 Tung oil 5% w/w Tung oil 10% w/w Tung oil 15% w/w 91 Table 7 TGA and DTG Curve Parameters of the Films Lipid Type Conc. (% w / w ) T d max ( o C) R esidue (%) 1 o stage 2 o stage 3 o stage 4 o stage 5 o stage Control 0 81.5 - 226.6 336.6 504.8 27.1 CWO 5 81.8 - 226.8 337.7 505.0 23.4 10 83.1 - 227.6 337.4 504.6 23.8 15 82.9 - 228.2 339.1 504.6 23.9 Tung oil 5 82.1 180.7 226.5 337.1 505.1 28.8 10 82.6 180.8 227.3 337.9 505.9 25.3 15 83.1 180.5 228.7 339.4 506.1 18.7 A second thermal degradation stage with weight loss from 7.3 to 12.5% was noticeable in temperature range from 120 to 250 o C corresponding water evaporation, degradation of hemicellulose, fatty acids of tung oils and molecules with boiling point and degradation temperature in this range. The third stage between 250 to 350 o C was associated with the partial degradation of glycerol, span 80, hemicelluloses, cellulose in agreement with work by Yang et al. (2007) . The weight loss in this third stage represented 41 to 49 percent loss of the initial weight of the film. The fourth stage, T d max at temperature ranging from 350 to 550 o C, is most likely due to the degradation or decomposition of cellulose components and some oils compo nents (Ahmad et al. 2012; Lin et al. 2008) . 92 The fifth stage, T d max around 5 50 to 650 o C, is attribu ted to aromatic components from lignin and oils (Tongnuanchan et al. 2013; Yang et al. 2007) . About 18 to 27% percent of remaining after temperature degradation of 650 o C was considered as residues rich in calcium, charred compounds impurities from SBL an d oils such as calcium, carbonates and oxalates. Films containing Tung oil or CWO at levels used in this study did not show an important thermal behavior as evidenced with their similar maximum temperature degradation at 333 ± 1ºC and their percentage of weight loss per unit temperature at 0.21 ± 0.02% per unit temperature. In comparison to film without oil at 0.19, it is prudent to suggest that addition of tung oil or CWO may decrease the stability of SBL films. Thermal behavior of films with and without lipids was investigated by DSC. As shown in Appendix - 3 , DSC of all SB L films exhibited broad endothermic peaks at approximately 35 - 140 o C, mainly associated with the removal of moisture when the sample was heated up. When temperature increas ed further ( > 200 o C), the DSC profile of films showed exothermic peaks at 234 - 350 o C, mostly attributed to the primary pyrolysis of cellulose components in SB L . 5.3.6. Antibacterial activity Table 8 shows the inhibition zone in mm 2 of the films after exposure to bacterium. Films containing CWO inhibition all three test ed bacteria , while control f ilm without oil and films with t ung oil were not effective against any of the tested bacteria confirming and validating the robustness of t he bacteria growth in this study. Fig ure 17 shows the inhibitory effect of SB L films with CWO as a dose response against the three tested bacteria. L. innocua (Gram - positive) was observed to be more sensitive to CWO as compared to E. coli and S. enteria (Gram - negative). 93 Fig ure 17 Inhibition Index of SBL films incorporated with various concentration of CWO It is clear that the antimicrobial activity of the SBL film improved significantly (p < 0.05) with the increased adding amount of CWO. The inhibition index of SBL film containing 20% w/w of CWO increased to 23% for L. innocua , 20% for E.coli , and 20% for S. enteria , respectively. Data obtained in this work are similar to what was reported ea rlier on the performance of oils from plant origin on better in inhibiti on of Gram - positive as compare to Gram - negative bacteria (Couladis et al. 2003) . They attributed this phenomenon to the presence of an extra external membrane outside of the cell wall in Gram - negative bacteria, known to retard diffusion of lipophilic compounds through the lipopolysaccharide covering (Burt 2004) . As the CWO concentration increased, the zone of inhibition indicated by the absence of bacterial growth around the film strip s increased significantly for all tested bacteria. 0 5 10 15 20 25 0 5 10 15 20 Inhibition Index (%) Oil Conc. (% w/w) L. innocua S. enteria E. coli 94 Table 8 Antimicrobial activity of SBL films incorporated with CWO Bacteria Percentage concentration ( % w / w ) in film emulsion Inhibitory zone (mm 2 ) Inhibition Index (%) L. innocua 0 0 0 a 0 5 4.77 0.63 a 0.82 10 42.17 1.27 b 7.22 15 71.50 0.85 c 12.24 20 136.01 0.93 d 23.29 Tetracycline 584.06 7.65 100 S. enteria 0 0 0 a 0 5 0 0 a 0 10 32.88 0.80 b 6.92 15 49.85 0.56 c 10.49 20 94.81 0.81 d 20.00 Tetracycline 475.34 8.45 100 E. coli O157:H7 0 0 0 a 0 5 0 0 a 0 10 36.12 0.71 b 6.79 15 61.77 0.77 c 11.61 20 107.45 0.97 d 20.20 Tetracycline 532 10.34 100 Values (n=6) with different superscript letters in each row are significantly different (P < 0.05). 95 The performance of CWO reported here is similar to results reported by Ghanem and Olama (2014) on the stem methanolic extract from Lebanese Cedar ( Cedrus linani ) on E. coli , Listeria monocytogenes , and Candida albicans due to the presence of - - - - cedrene, and cedrol (Adams 1991; Guerrini 2011) . According to Hall et al. (1977) , the proposed mechanism of antimicrobial activity of sesquiterpenes compounds of CWO is in their react ion with thiol groups of enzymes necessary for D NA replication, which greatly hindered the production of bacterial. 5.4. Conclusion Laboratory SBL films containing 5, 10, 15, and 20% (w/w) CWO or Tung oil were made by casting method. SBL containing 20% (w/w) of oil addition presented inhomogeneous film which would not be suitable for single using but coating. The properties of films containing 20% (w/w) oil addition did not be evaluated excepting antimicrobial activity for this result. The functional properties of films containing 5, 10, and 15% CWO or tung oil were affected. The composite films containing oils exhibit a less water absorption and lower tensile strength in comparison to film without oil absorbed less water. However, the thermal properties were not impacted by the addition of oils. FTIR sp ectra demonstrated introduce and good interaction between polysaccharide components in SBL and hydrophobic bonding in lipids. Films containing CWO exhibited significant antibacterial activity against the three bacteria studied. The films were more effectiv e against Gram - positive bacteria ( L. innocua ) than Gram - negative bacteria ( E. coli and S. enteria ). These results suggest that physical, tensile, and antimicrobial properties of SBL films can be modified by controlling the level of oils concentration. Tung oil has provided 96 hydrophobic properties to the film but rather than the components in CWO, that of Tung oil was not efficient as antimicrobials. 97 Chapter 6 General Conclusions and Future W ork 6.1 General conclusions Development of antimicrobial flexible films from SBL has been a challenge. Many critical factors such as mechanical strength, water vapor barrier, and antimicrobial properties have to be optimized in order to successfully develop an antimicrobial film. Dilute sulfuric ac id followed by peracetic acid delignification can effectively remove lignin, hemicelluloses, and pectin from SBL, which resulted in an increase of crystallinity of lignocellulose. The pretreatment o f SBL enhanced thermal stability, which makes them as a promising candidate for use in thermoplastic s . Among many factors, the tensile properties, water vapor permeability, and thermal properties of films are very critical in selecting film formulations for packaging requirement. The properties of SBL film w ere significantly influenced by add ed amount of PVOH. Introduc tion of PVOH into SBL resulted in improvement in tensile properties and water vapor permeability of the film. Antimicrobial activity is an im portant consideration for flexible films for active packaging. Besides improv ing the water repellency of SBL film, some plant oil s can be used as antimicrobial agent. The SBL film incorporated with cedarwood oil (CWO) was effective against both Gram - positi ve and Gram - negative pathogenic food bacteria. The films show better inhibition o f Gram - positive compared to Gram - negative bacteria. This study indicated the possibility of application of SBL. Its workable mechanical and water vapor barrier properties, co mbined with some active additives, would be recommended for the formation of packaging material. 98 6. 2 Future work This research has addressed some work on SBL used as raw material for flexible film, including pretreatment, additives, and formulations. Howe v er, there are still issues on the SBL application, and more efforts need to be done before beneficial use in industry. Chemical pretreatment of SBL is one of the most expensive processing steps in film making. A promising method combin ing biological, chemi cal, and physical need s to be considered to increase the yield as well as economic efficiency of SBL. In combination with PVOH, the SBL films show ed excellent water vapor barrier properties improvement, but did not contribute to improved tensile propertie s. In a future study, effort s should be done to introduce crosslinker s to achieve better tensile properties. SBL films enriched with plant oil s show ed antimicrobial activity. However, the amount of additives was too high to maintain flexibility of the film . Future work will focus on SBL films incorporated with major antimicrobial constituents of plant oil, such as carvacrol, citral, and cinnamaldehyde, to maintain the properties of the film. 99 APPENDIX 100 Fig ure 18 Typical DSC thermograms of SB L films incorporated with different concentration of (a) CWO and (b) Tung oil 0 50 100 150 200 250 300 350 400 Endo Temperature ( o C) SBL SBL Span 80 CWO 5 % w/w CWO 10 % w/w CWO 15 % w/w 0 50 100 150 200 250 300 350 400 Endo Temperature ( o C) SBL SBL Span80 Tung oil 5 % w/w Tung oil 10 % w/w Tung oil 15 % w/w a b 101 Fig ure 19 Volume kinetics of water droplets deposited on surface of SB L films with different levels of CWO and Tung oil 102 Fig ure 20 Petri dishes circular disks of films incorporated with SB L films incorporated with different contents of CWO showing the inhibitory zone against three types of bacteria 103 R EFERENCES 104 R EFERENCES Abdollahi, M., Rezaei, M., & Farzi, G. (2012). A novel active bionanocomposite film incorporating rosemary essential oil and nanoclay into chitosan. Journal of Food Engineering, 111 (2), 343 - 350. Adams, R. (1991). Cedar wood oil Analyses and properties Ess ential oils and waxes (pp. 159 - 173): Springer. Agrawal, A. D. (2013). Green Marketing: sustainable marketing strategy. Indira Management Review , 17. Ahmad, M., Benjakul, S., Prodpran, T., & Agustini, T. W. (2012). Physico - mechanical and antimicrobial prop erties of gelatin film from the skin of unicorn leatherjacket incorporated with essential oils. Food hydrocolloids, 28 (1), 189 - 199. Alekhina, M., Mikkonen, K. S., Alén, R., Tenkanen, M., & Sixta, H. (2014). Carboxymethylation of alkali extracted xylan for preparation of bio - based packaging films. Carbohydrate Polymers, 100 , 89 - 96. Alemdar, A., & Sain, M. (2008a). Biocomposites from wheat straw nanofibers: Morphology, thermal and mechanical properties. Composites Science and technology, 68 (2), 557 - 565. Al emdar, A., & Sain, M. (2008b). Isolation and characterization of nanofibers from agricultural residues Wheat straw and soy hulls. Bioresource Technology, 99 (6), 1664 - 1671. Anker, M., Berntsen, J., Hermansson, A. - M., & Stading, M. (2002). Improved water va por barrier of whey protein films by addition of an acetylated monoglyceride. Innovative Food Science & Emerging Technologies, 3 (1), 81 - 92. Appendini, P., & Hotchkiss, J. H. (2002). Review of antimicrobial food packaging. Innovative Food Science & Emergin g Technologies, 3 (2), 113 - 126. Arzt, E. (1998). Size effects in materials due to microstructural and dimensional constraints: a comparative review. Acta materialia, 46 (16), 5611 - 5626. 105 ASTM Standard D 1105 , 2013 , "Standard Test Method for Preparation of Ex tractive - Free Wood," ASTM International, West Conshohocken, PA, 2003, DOI : 10.1520/D1105, www.astm.org . ASTM Standard D882, 2010, "Standard Test Method for Tensile Properties of Thin Plastic Sheeting," ASTM Internationa l, West Conshohocken, PA, 2003, DOI : 10.1520/D0882 - 12, www.astm.org . ASTM Standard E96/E96M , 201 4 , "Standard Test Methods for Water Vapor Transmission of Materials," ASTM International, West Conshohocken, PA, 2003, DOI: 10.1520/E0096 - E0096M - 14, www.astm.org . ASTM Standard E1690 , 20 08 , "Standard Test Method for Determination of Ethanol Extractives in Biomass," ASTM International, West Conshohocken, PA, 2003, DOI : 10.1520/E1690 - 08, www.astm.org . Aulin, C., Salazar - Alvarez, G., & Lindström, T. (2012). High strength, flexible and transparent nanofibrillated cellulose nanoclay biohybrid films with tunable oxygen and water vapor permeability. Nanoscale, 4 (20), 6622 - 6628. Auras, R., Harte, B., & Selke, S. (2004). An overview of polylactides as packaging materials. Macromolecular bioscience, 4 (9), 835 - 864. Azeredo, H., Mattoso, L. H. C., Avena Bustillos, R. J., Munford, M. L., Wood, D., & McHugh, T. H. ( 2010). Nanocellulose reinforced chitosan composite films as affected by nanofiller loading and plasticizer content. Journal of Food Science, 75 (1), N1 - N7. Azizi Samir, M. A. S., Alloin, F., & Dufresne, A. (2005). Review of recent research into cellulosic whiskers, their properties and their application in nanocomposite field. Biomacromolecules, 6 (2), 612 - 626. Bahrami, S. B., Kordestani, S. S., Mirzadeh, H., & Mansoori, P. (2003). Poly (vinyl alcohol) - chitosan blends: preparation, mechanical and physical p roperties. Iranian Polymer Journal, 12 , 139 - 146. Bari, S. B., Kadam, B. R., Jaiswal, Y. S., & Shirkhedkar, A. A. (2007). Impurity profile: Significance in active pharmaceutical ingredient. Eurasian journal of analytical chemistry, 2 (1), 32 - 53. 106 Barry, A., Kamdem, P., Riedl, B., & Kaliaguine, S. (1989). Infrared study of grafted CTMP fibers. Journal of wood chemistry and technology, 9 (3), 341 - 364. Bertan, L., Tanada - e Food hydrocolloids, 19 (1), 73 - 82. Bertuzzi, M., Castro Vidaurre, E., Armada, M., & Gottifredi, J. (2007). Water vapor permeability of edible starch based films. Journal of Food Engineering, 80 (3), 972 - 978. Bierma nn, C. J. (1996). Handbook of pulping and papermaking : Academic press. Bilbao - Sainz, C., Bras, J., Williams, T., Sénechal, T., & Orts, W. (2011). HPMC reinforced with different cellulose nano - particles. Carbohydrate Polymers, 86 (4), 1549 - 1557. Björkman, A . (1956). Studies on finely divided wood. Part 1. Extraction of lignin with neutral solvents. Svensk papperstidning, 59 (13), 477 - 485. Bodirlau, R., & Teaca, C. (2009). Fourier transform infrared spectroscopy and thermal analysis of lignocellulose fillers treated with organic anhydrides. Rom J Phys, 54 (1 - 2), 93 - 104. Bodirlau, R., Teaca, C. A., & Spiridon, I. (2009). Preparation and characterization of composites comprising modified hardwood and wood polymers/poly (vinyl chloride). BioResources, 4 (4), 1285 - 1304. Bommarius, A. S., Katona, A., Cheben, S. E., Patel, A. S., Ragauskas, A. J., Knudson, K., & Pu, Y. (2008). Cellulase kinetics as a function of cellulose pretreatment. Metabolic engineering, 10 (6), 370 - 381. Bonilla, J., Atarés, L., Vargas, M., & Chi ralt, A. (2012). Effect of essential oils and homogenization conditions on properties of chitosan - based films. Food hydrocolloids, 26 (1), 9 - 16. Bonilla, J., Fortunati, E., Atarés, L., Chiralt, A., & Kenny, J. (2014). Physical, structural and antimicrobial properties of poly vinyl alcohol chitosan biodegradable films. Food hydrocolloids, 35 , 463 - 470. Brown, K., & Keeler, W. (2005). The history of tung oil. Wildland weeds, 9 (1), 4 - 24. 107 Burt, S. (2004). Essential oils: their antibacterial properties and pote ntial applications in foods a review. International journal of food microbiology, 94 (3), 223 - 253. Castañón - Rodríguez, J., Torrestiana - Sánchez, B., Montero - Lagunes, M., Portilla - Arias, J., Ramírez de León, J., & Aguilar - Uscanga, M. (2013). Using high press ure processing (HPP) to pretreat sugarcane bagasse. Carbohydrate Polymers, 98 (1), 1018 - 1024. Cha, D. S., & Chinnan, M. S. (2004). Biopolymer - based antimicrobial packaging: a review. Critical Reviews in Food Science and Nutrition, 44 (4), 223 - 237. Chang, Y ., Cheah, P., & Seow, C. (2000). Plasticizing Antiplasticizing Effects of Water on Physical Properties of Tapioca Starch Films in the Glassy State. Journal of Food Science, 65 (3), 445 - 451. Chen, F., Liu, L., Cooke, P. H., Hicks, K. B., & Zhang, J. (2008). Performance enhancement of poly (lactic acid) and sugar beet pulp composites by improving interfacial adhesion and penetration. Industrial & Engineering Chemistry Research, 47 (22), 8667 - 8675. Chiellini, E., Cinelli, P., Corti, A., & Kenawy, E. R. (2001). Composite films based on waste gelatin: thermal mechanical properties and biodegradation testing. Polymer Degradation and Stability, 73 (3), 549 - 555. Chiellini, E., Corti, A., D'Antone, S., & Solaro, R. (2003). Biodegradation of poly (vinyl alcohol) based materials. Progress in Polymer Science, 28 (6), 963 - 1014. - acid) laminates. Journal of agricultural and food chemistry, 58 (12), 7344 - 7350. Chollet, E., Sebti, I., Martial - Gros, A., & Degraeve, P. (2008). Nisin preliminary study as a potential preservative for sliced ripened cheese: NaCl, fat and enzymes influence on nisin concentration and its antimicrobial activity. Food Control, 19 (10), 9 82 - 989. Cinelli, P., Schmid, M., Bugnicourt, E., Wildner, J., Bazzichi, A., Anguillesi, I., & Lazzeri, A. (2014). Whey protein layer applied on biodegradable packaging film to improve barrier properties while maintaining biodegradability. Polymer Degradat ion and Stability, 108 , 151 - 157. 108 Colom, X., Carrasco, F., Pages, P., & Canavate, J. (2003). Effects of different treatments on the interface of HDPE/lignocellulosic fiber composites. Composites Science and technology, 63 (2), 161 - 169. Coma, V., Sebti, I., Pardon, P., Pichavant, F., & Deschamps, A. (2003). Film properties from crosslinking of cellulosic derivatives with a polyfunctional carboxylic acid. Carbohydrate Polymers, 51 (3), 265 - 271. Combo, A. M. M., Aguedo, M., Quiévy, N., Danthine, S. , Goffin, D. , Jacquet, N., Paquot, M. (2013). Characterization of sugar beet pectic - derived oligosaccharides obtained by enzymatic hydrolysis. International journal of biological macromolecules, 52 , 148 - 156. Concha Olmos, J., & Zúñiga Hansen, M. (2012). Enzymatic dep olymerization of sugar beet pulp: Production and characterization of pectin and pectic - oligosaccharides as a potential source for functional carbohydrates. Chemical Engineering Journal, 192 , 29 - 36. Couladis, M., Chinou, I., Tzakou, O., & Petrakis, P. (200 3). Composition and antimicrobial activity of the essential oil of Hypericum rumeliacum subsp. apollinis (Boiss. & Heldr.). Phytotherapy Research, 17 (2), 152 - 154. de Souza Lima, M. M., & Borsali, R. (2004). Rodlike cellulose microcrystals: structure, prop erties, and applications. Macromolecular rapid communications, 25 (7), 771 - 787. Deegan, L. H., Cotter, P. D., Hill, C., & Ross, P. (2006). Bacteriocins: biological tools for bio - preservation and shelf - life extension. International Dairy Journal, 16 (9), 1058 - 1071. Delaquis, P. J., Stanich, K., Girard, B., & Mazza, G. (2002). Antimicrobial activity of individual and mixed fractions of dill, cilantro, coriander and eucalyptus essential oils. International journal of food microbiology, 74 (1), 101 - 109. Delves - Broughton, J., Blackburn, P., Evans, R., & Hugenholtz, J. (1996). Applications of the bacteriocin, nisin. Antonie van Leeuwenhoek, 69 (2), 193 - 202. Denyer, S. P., & Hugo, W. B. (1991). Mechanisms of action of chemical biocides. Their study and expl oitation : Blackwell Scientific Publications. Dikobe, D., & Luyt, A. (2007). Effect of filler content and size on the properties of ethylene vinyl acetate copolymer wood fiber composites. Journal of Applied Polymer Science, 103 (6), 3645 - 3654. 109 Dinand, E., C hanzy, H., & Vignon, M. (1996). Parenchymal cell cellulose from sugar beet pulp: preparation and properties. Cellulose, 3 (1), 183 - 188. Dinand, E., Chanzy, H., & Vignon, R. (1999). Suspensions of cellulose microfibrils from sugar beet pulp. Food hydrocollo ids, 13 (3), 275 - 283. Dufresne, A., Cavaillé, J. Y., & Vignon, M. R. (1997). Mechanical behavior of sheets prepared from sugar beet cellulose microfibrils. Journal of Applied Polymer Science, 64 (6), 1185 - 1194. Ebringerova, A., & Heinze, T. (2000). Xylan a nd xylan derivatives biopolymers with valuable properties, 1. Naturally occurring xylans structures, isolation procedures and properties. Macromolecular rapid communications, 21 (9), 542 - 556. Ebringerova, A., Hromadkova, Z., & Heinze, T. (2005). Hemicellul ose Polysaccharides i (pp. 1 - 67): Springer. El - Sayed, S., Mahmoud, K., Fatah, A., & Hassen, A. (2011). DSC, TGA and dielectric properties of carboxymethyl cellulose/polyvinyl alcohol blends. Physica B: Condensed Matter, 406 (21), 4068 - 4076. Epstein, S., Ya pp, C. J., & Hall, J. H. (1976). The determination of the D/H ratio of non - exchangeable hydrogen in cellulose extracted from aquatic and land plants. Earth and Planetary Science Letters, 30 (2), 241 - 251. Espitia, P. J. P., Du, W. - X., de Jesús Avena - Bustill os, R., Soares, N. d. F. F., & McHugh, T. H. (2014). Edible films from pectin: Physical - mechanical and antimicrobial properties - a review. Food hydrocolloids, 35 , 287 - 296. Esteghlalian, A., Hashimoto, A. G., Fenske, J. J., & Penner, M. H. (1997). Modeling and optimization of the dilute - sulfuric - acid pretreatment of corn stover, poplar and switchgrass. Bioresource Technology, 59 (2), 129 - 136. Fernández Pan, I., Mendoza, M., & Maté, J. I. (2013). Whey protein isolate edible films with essential oils incorpora ted to improve the microbial quality of poultry. Journal of the Science of Food and Agriculture, 93 (12), 2986 - 2994. Finkenstadt, V. L. A Review on the Complete Utilization of the Sugarbeet. Sugar Tech , 1 - 8. 110 fluence of sugar beet pulp on bond strength and structure of paper. Wood Research, 52 (3), 59 - 74. Fuentes, C. (2014). Green Materialities: Marketing and the Socio material Construction of Green Products. Business Strategy and the Environment, 23 (2), 105 - 11 6. Furda, I. (1981). Simultaneous analysis of soluble and insoluble dietary fiber (Vol. 163): Marcell Dekker. Gennadios, A., Park, H., & Weller, C. L. (1993). Relative humidity and temperature effects on tensile strength of edible protein and cellulose et her films. Biological Systems Engineering: Papers and Publications , 91. Gennadios, A., Weller, C. L., & Testin, R. (1993). Property modification of edible wheat, gluten - based films. Ghanbarzadeh, B., Almasi, H., & Entezami, A. A. (2011). Improving the ba rrier and mechanical properties of corn starch - based edible films: Effect of citric acid and carboxymethyl cellulose. Industrial Crops and Products, 33 (1), 229 - 235. Ghanbarzadeh, B., & Oromiehi, A. (2008). Biodegradable biocomposite films based on whey pr otein and zein: Barrier, mechanical properties and AFM analysis. International journal of biological macromolecules, 43 (2), 209 - 215. Ghanem, S., & Olama, Z. Antimicrobial petential of lebanese cedar extract against human pathogens and food spoilage microo rganisms. Ghasemlou, M., Aliheidari, N., Fahmi, R., Shojaee - Aliabadi, S., Keshavarz, B., Cran, M. J., & Khaksar, R. (2013). Physical, mechanical and barrier properties of corn starch films incorporated with plant essential oils. Carbohydrate Polymers, 98 (1 ), 1117 - 1126. Ghasemlou, M., Khodaiyan, F., & Oromiehie, A. (2011). Physical, mechanical, barrier, and thermal properties of polyol - plasticized biodegradable edible film made from kefiran. Carbohydrate Polymers, 84 (1), 477 - 483. Ghasemlou, M., Khodaiyan, F., Oromiehie, A., & Yarmand, M. S. (2011). Development and characterisation of a new biodegradable edible film made from kefiran, an exopolysaccharide obtained from kefir grains. Food Chemistry, 127 (4), 1496 - 1502. 111 Gierer, J. (1980). Chemical aspects of k raft pulping. Wood Science and Technology, 14 (4), 241 - 266. Grover, P., & Mishra, S. (1996). Biomass briquetting: technology and practices : Food and Agriculture Organization of the United Nations. Guerrini, A. (2011). Antiproliferative and erythroid differentiation activities of Cedrus libani seed extracts against K562 human chronic myelogenus leukemia cells. International Journal of Pharmaceutical & Biological Archive, 2 (6). Gullichsen, J., & Foge lholm, C. (1999). Book 6: Chemical pulping : Helsinki: Fapet Oy . Gurbuz, E., & Coskun, B. (2011). Effect of Dried Sugar Beet Pulp on Some Blood Parameters and Heart Rate in Exercised Horses. KAFKAS UNIVERSITESI VETERINER FAKULTESI DERGISI, 17 (2), 191 - 195. Hall, I. H., Lee, K. - H., Mar, E. C., Starnes, C. O., & Waddell, T. G. (1977). Antitumor agents. 21. A proposed mechanism for inhibition of cancer growth by tenulin and helenalin and related cyclopentenones. Journal of medicinal chemistry, 20 (3), 333 - 337. Hall, M., Bansal, P., Lee, J. H., Realff, M. J., & Bommarius, A. S. (2010). Cellulose crystallinity a key predictor of the enzymatic hydrolysis rate. FEBS journal, 277 (6), 1571 - 1582. Hallac, B. B., & Ragauskas, A. J. (2011). Analyzing cellulose degree of polymerization and its relevancy to cellulosic ethanol. Biofuels, Bioproducts and Biorefining, 5 (2), 215 - 225. Hammer, K. A., Carson, C., & Riley, T. (1999). Antimicrobial activity of essential oils and other plant extracts. Journal of applied microbiology , 86 (6), 985 - 990. Haq, M., Burgueño, R., Mohanty, A. K., & Misra, M. (2008). Hybrid bio - based composites from blends of unsaturated polyester and soybean oil reinforced with nanoclay and natural fibers. Composites Science and technology, 68 (15), 3344 - 3351 . Harmsen, P., Huijgen, W., Bermudez, L., & Bakker, R. (2010). Literature review of physical and chemical pretreatment processes for lignocellulosic biomass : Wageningen UR, Food & Biobased Research. 11 2 Helander, I. M., Alakomi, H. - L., Latva - Kala, K., Mattila - Sandholm, T., Pol, I., Smid, E. J., von Wright, A. (1998). Characterization of the action of selected essential oil components on Gram - negative bacteria. Journal of agricultural and food chemistry, 46 (9), 3590 - 3595. Hernandez, E. (1994). Edible coatings from lipids and resins. Edible coatings and films to improve food quality , 279 - 303. Heux, L., Dinand, E., & Vignon, M. (1999). Structural aspects in ultrathin cellulose microfibrils followed by 13 C CP - MAS NMR. Carbohydrate Polymers, 40 (2), 115 - 124. Huang , M., Yu, J., & Ma, X. (2006). High mechanical performance MMT - urea and formamide - plasticized thermoplastic cornstarch biodegradable nanocomposites. Carbohydrate Polymers, 63 (3), 393 - 399. Hussain, F., Hojjati, M., Okamoto, M., & Gorga, R. E. (2006). Revie w article: polymer - matrix nanocomposites, processing, manufacturing, and application: an overview. Journal of composite materials, 40 (17), 1511 - 1575. Ivanov, V. (1957). Structure of the cellulose molecule and its degradation. Russian Chemical Bulletin, 6 ( 3), 367 - 374. Jääskeläinen, A., Sun, Y., Argyropoulos, D., Tamminen, T., & Hortling, B. (2003). The effect of isolation method on the chemical structure of residual lignin. Wood Science and Technology, 37 (2), 91 - 102. Jegal, J., Oh, N. W., Park, D. S., & L ee, K. H. (2001). Characteristics of the nanofiltration composite membranes based on PVA and sodium alginate. Journal of Applied Polymer Science, 79 (13), 2471 - 2479. Jia, D., Fang, Y., & Yao, K. (2009). Water vapor barrier and mechanical properties of konj ac glucomannan chitosan soy protein isolate edible films. Food and bioproducts processing, 87 (1), 7 - 10. Jideani, V., & Vogt, K. (2014). Antimicrobial Packaging for Extending the Shelf Life of Bread A Review. Critical Reviews in Food Science and Nutrition ( just - accepted), 00 - 00. Johansson, C., Bras, J., Mondragon, I., Nechita, P., Plackett, D., Simon, P., Breen, C. (2012). Renewable fibers and bio - based materials for packaging applications a review of recent developments. BioResources, 7 (2), 2506 - 2552. 113 Joh nston, W., Karchesy, J., Constantine, G., & Craig, A. (2001). Antimicrobial activity of some Pacific Northwest woods against anaerobic bacteria and yeast. Phytotherapy Research, 15 (7), 586 - 588. Jouki, M., Khazaei, N., Ghasemlou, M., & HadiNezhad, M. (2013 ). Effect of glycerol concentration on edible film production from cress seed carbohydrate gum. Carbohydrate Polymers, 96 (1), 39 - 46. Juven, B., Kanner, J., Schved, F., & Weisslowicz, H. (1994). Factors that interact with the antibacterial action of thyme essential oil and its active constituents. Journal of applied bacteriology, 76 (6), 626 - 631. Jwanny, E., Montanari, L., & Fantozzi, P. (1993). Protein production for human use from Sugarbeet: Byproducts. Bioresource Technology, 43 (1), 67 - 70. Kacurakova, M ., Capek, P., Sasinkova, V., Wellner, N., & Ebringerova, A. (2000). FT - IR study of plant cell wall model compounds: pectic polysaccharides and hemicelluloses. Carbohydrate Polymers, 43 (2), 195 - 203. hemical composition of the wood and JA Marsh): A critically endangered species. South African Journal of Botany, 76 (4), 652 - 654. Kanatt, S. R., Rao, M., Chawla, S., & Sharma, A. (2012). A ctive chitosan polyvinyl alcohol films with natural extracts. Food hydrocolloids, 29 (2), 290 - 297. Kawa - Rygielska, J., Pietrzak, W., Regiec, P., & Stencel, P. (2013). Utilization of concentrate after membrane filtration of sugar beet thin juice for ethanol production. Bioresource Technology, 133 , 134 - 141. by - product, in wheat gluten based biodegradable films: mechanical, solubility and water vapor transfer rate properties. Bioresource Technology, 87 (3), 239 - 246. Kester, J., & Fennema, O. (1986). Edible films and coatings: a review. Food technology (USA) . Khazaei, N., Esmaiili, M., Djomeh, Z. E., Ghasemlou, M., & Jouki, M. (2014). Characterization of new biodegradable edible film made from basil seed (Ocimum basilicum L.) gum. Carbohydrate Polymers, 102 , 199 - 206. 114 Kim, D. - Y., Nishiyama, Y., Wada, M., & Kug a, S. (2001). High - yield carbonization of cellulose by sulfuric acid impregnation. Cellulose, 8 (1), 29 - 33. Kim, K. M., Son, J. H., Kim, S. K., Weller, C. L., & Hanna, M. A. (2006). Properties of chitosan films as a function of pH and solvent type. Journal of Food Science, 71 (3), E119 - E124. Kobayashi, M., Funane, K., Ueyama, H., Ohya, S., Tanaka, M., & Kato, Y. (1993). Sugar composition of beet pulp polysaccharides and their enzymatic hydrolysis. Biosci. Biotech. Biochem, 57 (6), 998 - 1000. Kracher, D., Oro s, D., Yao, W., Preims, M., Rezic, I., Haltrich, D., Ludwig, R. (2014). Fungal secretomes enhance sugar beet pulp hydrolysis. Biotechnology journal, 9 (4), 483 - 492. Kumar, R., Hu, F., Hubbell, C. A., Ragauskas, A. J., & Wyman, C. E. (2013). Comparison of l aboratory delignification methods, their selectivity, and impacts on physiochemical characteristics of cellulosic biomass. Bioresource Technology, 130 , 372 - 381. Kumar, R., Mago, G., Balan, V., & Wyman, C. E. (2009). Physical and chemical characterizations of corn stover and poplar solids resulting from leading pretreatment technologies. Bioresource Technology, 100 (17), 3948 - 3962. Lapp, T., & Shrager, B. (1996). Emission Factor Development for the Malt Beverage, Wine, and Distilled Spirits Industries. The Emission Inventory: Key to Planning, Permits, Compliance, and Reporting, 65 , 406. Le Tien, C., Letendre, M., Ispas - Szabo, P., Mateescu, M., Delmas - Patterson, G., Yu, H. - L., & Lacroix, M. (2000). Development of biodegradable films from whey proteins by cro ss - linking and entrapment in cellulose. Journal of agricultural and food chemistry, 48 (11), 5566 - 5575. Lee, S. H., Doherty, T. V., Linhardt, R. J., & Dordick, J. S. (2009). Ionic liquid mediated selective extraction of lignin from wood leading to enhanced enzymatic cellulose hydrolysis. Biotechnology and Bioengineering, 102 (5), 1368 - 1376. Leitner, J., Hinterstoisser, B., Wastyn, M., Keckes, J., & Gindl, W. (2007). Sugar beet cellulose nanofibril - reinforced composites. Cellulose, 14 (5), 419 - 425. 115 Leontowic z, M., Gorinstein, S., Bartnikowska, E., Leontowicz, H., Kulasek, G., & Trakhtenberg, S. (2001). Sugar beet pulp and apple pomace dietary fibers improve lipid metabolism in rats fed cholesterol. Food Chemistry, 72 (1), 73 - 78. Li, C., Knierim, B., Manisseri , C., Arora, R., Scheller, H. V., Auer, M., Singh, S. (2010). Comparison of dilute acid and ionic liquid pretreatment of switchgrass: biomass recalcitrance, delignification and enzymatic saccharification. Bioresource Technology, 101 (13), 4900 - 4906. Li, F. , & Larock, R. C. (2000). Thermosetting polymers from cationic copolymerization of tung oil: synthesis and characterization. Journal of Applied Polymer Science, 78 (5), 1044 - 1056. Li, M., Wang, L. - j., Li, D., Cheng, Y. - L., & Adhikari, B. (2014). Preparatio n and characterization of cellulose nanofibers from de - pectinated sugar beet pulp. Carbohydrate Polymers, 102 , 136 - 143. Li, R., Fei, J., Cai, Y., Li, Y., Feng, J., & Yao, J. (2009). Cellulose whiskers extracted from mulberry: A novel biomass production. C arbohydrate Polymers, 76 (1), 94 - 99. Li, W., Coffin, D. R., Jin, T. Z., Latona, N., Liu, C. K., Liu, B., Liu, L. (2012). Biodegradable composites from polyester and sugar beet pulp with antimicrobial coating for food packaging. Journal of Applied Polymer S cience, 126 (S1), E362 - E373. Limpan, N., Prodpran, T., Benjakul, S., & Prasarpran, S. (2010). Properties of biodegradable blend films based on fish myofibrillar protein and polyvinyl alcohol as influenced by blend composition and pH level. Journal of Food Engineering, 100 (1), 85 - 92. Lin, M., Sosulski, F., & Humbert, E. (1978). Acidic isolation of sunflower pectin. Canadian Institute of Food Science and Technology Journal, 11 (2), 75 - 77. Lin, Z., Renneckar, S., & Hindman, D. P. (2008). Nanocomposite - based l ignocellulosic fibers 1. Thermal stability of modified fibers with clay - polyelectrolyte multilayers. Cellulose, 15 (2), 333 - 346. Liu, B., Bhaladhare, S., Zhan, P., Jiang, L., Zhang, J., Liu, L., & Hotchkiss, A. T. (2011). Morphology and properties of therm oplastic sugar beet pulp and poly (butylene adipate - co - terepthalate) blends. Industrial & Engineering Chemistry Research, 50 (24), 13859 - 13865. 116 Liu, B., Zhang, J., Liu, L., & Hotchkiss, A. T. (2011). Preparation and properties of water and glycerol - plastic ized sugar beet pulp plastics. Journal of Polymers and the Environment, 19 (3), 559 - 567. Liu, L., Liu, L., Fishman, M. L., Hicks, K. B., & Liu, C. - K. (2005). Biodegradable composites from sugar beet pulp and poly (lactic acid). Journal of agricultural and food chemistry, 53 (23), 9017 - 9022. Luo, X., Li, J., & Lin, X. (2012). Effect of gelatinization and additives on morphology and thermal behavior of corn starch/PVA blend films. Carbohydrate Polymers, 90 (4), 1595 - 1600. Magana, C., Núñez - Sánchez, N., Fernán dez - Cabanás, V., García, P., Serrano, A., Pérez - Marín, D., Alcalde, E. (2011). Direct prediction of bioethanol yield in sugar beet pulp using Near Infrared Spectroscopy. Bioresource Technology, 102 (20), 9542 - 9549. McAuliffe, O., Hill, C., & Ross, R. (1999). Inhibition of Listeria monocytogenes in cottage cheese manufactured with a lacticin 3147 producing starter culture. Journal of applied microbiology, 86 (2), 251 - 256. McCabe, D. L., & Block, W. E. (2014). The Plastic Problem: A Free Market Solution. Journal of Accounting, Ethics and Public Policy, 15 (2). McHugh, T. H., Avena Bustillos, R., & Krochta, J. (1993). Hydrophilic edible films: modified procedure for water vapor permeability and explanation of thickness effects. Journal of Food Science, 58 ( 4), 899 - 903. McMillin, K. W. (2008). Where is MAP going? A review and future potential of modified atmosphere packaging for meat. Meat science, 80 (1), 43 - 65. . Incorporation of partially purified hen egg white lysozyme into zein films for antimicrobial food packaging. Food Research International, 39 (1), 12 - 21. Mi, F. - L., Huang, C. - T., Liang, H. - F., Chen, M. - C., Chiu, Y. - L., Chen, C. - H., & Sung, H. - W. (2006). P hysicochemical, antimicrobial, and cytotoxic characteristics of a chitosan film cross - linked by a naturally occurring cross - linking agent, aglycone geniposidic acid. Journal of agricultural and food chemistry, 54 (9), 3290 - 3296. 117 Michel, F., Thibault, J. F. , Barry, J. L., & de Baynast, R. (1988). Preparation and characterisation of dietary fibre from sugar beet pulp. Journal of the Science of Food and Agriculture, 42 (1), 77 - 85. Mikkonen, K. S., Yadav, M. P., Cooke, P., Willför, S., Hicks, K. B., & Tenkanen, M. (2008). Films from spruce galactoglucomannan blended with poly (vinyl alcohol), corn arabinoxylan, and konjac glucomannan. BioResources, 3 (1), 178 - 191. Minelli, M., Baschetti, M. G., Doghieri, F., Ankerfors, M., Lindström, T., Siró, I., & Plackett, D. (2010). Investigation of mass transport properties of microfibrillated cellulose (MFC) films. Journal of Membrane Science, 358 (1), 67 - 75. Mishra, R., Banthia, A., & Majeed, A. (2012). Pectin based formulations for biomedical applications: a review. Asian Journal of Pharmaceutical and Clinical Research, 5 (4), 1 - 7. Mohdaly, A. A., Sarhan, M. A., Smetanska, I., & Mahmoud, A. (2010). Antioxidant properties of various solvent extracts of potato peel, sugar beet pulp and sesame cake. Journal of the Science of Food and Agriculture, 90 (2), 218 - 226. Mojtahedi, M., & Mesgaran, M. D. (2009). Variability in the chemical composition and in situ ruminal degradability of sugar beet pulp produced in North - East Iran. Res J Biol Sci, 4 , 1262 - 1266. Mondal, S., & Hu, J. (2 007). Water vapor permeability of cotton fabrics coated with shape memory polyurethane. Carbohydrate Polymers, 67 (3), 282 - 287. Monsoor, M. A. (2005). Effect of drying methods on the functional properties of soy hull pectin. Carbohydrate Polymers, 61 (3), 3 62 - 367. Moon, R. J., Martini, A., Nairn, J., Simonsen, J., & Youngblood, J. (2011). Cellulose nanomaterials review: structure, properties and nanocomposites. Chemical Society Reviews, 40 (7), 3941 - 3994. Moosavi, A., & Karbassi, A. (2010). Bioconversion of sugar - beet molasses into xanthan gum . Journal of food processing and preservation, 34 (2), 316 - 322. Mosiewicki, M. A., & Aranguren, M. I. (2013). A short review on novel biocomposites based on plant oil precursors. European Polymer Journal, 49 (6), 1243 - 12 56. 118 Müller, C. M., Laurindo, J. B., & Yamashita, F. (2009). Effect of cellulose fibers addition on the mechanical properties and water vapor barrier of starch - based films. Food hydrocolloids, 23 (5), 1328 - 1333. Müller, C. M., Laurindo, J. B., & Yamashita, F. (2011). Effect of nanoclay incorporation method on mechanical and water vapor barrier properties of starch - based films. Industrial Crops and Products, 33 (3), 605 - 610. Nguyen, H. D., Mai, T. T. T., Nguyen, N. B., Dang, T. D., Le, M. L. P., & Dang, T. T . (2013). A novel method for preparing microfibrillated cellulose from bamboo fibers. Advances in Natural Sciences: Nanoscience and Nanotechnology, 4 (1), 015016. Norsker, M., Jensen, M., & Adler - Nissen, J. (2000). Enzymatic gelation of sugar beet pectin i n food products. Food hydrocolloids, 14 (3), 237 - 243. O'SULLIVAN, A. C. (1997). Cellulose: the structure slowly unravels. Cellulose, 4 (3), 173 - 207. Oh, S. Y., Yoo, D. I., Shin, Y., Kim, H. C., Kim, H. Y., Chung, Y. S., Youk, J. H. (2005). Crystalline structure analysis of cellulose treated with sodium hydroxide and carbon dioxide by means of X - ray diffraction and FTIR spectroscopy. Carbohydrate resear ch, 340 (15), 2376 - 2391. Ojagh, S. M., Rezaei, M., Razavi, S. H., & Hosseini, S. M. H. (2010). Development and evaluation of a novel biodegradable film made from chitosan and cinnamon essential oil with low affinity toward water. Food Chemistry, 122 (1), 16 1 - 166. Olsson, A. - M., & Salmén, L. (2004). The association of water to cellulose and hemicellulose in paper examined by FTIR spectroscopy. Carbohydrate research, 339 (4), 813 - 818. er Fruit Stem as Reinforcing Filler in Plastic Composites. BioResources, 8 (4), 5299 - 5308. Padgett, T., Han, I., & Dawson, P. (1998). Incorporation of food - grade antimicrobial compounds into biodegradable packaging films. Journal of Food Protection , 61 (10) , 1330 - 1335. Palmowski, L., & Mller, J. (2000). Influence of the size reduction of organic waste on their anaerobic digestion. Water science and technology, 41 (3), 155 - 162. 119 Pan, X., & Sano, Y. (2005). Fractionation of wheat straw by atmospheric acetic acid process. Bioresource Technology, 96 (11), 1256 - 1263. Pandey, K., & Pitman, A. (2004). Examination of the lignin content in a softwood and a hardwood decayed by a brown rot fungus wit h the acetyl bromide method and Fourier transform infrared spectroscopy. Journal of Polymer Science Part A: Polymer Chemistry, 42 (10), 2340 - 2346. Panten, J., Bertram, H. J., & Surburg, H. (2004). New woody and ambery notes from cedarwood and turpentine oi l. Chemistry & biodiversity, 1 (12), 1936 - 1948. Park, S., Baker, J. O., Himmel, M. E., Parilla, P. A., & Johnson, D. K. (2010). Research cellulose crystallinity index: measurement techniques and their impact on interpreting cellulase performance. Biotechno l Biofuels, 3 (10). Pearson , M. Future Trends in Global Packaging, 2009. World Packaging Organization . Pelissari, F. M., Grossmann, M. V., Yamashita, F., & Pineda, E. A. G. (2009). Antimicrobial, san films incorporated with oregano essential oil. Journal of agricultural and food chemistry, 57 (16), 7499 - 7504. Pereda, M., Amica, G., & Marcovich, N. E. (2012). Development and characterization of edible chitosan/olive oil emulsion films. Carbohydrate Polymers, 87 (2), 1318 - 1325. Pereda, M., Aranguren, M. I., & Marcovich, N. E. (2010). Caseinate films modified with tung oil. Food hydrocolloids, 24 (8), 800 - 808. Pérez, J., Munoz - Dorado, J., de la Rubia, T., & Martinez, J. (2002). Biodegradation and biolo gical treatments of cellulose, hemicellulose and lignin: an overview. International Microbiology, 5 (2), 53 - 63. Phan, N. H., Rio, S., Faur, C., Le Coq, L., Le Cloirec, P., & Nguyen, T. H. (2006). Production of fibrous activated carbons from natural cellulo se (jute, coconut) fibers for water treatment applications. Carbon, 44 (12), 2569 - 2577. Phatak, L., Chang, K., & Brown, G. (1988). Isolation and Characterization of Pectin in Sugar Beet Pulp. Journal of Food Science, 53 (3), 830 - 833. 120 Piyada, K., Waranyou, S., & Thawien, W. (2013). Mechanical, thermal and structural properties of rice starch films reinforced with rice starch nanocrystals. International Food Research Journal, 20 (1), 439 - 449. Pol, H., Dawson, P., Acton, J., & Ogale, A. (2002). Soy Protein Iso late/Corn Zein Laminated Films: Transport and Mechanical Properties. Journal of Food Science, 67 (1), 212 - 217. Postek, M. T., Vladár, A., Dagata, J., Farkas, N., Ming, B., Wagner, R., Wegner, T. H. (2011). Development of the metrology and imaging of cellul ose nanocrystals. Measurement Science and Technology, 22 (2), 024005. Prabuseenivasan, S., Jayakumar, M., & Ignacimuthu, S. (2006). In vitro antibacterial activity of some plant essential oils. BMC Complementary and Alternative Medicine, 6 (1), 39. Pranoto , Y., Rakshit, S., & Salokhe, V. (2005). Enhancing antimicrobial activity of chitosan films by incorporating garlic oil, potassium sorbate and nisin. LWT - Food Science and Technology, 38 (8), 859 - 865. Proctor, V. A., Cunningham, F., & Fung, D. Y. (1988). Th e chemistry of lysozyme and its use as a food preservative and a pharmaceutical. Critical Reviews in Food Science & Nutrition, 26 (4), 359 - 395. Puri, V. P. (1984). Effect of crystallinity and degree of polymerization of cellulose on enzymatic saccharificat ion. Biotechnology and Bioengineering, 26 (10), 1219 - 1222. Ralet, M. - C., Lerouge, P., & Quéméner, B. (2009). Mass spectrometry for pectin structure analysis. Carbohydrate research, 344 (14), 1798 - 1807. Ralet, M. - C., Thibault, J. - F., Faulds, C. B., & Willia mson, G. (1994). Isolation and purification of feruloylated oligosaccharides from cell walls of sugar - beet pulp. Carbohydrate research, 263 (2), 227 - 241. Ramos, M., Jiménez, A., Peltzer, M., & Garrigós, M. C. (2012). Characterization and antimicrobial acti vity studies of polypropylene films with carvacrol and thymol for active packaging. Journal of Food Engineering, 109 (3), 513 - 519. Reddy, N., & Yang, Y. (2005). Biofibers from agricultural byproducts for industrial applications. TRENDS in Biotechnology, 23 (1), 22 - 27. 121 REGNAULT - ROGER, C. (1997). The potential of botanical essential oils for insect pest control. Integrated Pest Management Reviews, 2 (1), 25 - 34. Rhim, J. - W., Park, H. B., Lee, C. - S., Jun, J. - H., Kim, D. S., & Lee, Y. M. (2004). Crosslinked poly (vinyl alcohol) membranes containing sulfonic acid group: proton and methanol transport through membranes. Journal of Membrane Science, 238 (1), 143 - 151. Rojas - Graü, M. A., Avena - Bustillos, R. J., Olsen, C., Friedman, M ., Henika, P. R., Martin - Belloso, O., McHugh, T. H. (2007). Effects of plant essential oils and oil compounds on mechanical, barrier and antimicrobial properties of alginate apple puree edible films. Journal of Food Engineering, 81 (3), 634 - 641. Roncero, M . B., Torres, A. L., Colom, J. F., & Vidal, T. (2005). The effect of xylanase on lignocellulosic components during the bleaching of wood pulps. Bioresource Technology, 96 (1), 21 - 30. Rouilly, A., Geneau - Sbartaï, C., & Rigal, L. (2009). Thermo - mechanical pr ocessing of sugar beet pulp. III. Study of extruded films improvement with various plasticizers and cross - linkers. Bioresource Technology, 100 (12), 3076 - 3081. Rouilly, A., Jorda, J., & Rigal, L. (2006). Thermo - mechanical processing of sugar beet pulp. I. Twin - screw extrusion process. Carbohydrate Polymers, 66 (1), 81 - 87. Rowell, R. (1980). Distribution of reacted chemicals in southern pine modified with methyl isocyanate. Wood Science, 13 (2), 102 - 110. Ruiz, H. A., Cerqueira, M. A., Silva, H. D., Rodríguez - Jasso, R. M., Vicente, A. A., & Teixeira, J. A. (2013). Biorefinery valorization of autohydrolysis wheat straw hemicellulose to be applied in a polymer - blend film. Carbohydrate Polymers, 92 (2), 2154 - 21 62. Salarbashi, D., Tajik, S., Ghasemlou, M., Shojaee - Aliabadi, S., Shahidi Noghabi, M., & Khaksar, R. (2013). Characterization of soluble soybean polysaccharide film incorporated essential oil intended for food packaging. Carbohydrate Polymers, 98 (1), 11 27 - 1136. Salman, F., El - Kadi, R., Abdel - Rahman, H., Ahmed, S., Mohamed, M., & Shoukry, M. (2008). Biologically treated sugar beet pulp as a supplement in goat rations. International Journal of Agriculture and Biology, 10 (4), 412 - 416. 122 Samal, R., & Ray, M. C. (1997). Effect of chemical modifications on FTIR spectra. II. Physicochemical behavior of pineapple leaf fiber (PALF). Journal of Applied Polymer Science, 64 (11), 2119 - 2125. Saxena, A., & Ragauskas, A. J. (2009). Water transmission barrier properties of biodegradable films based on cellulosic whiskers and xylan. Carbohydrate Polymers, 78 (2), 357 - 360. Scholze, B., & Meier, D. (2001). Characterization of the water - insoluble fraction from pyrolysis oil (pyrolytic lignin). Part I. PY GC/MS, FTIR, and func tional groups. Journal of Analytical and Applied Pyrolysis, 60 (1), 41 - 54. Schultz, T. P., Templeton, M. C., & McGinnis, G. D. (1985). Rapid determination of lignocellulose by diffuse reflectance Fourier transform infrared spectrometry. Analytical chemistr y, 57 (14), 2867 - 2869. Segal, L., Creely, J., Martin, A., & Conrad, C. (1959). An empirical method for estimating the degree of crystallinity of native cellulose using the X - ray diffractometer. Textile Research Journal, 29 (10), 786 - 794. Sen, C., Mishra, H ., & Srivastav, P. (2012). Modified atmosphere packaging and active packaging of banana (Musa spp.): a review on control of ripening and extension of shelf life. Journal of Stored Products and Postharvest Research, 3 (9), 122 - 132. Seydim, A., & Sarikus, G. (2006). Antimicrobial activity of whey protein based edible films incorporated with oregano, rosemary and garlic essential oils. Food Research International, 39 (5), 639 - 644. Shafie, A. E., Fouda, M. M., & Hashem, M. (2009). One - step process for bio - scour ing and peracetic acid bleaching of cotton fabric. Carbohydrate Polymers, 78 (2), 302 - 308. Sharma, V., & Kundu, P. (2006). Addition polymers from natural oils a review. Progress in Polymer Science, 31 (11), 983 - 1008. Shen, Z., & Kamdem, D. P. (2015). Devel opment and characterization of biodegradable chitosan films containing two essential oils. International journal of biological macromolecules, 74 , 289 - 296. Shi, W., & Dumont, M. - J. (2014). Review: Bio - based films from zein, keratin, pea, and rapeseed prot ein feedstocks. Journal of Materials Science, 49 (5), 1915 - 1930. 123 Shin, E. W., & Rowell, R. M. (2005). Cadmium ion sorption onto lignocellulosic biosorbent modified by sulfonation: the origin of sorption capacity improvement. Chemosphere, 60 (8), 1054 - 1061. Shojaee - Aliabadi, S., Hosseini, H., Mohammadifar, M. A., Mohammadi, A., Ghasemlou, M., Ojagh, S. M., Khaksar, R. (2013). Characterization of antioxidant - - carrageenan films containing Satureja hortensis essential oil. International journal o f biological macromolecules, 52 , 116 - 124. Sikkema, J., De Bont, J., & Poolman, B. (1995). Mechanisms of membrane toxicity of hydrocarbons. Microbiological reviews, 59 (2), 201 - 222. Silvério, H. A., Flauzino Neto, W. P., Dantas, N. O., & Pasquini, D. (2013 ). Extraction and characterization of cellulose nanocrystals from corncob for application as reinforcing agent in nanocomposites. Industrial Crops and Products, 44 , 427 - 436. Sivarooban, T., Hettiarachchy, N., & Johnson, M. (2008). Physical and antimicrobi al properties of grape seed extract, nisin, and EDTA incorporated soy protein edible films. Food Research International, 41 (8), 781 - 785. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., & Crocker, D. (2008). Determination of str uctural carbohydrates and lignin in biomass. Laboratory analytical procedure . Smith, S. (1986). Polyethylene, low density (pp. 514 - 523): John Wiley & Sons, New York. Snyder, J., & Timell, T. (1955). Molecular properties of native balsam fir cellulose. Sve n papperstidn, 58 , 851 - 859. Spence, K. L., Venditti, R. A., Rojas, O. J., Habibi, Y., & Pawlak, J. J. (2010). The effect of chemical composition on microfibrillar cellulose films from wood pulps: water interactions and physical properties for packaging ap plications. Cellulose, 17 (4), 835 - 848. Stamboulis, A., Baillie, C., & Peijs, T. (2001). Effects of environmental conditions on mechanical and physical properties of flax fibers. Composites Part A: Applied Science and Manufacturing, 32 (8), 1105 - 1115. Sun, J., Sun, X., Zhao, H., & Sun, R. (2004). Isolation and characterization of cellulose from sugarcane bagasse. Polymer Degradation and Stability, 84 (2), 331 - 339. 124 Sun, L., Hu, R., Shen, G., & Zhang, H. (2013). Genetic Engineering Peanut for Higher Drought - a nd Salt - Tolerance. Food and Nutrition Sciences, 4 , 1. Sun, R., & Hughes, S. (1998). Fractional extraction and physico - chemical characterization of hemicelluloses and cellulose from sugar beet pulp. Carbohydrate Polymers, 36 (4), 293 - 299. Sun, R., & Hughes, S. (1999). Fractional isolation and physico - chemical characterization of alkali - soluble polysaccharides from sugar beet pulp. Carbohydrate Polymers, 38 (3), 273 - 281. Sun, X., Sun, R., Fowler, P., & Baird, M. (2004). Isolation and characterisation of cellulose obtained by a two - stage treatment with organosolv and cyanamide activated hydrogen peroxide from wheat straw. Carbohydrate Polymers, 55 (4), 379 - 391. Sun, X., Xu, F., Sun, R., Fowler, P., & Baird, M. (2005). Characteristics of degraded cellulo se obtained from steam - exploded wheat straw. Carbohydrate research, 340 (1), 97 - 106. Sundholm, J., Gullichsen, J., & Paulerpo, H. (1999). Book 5 Mechanical pulping. Papaermaking Science and Technology; Helsinki Fapet Oy . Suppakul, P., Miltz, J., Sonneveld , K., & Bigger, S. W. (2003). Active packaging technologies with an emphasis on antimicrobial packaging and its applications. Journal of Food Science, 68 (2), 408 - 420. Sutton, M. D., & Peterson, J. D. (2001). Fermentation of sugarbeet pulp for ethanol prod uction using bioengineered Klebsiella oxytoca strain P2. Journal of sugar beet research, 38 (1), 19 - 34. - Chargot, M., Cybulska, J., & Zdunek, A. (2011). Sensing the structural differences in cellulose from apple and bacterial cell wall materials b y Raman and FT - IR spectroscopy. Sensors, 11 (6), 5543 - 5560. Tajik, S., Maghsoudlou, Y., Khodaiyan, F., Jafari, S. M., Ghasemlou, M., & Aalami, M. (2013). Soluble soybean polysaccharide: A new carbohydrate to make a biodegradable film for sustainable green packaging. Carbohydrate Polymers, 97 (2), 817 - 824. Tang, S., Zou, P., Xiong, H., & Tang, H. (2008). Effect of nano - SiO 2 on the performance of starch/polyvinyl alcohol blend films. Carbohydrate Polymers, 72 (3), 521 - 526. 125 Teimouri Yansari, A. (2014). Physically effectiveness of beet pulp based diets in dairy cows as assessed by responses of feed intake, digestibility, chewing activity and milk production. Journal of animal physiology and animal nutrition, 98 (1), 158 - 168. Tian, Z., Chauliac, D., & Pull ammanappallil, P. (2013). Comparison of non - agitated and agitated batch, thermophilic anaerobic digestion of sugarbeet tailings. Bioresource Technology, 129 , 411 - 420. Timell, T. E. (1955). Chain Length and Chain - length Distribution of Native White Spruce Cullulose : Pulp and Paper Research Institut of Canada. Togrul, H., & Arslan, N. (2005). Carboxymethyl cellulose from sugar beet pulp cellulose as a hydrophilic polymer in coating of apples. Journal of food science and technology - mysore , 42 (2), 139 - 144. To rul, H., & Arslan, N. (2004). Carboxymethyl cellulose from sugar beet pulp cellulose as a hydrophilic polymer in coating of mandarin. Journal of Food Engineering, 62 (3), 271 - 279. - life of peach and pear by using CMC from sugar beet pulp cellulose as a hydrophilic polymer in emulsions. Food hydrocolloids, 18 (2), 215 - 226. Tongnuanchan, P., Benjakul, S., & Prodpran, T. (2013). Physico - chemical properties, morphology and antioxidant activity of film from fish s kin gelatin incorporated with root essential oils. Journal of Food Engineering, 117 (3), 350 - 360. Tripathi, S., Mehrotra, G., & Dutta, P. (2010). Preparation and physicochemical evaluation of chitosan/poly (vinyl alcohol)/pectin ternary film for food - packa ging applications. Carbohydrate Polymers, 79 (3), 711 - 716. Trumbo, D., & Mote, B. (2001). Synthesis of tung oil diacrylate copolymers via the Diels Alder reaction and properties of films from the copolymers. Journal of Applied Polymer Science, 80 (12), 2369 - 2375. Türünç, O., & Meier, M. A. (2013). The thiol ene (click) reaction for the synthesis of plant oil derived polymers. European Journal of Lipid Science and Technology, 115 (1), 41 - 54. 126 Valenta, C., Schwarz, E., & Bernkop - Schnürch, A. (1998). Lysozyme - c affeic acid conjugates: possible novel preservatives for dermal formulations. International journal of pharmaceutics, 174 (1), 125 - 132. Van Soest, P. u., & Wine, R. (1967). Use of detergents in the analysis of fibrous feeds. IV. Determination of plant cell - wall constituents. J. Assoc. Off. Anal. Chem, 50 (1), 50 - 55. Vertuccio, L., Gorrasi, G., Sorrentino, A., & Vittoria, V. (2009). Nano clay reinforced PCL/starch blends obtained by high energy ball milling. Carbohydrate Polymers, 75 (1), 172 - 179. Villaverde , J. J., Ligero, P., & Vega, A. d. (2009). Bleaching Miscanthus x giganteus Acetosolv pulps with hydrogen peroxide/acetic acid. Part 1: Behaviour in aqueous alkaline media. Bioresource Technology, 100 (20), 4731 - 4735. Warren, H., Scollan, N., Nute, G., Hug hes, S., Wood, J., & Richardson, R. (2008). Effects of breed and a concentrate or grass silage diet on beef quality in cattle of 3 ages. II: Meat stability and flavour. Meat science, 78 (3), 270 - 278. Wen, L. - F., Chang, K., Brown, G., & Gallaher, D. (1988). Isolation and characterization of hemicellulose and cellulose from sugar beet pulp. Journal of Food Science, 53 (3), 826 - 829. Willats, W. G., McCartney, L., Mackie, W., & Knox, J. P. (2001). Pectin: cell biology and prospects for functional analysis Plant Cell Walls (pp. 9 - 27): Springer. Xiao, C., & Anderson, C. T. (2013). Roles of pectin in biomass yield and processing for biofuels. Frontiers in plant science, 4 . Xiao, C., Liu, H., Gao, S., & Zhang, L. (2000). Characterization of poly (vinyl alcohol) - kon jac glucomannan blend films. Xu, Y., Kim, K. M., Hanna, M. A., & Nag, D. (2005). Chitosan starch composite film: preparation and characterization. Industrial Crops and Products, 21 (2), 185 - 192. Yang, H., Yan, R., Chen, H., Lee, D. H., & Zheng, C. (2007). Characteristics of hemicellulose, cellulose and lignin pyrolysis. Fuel, 86 (12), 1781 - 1788. 127 Yang, X., Yang, K., Wu, S., Chen, X., Yu, F., Li, J., Zhu, Z. (2010). Cytotoxicity and wound healing properties of PVA/ws - chitosan/glycerol hydro gels made by irradiation followed by freeze thawing. Radiation Physics and Chemistry, 79 (5), 606 - 611. - pyrolysis of lignite and sugar beet pulp. Energy Conversion and Management, 51 (5), 1060 - 1064. Yoon, S. - D., Park, M. - H., & Byun, H. - S. (2012). Mechanical and water barrier properties of starch/PVA composite films by adding nano - sized poly (methyl methacrylate - co - acrylamide) particles. Carbohydrate Polymers, 87 (1), 676 - 686. Y ousef , A. E. (1999). Cha racteristics of Listeria monocytogenes important to food processors. Listeria: Listeriosis, and Food Safety , 131. Zhang 1, Y., Sjögren, B., Engstrand 2, P., & Htun, M. (1994). Determination of charged groups in mechanical pulp fibres and their influence o n pulp properties. Journal of wood chemistry and technology, 14 (1), 83 - 102. Zhang, X., Burgar, I., Lourbakos, E., & Beh, H. (2004). The mechanical property and phase structures of wheat proteins/polyvinyl alcohol blends studied by high - resolution solid - st ate NMR. Polymer, 45 (10), 3305 - 3312. Zhao, H., & Baker, G. A. (2013). Ionic liquids and deep eutectic solvents for biodiesel synthesis: a review. Journal of Chemical Technology and Biotechnology, 88 (1), 3 - 12. Zhao, H., Kwak, J. H., Conrad Zhang, Z., Brown, H. M., Arey, B. W., & Holladay, J. E. (2007). Studying cellulose fiber structure by SEM, XRD, NMR and acid hydrolysis. Carbohydrate Polymers, 68 (2), 235 - 241. Zheng, Y., Yu, C., Cheng, Y. - S., Lee, C., Simmons , C. W., Dooley, T. M., VanderGheynst, J. S. (2012). Integrating sugar beet pulp storage, hydrolysis and fermentation for fuel ethanol production. Applied Energy, 93 , 168 - 175. Zhong, Y., Song, X., & Li, Y. (2011). Antimicrobial, physical and mechanical pr operties of kudzu starch chitosan composite films as a function of acid solvent types. Carbohydrate Polymers, 84 (1), 335 - 342. 128 Zhou, G., Taylor, G., & Polle, A. (2011). FTIR - ATR - based prediction and modelling of lignin and energy contents reveals independe nt intra - specific variation of these traits in bioenergy poplars. Plant Methods, 7 (9), 1 - 10. lignocellulosic wastes to improve biogas production. Waste management, 32 (6), 1131 - 1137.