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DEVELOPMENT AND CHARACTERIZATION OF FLEXIBLE FILMS MADE OF
SUGAR BEET LIGNOCELLULOSE
By
Zhu Shen
A DISSERTATION
Submitted to
Michigan State University
in 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 applications 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 flexible films with antimicrobial activity.
The first 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 lignocellulose (SBL). Cellulose content of SBL was increased by sulfuric
acid pretreatment 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
spectroscopic 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 thermal gravimetric
analysis (TGA) was used to demonstrate the increased thermal stability after acid pretreatment
due to the increased contribution of stable cellulose crystals. TGA curves after sulfuric acid
pretreatment demonstrated a two-stage thermal degradation 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 composites 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 dispersions 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 × 10−10 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 studied variables into account, composite films formulated with
50% PVOH are most suitable for various packaging applications.
In the third objective, SBL films were developed with cedarwood oil (CWO) and tung oil to
improve the water barrier properties and antimicrobial activity. The microstructure of the
composite films was characterized through Fourier transform infrared spectroscopy (FTIR). The
results showed that cedar and tung 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 tung oil. These results showed the hydrophobicity of the SBL
films was increased by adding oils. Antimicrobial properties of the films were improved 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
introduction of oils showed no obvious change in 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 so 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 would 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 States.
I thank my wife Mingwei Yan for without her editing, insight, encouragement, love and support,
I would be empty, without form, and void.
Finally, I dedicate this project to my grandfather, Zhenguang Shen; my parents, Guozheng Shen
and Yaping Zhou; my parents in law, Xiping Yan and Shanhua Wei; my uncle and aunt,
Xiaoping Shen and Mingxia Ding, who provided a support system that I could not do without.
v
TABLE OF CONTENTS
LIST OF TABLES ................................................................................................................. viii
LIST OF FIGURES ................................................................................................................. ix
Chapter 1 Introduction ...............................................................................................................1
1.1. Introduction ..........................................................................................................................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 characterization 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
3.2.3.3. α-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. Thermogravimetric 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 (vinyl 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. Preparation of SBL ......................................................................................................53
4.2.3. Chemical composition of SBL .....................................................................................54
4.2.4. Preparation of films .....................................................................................................54
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. Materials 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 oC .............................23
Table 3 Water vapor permeability (WVP) of biomass based film at 25 oC ................................25
Table 4 Chemical composition of SBL powders at different stages ...........................................42
Table 5 Main functional 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 (Postek 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) I1550/I1315, (2) I1550/1157 .............................................................................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
significant differences (p < 0.05) between the means obtained in Duncan’s test. ........................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 < 0.05) between the means
obtained in Duncan’s test. .........................................................................................................63
Figure 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. .................................64
Figure 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. ..............................................................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/PVOH 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 incorporated 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, consumers 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 human 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 bio-
refinery platform to extract cellulose and hemicellulose for the production of flexible packaging
alternatives (Björkman, 1956; Epstein et al., 1976; Lee et al., 2009). Today, competition from
different sectors, such as the construction industry, furniture manufacturing, solid wood
packaging, and the pulp and paper industry, has made this option less cost competitive. Several
other potential bio-products from agricultural residues and industries are available including
starch from rice and potatoes (Piyada et al., 2013), corn (Ghasemlou et al., 2013); proteins from
peas and peanuts (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 jute
(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 (SBL). SBL 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 SBL generated after the extraction of sugar is used for low
value animal food and energy production through combustion or fermentation to generate
alcohol. SBL contains about one-third cellulose, one third hemicelluloses, 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 SBL 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 successful, 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
sectors of the business in US and in the world. Some efforts have been reported on the use of
SBL for the formulation of flexible film. Dufresne et al. (1997) noted that cellulose extracted
3
from SBL 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
SBL film in packaging. Leitner et al. (2007) investigated a thin film with tensile strength higher
than 100 MPa made from SBL nano-cellulose. A serial of chemical and high pressure
pretreatment was used to extract less than 10% nano-cellulose from SBL resulting in relatively
high cost, which is a well-known limiting 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 environmentally acceptable film from the
processing perspective.
As a bio-based polymer, films made from SBL are likely 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
lignocellulosic based materials. One simple and economical way to improve the properties of
biopolymers 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 organic lipids that naturally occur in wood and
plants contain saturated and unsaturated aromatic compounds, such as terpenes, monoterpenes,
thujone, polyphenols, tannins, alkenes, flavonoids, cedrol, and phenolic acids (Fernández‐Pan
4
et al. 2013; Türünç and Meier 2013). Most of these compounds are typically 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 effect of pretreatment on the chemical composition of SBL 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 tung oil into SBL films to improve the hydrophobicity
and antimicrobial activity.
5
Chapter 2 Literature Review
2.1. Sugar beet lignocellulose (SBL)
Sugar beet (Beta vulgaris), is used to extract sugar and the byproduct after the removal of sugar
is known as sugar beet lignocellulose (SBL). SBL 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
past decade, the chemical composition of SBL has been well documented. SBL contains
relatively low lignin content ranging 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 SBL cell wall
2.2.1 Chemical composition of SBL cell wall
The major components of the SBL 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
processing (Mojtahedi and Mesgaran, 2009).
6
It is reported that cellulose makes up about 30% of the dry weight of the SBL cell (Dinand et al.,
1996; Dinand et al., 1999). This line carbohydrate consists of a linear chain of 𝛽 -D-
glucopyranose units linked by glucoside bonds between their C-1 and C-4 hydroxyl groups.
Figure 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
cellobiose constitute the microfibrils, which are grouped 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 regions, 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 regions
besides the crystalline regions, which is often referred to as amorphous (O'Sullivan, 1997). In
these areas, 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).
Figure 1 Molecular structure of cellulose
In most biomass, such as SBL, the cellulose microfibrils are linked to hemicelluloses, pectin, and
lignin. Hemicelluloses are a series of complex carbohydrate polyols which make up 25-30% of
the total dry weight. Compared to cellulose, hemicellulose is a carbohydrate with lower
molecular weight. The major hemicelluloses type in SBL is reported to be arabinan, a branched
polymer composed of α-1,5-linked L-arabinose and α-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
SBL (Phatak et al., 1988). The highest 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 SBL
The cell wall of SBL has both a primary cell wall, which accommodates the cell as it grows; and
a secondary cell wall, which develops 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 SBL is normally thinner than 100 nm, in sharp contrast with the
secondary cell wall, which can reach several micrometers (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
SBL 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 in a tight network (Dinand et al., 1999). The structure of the SBL cell
wall is shown in Figure 2.
2.3. Isolation and characterization of components from SBL
After harvesting of sugar beet, SBL 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 (SBL) (Lapp and Shrager, 1996).
Figure 2 Structure of SBL from plant to the fiber (Postek et al., 2011; Xiao and Anderson, 2013)
It was reported total approximately 67% of SBL 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). Several 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 is to reduce the
9
particle size of the biomass and therefore increase the available surface to the chemicals
(Palmowski and Mller, 2000). Then, extractives and protein can be easily removed from SBL
before isolation.
2.3.1. Isolation and characterization of pectin from SBL
Pectin can be extracted from biomass by hot water, weak acids or chelating agents like
ethylenediaminetetraacetic acid (EDTA) or ethylenediaminetetraacetic acid (CDTA)
(Ebringerova et al., 2005). Lin et al. (1978) investigated an acidic method to isolate pectin from
sunflower heads. This method was modified by Phatak et al. (1988) and they were able to isolate
pectin from SBL. In detail, SBL 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 centrifuged, filtered,
and washed with 45% ethanol. The resulting solid was pectin. It was also reported that pectin in
SBL can be isolated by chelating agents (Furda, 1981). In this method, ethylenediamine
tetraacetic acid (EDTA) and disodium hydrogen phosphate were used to dissolve pectin into
chelating agents and then precipitated and isolated using ethanol. The pectins from SBL 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 authors also investigated the molecular weight distribution of the isolated pectin (Furda,
1981). Pectin isolated using the EDTA 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 acidic treatment is more included to modify/degrade pectin from SBL.
10
2.3.2. Isolation and characterization of hemicellulose from SBL
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 fractionate
and isolate hemicelluloses from wood and SBL (Sun and Hughes, 1998; Wen et al., 1988). In
general, KOH and NaOH are used to dissolve hemicellulose from pectin-free SBL. After
filtration, the hemicelluloses are precipitated in 95% ethanol solution. Sun and Hughes (1998,
1999) reported on the properties of SBL hemicelluloses isolated using this method. They
indicated that the main sugar components of isolated hemicelluloses from SBL are arabinose,
glucose, galactose, xylose, and minor quantities of rhamnose. Some scientists compared the
effect of delignification raw SBL on the range of molecular weight of hemicelluloses.
Hemicelluloses isolated from delignificated SBL have a much lower molecular weight (21,620 to
21,990) compared to those from SBL without delignification. This confirmed that the
delignification triggered degradation of hemicelluloses in SBL.
2.3.3. Isolation and characterization of cellulose from SBL
After the removal of extractives, lignin, and hemicelluloses from SBL, the remainder is assumed
to be cellulose and minerals. After alkaline treatments to dissolve hemicelluloses, the insoluble
products were immersed in a sodium chlorite solution for delignification. Then, the minerals
were removed by washing with abundant running water using a nylon sieve filter.
Physical, mechanical and chemical properties of cellulose are strongly related to the degree of
polymerization (DP) and the crystalline index (CI) which depends on the process method used to
isolate the cellulose (Szymańska-Chargot 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-permeation chromatography (GPC). The
viscometry 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 SBL 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 index, defined as the relative amount of crystalline material in cellulose
(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 equation below is used to calculate the CI of
cellulose from the XRD spectra using the peak intensity method (Segal et al., 1959).
𝐶𝐼 =𝐼002 − 𝐼𝑎𝑚
𝐼002× 100%
where I002 is the intensity of the peak at 2θ = 22.5o and Iam is the intensity corresponding to the
amorphous content at 2θ = 18o 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 on 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):
𝐶𝐼 =𝐴86−92 𝑝.𝑝.𝑚.
𝐴79−92 𝑝.𝑝.𝑚.× 100%
where 𝐴79−86 𝑝.𝑝.𝑚. is the area of the crystalline peak (79 to 86 ppm) and 𝐴79−92 𝑝.𝑝.𝑚.the total
area (crystalline and amorphous) assigned to the C4 peak (79-92 ppm). Heux et al. (1999)
investigated the CI of cellulose from SBL before and after the removal of pectin and
hemicelluloses by the NMR method. They indicated that the CI of cellulose in SBL before pectin
and hemicelluloses removal was around 40%. The CI increased 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 from 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 utilizations of sugar beet residues bring little economic benefit to farmers
(Finkenstadt, 2013). Several attempts have been made to create high value-added products from
agricultural residues that will improve farmers’ revenues and increase US agriculture
competitiveness while protecting our natural resources and environment. Uses of SBL will
contribute to the above objective.
2.4.1. Food ingredients
It is reported that sugar beet fibers contain about 8% protein and 67% carbohydrate including
hemicellulose, cellulose 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
positive results of diet rich in sugar beet fibers on lowering humans’ cholesterol levels. Protein
isolated from SBL was evaluated as a food component in comparison to other leafy green matter
and the outcome is positive (Jwanny et al., 1993). Moreover, SBL can be used as the
carbohydrate sources to produce xanthan, 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 phenolic 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 SBL. Ziemiński et al. (2012) reported producing
biogases from SBL after a variety of pre-treatments.
2.4.3. Sources for polymers and composites
A great deal of research efforts have been focused on the use of SBL as raw materials for
biopolymers. SBL was chemically and/or physically pretreated before uses to improve some
properties. Rouilly et al. (2006) reported on the mechanical properties of composite 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 oC and an injection
pressure of 1500kg·cm-2
, they successfully produced SBL based thermoplastic. This SBL
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 SBL film made through extrusion methods at 100 oC by adding plasticizers or
cross-linkers at a concentration of 30% w/w. They reported that the addition of xylitol increases
the elongation at break from 1 to 11.3%. The authors attributed this phenomenon to the strong
hydrogen bonding interactions between the cellulose microfibrils and the xylitols.
15
Liu et al. (2011b) used SBL without pretreatment using various concentrations 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, SBL can be used to make sheet with
tensile strength of 9.3 MPa. It was further found that lower strength and modulus of elastic
occurred at a higher concentration of glycerol.
In addition, SBL were shown to be useful as an additive to reinforce other materials. Fišerová et
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 properties 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 SBL.
Chen et al. (2008) reported using a twin screw extruder to make poly (lactic acid) (PLA) and
SBL composites at various concentrations. They found that the tensile strength of the
combination of SBL and polylactic acid (PLA) at a ratio of 30/70 (w/w) approached that of neat
PLA with an addition of 2% of polymeric diphenylmethane diisocyanate (pMDI). They
attributed this phenomenon to the penetration of PLA into the SBL particles and the improved
interfacial adhesion produced by pMDI.
Liu et al. (2011a) demonstrated SBL can make thermoplastic sheets with polybutylene adipate-
co-terephthalate (PBAT) by extrusion. However, these resulting plastics have relatively low
tensile strength (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 worked as an interfacial modifier and improved the adhesion and the dispersion of SBL
phase in the blends, which resulted in increased tensile strength.
On the other hand, it has been shown that with some chemical treatments, SBL can be purified to
get plastics with higher properties. Dufresne et al. (1997) first addressed the mechanical
properties of film based on SBL before and after alkali extraction. In this paper, the authors using
a casting method produced SBL film with maximum tensile strength around 8 MPa. Moreover,
the authors indicated that at a high relative humidity, the tensile modulus of the resulting film
with purified SBL 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 film made with nano-cellulose from
SBL by the cast method. The 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 strength 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 industrial use.
In addition to making plastics and composites, a few attempts have been made to assess the
effect of SBL coatings on actual foods for human consumption. Most of the work has centered
on the barrier properties of SBL film.
Toğrul and Arslan (2004) evaluated the capacity of carboxymethyl cellulose (CMC) from SBL to
extend the shelf-life of peaches and pears at 25 oC and 75% relative humidity. The study showed
17
that the coated samples delayed the losses on soluble solids, titratable acidity and ascorbic acid in
comparison to the uncoated peaches and pears. Shelf-life of the coated sample had been extended
to 12 and 16 days for peaches and pears, respectively. Extensive investigation (Toǧrul and
Arslan, 2004) on mandarin oranges and apples (Togrul and Arslan, 2005) also showed similar
result, extending the shelf-life of mandarin oranges and apples up to 27 and 34 days, respectively,
without significantly (P < 0.05) loss of soluble solids, titratable acidity and ascorbic acid.
2.5. Lignocellulosic flexible 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 compare 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 strength (𝜎) 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
multiplied by the thickness. The values are expressed in pound-force per square inch (psi) in US
standard and in N/m2 in SI. It is also known as stress.
𝜎 =𝐹
𝐴
18
The change in length (L) of a film under tensile stress is the strain (ε ) calculated as the percent
change in length (∆𝐿) with the units in %. .
ε =∆𝐿
𝐿0
∆L is the stretch or change in the initial length from Lo to L under tensile stress.
A typical stress-strain curve is shown in Figure 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 deformation.
The relation between the modulus (E), the stress and the strain is also known as Hooke’s law
𝜎 = 𝐸휀
E is the Young’s modulus (elastic modulus), 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 tests 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 failure; the elastic modulus or Young’s modulus and the tensile energy to break (TEB)
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 process
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
fibers. This pulp type has high 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 with the
hydrogen bonding between fibers, yielding a relatively low strength film as in the case of
newsprint (Biermann, 1996).
σ
Ɛ
E
20
The chemi-mechanical pulping process consists of a particularly mild chemical treatment
followed by mechanical refining to liberate fibers. Some lignin, extractives and hemicellulose are
released in the pulping effluents (Biermann, 1996). The action of the mechanical process is less
intense compared to mechanical pulping, longer fibers and less lignin are present on fiber surface.
Yield of chemi-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 chemical 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 compared with that of other pulping process. The chemical
pulping process produces longer fibers due to the absence of grinding, which also leads to a
higher tensile strength (Gullichsen and Fogelholm, 1999).
2.5.2.2. Effect of moisture content
Moisture content also affects the tensile properties of lignocellulosic materials. This is attributed
to both the binding and lubricant functions of water, which helps develop Van der Waals’ forces
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 content 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
molecules, which resulted in improvement of the fiber’s tensile properties.
2.5.2.3. Effect 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
using wood fiber with a particle size lower than 150 μm to produce a copolymer with ethylene
vinyl acetate that will have higher tensile properties than that produced with larger particles.
They also demonstrated that smaller size composites had better filler dispersion and filler-matrix
interaction than composites made from larger 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 including
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 these 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)
investigated the plasticizers’ effect on the mechanical properties of composite films based on
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 found 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 to a higher efficiency of the stress transfer 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 mechanism
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 based film improves 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 prevent water sensitive products’ quality loss
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 oC
Sample TS (MPa) EB (%) Young’s Modulus (GPa) Reference
Gluten/xylan pH 4 2.3 130 0.04 Kayserilioğlu et al., 2003
Gluten/xylan pH11 7.1 26 0.14 Kayserilioğlu et al., 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 thermodynamics 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 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.
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 permeability 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 oC
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-SiO2 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 increased the possibility of intermolecular interactions of cellulose,
which led 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 based film. These
authors found that by forming ester bonds with cellulose, the polycarboxylic acid acted as a
cross-linker, which decreased cellulose chain mobility and increased the resistance to water
vapor transport (Kester and Fennema, 1986).
Moisture barrier 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 transmission 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 improvement 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 increased 2-3 times when relative humidity 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, including polysaccharides, protein, and lipids. For
development and applications 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 still 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.
Kayserilioğlu et al. (2003) investigated the mechanical properties of xylan-wheat gluten based
film at various pH. The authors found that a higher pH in the casting solvent resulted in a higher
tensile strength and Young’s modulus but reduced 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 various 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 properties 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 laminated film had higher TS and EB
than that of unlaminated film. Cho et al. (2010) also found the laminated wheat gluten/ poly
lactic acid had higher TS and EB than neat wheat gluten film.
By using intermolecular forces between different polymers to form composites, the mechanical
properties of the film can be improved. Xu et al. (2005) investigated the chitosan-starch
composite film at various ratios. They showed that the formation of inter-molecular hydrogen
bonds between NH3+ 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 intermolecular 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) determined
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 starch film improved by using
activated montmorillonite (MMT). This paper indicated that a low concentration of MMT
addition (up to 8%) improved the mechanical properties, including TS, EB and Young’s modulus,
of the starch film.
To date, oil addition, one of the most popular strategies, has 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. The acetylated monoglyceride
(AMG) addition reduced water vapor permeability of whey protein films by 50 %. Bertan et al.
(2005) also investigated the effect of fatty acids on water vapor barrier property improvement of
gelatin/triacetin film was positive results. Another possibility 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 crosslinking
and hydrogen bonds associations of cellulose-cellulose, cellulose-protein as well as protein-
protein. Rhim et al. (2004) determinated 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 was no abrupt change of WVP under 35 oC. However, the WVP of uncoated film
increased significantly from 25 oC to 35
oC. They related this phenomenon to the crystal phase of
SMPU under 35 oC. Adding reinforcing 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 property of starch/MMT composite
films. They noted that addition of MMT into starch films improved the water vapor barrier
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 humidities 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 vapor 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 flexible film and coating
One of the most important applications of flexible film and coating is active packaging materials.
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 versions (Suppakul et al., 2003). Many studies have
already been done in developing flexible film and coating with antimicrobial activity, 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 additives 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 Lactococcus lactis (Yousef,
1999). It has been widely suggested that nisin could be incorporated 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 antimicrobial 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 that 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 Ca2+
and Mg2+
)
and therefore improve the antimicrobial activity of nisin.
32
However, as a natural antimicrobial peptide, the widespread application of bacteriocins in food
packaging is limited due to their narrowly screen spectrum and the properties 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 activity 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 enzymes examined as an antimicrobial agent (Mecitoğlu et al., 2006).
This agent possesses enzymatic ability against the 𝜷 -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 films because of its stability 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 effective 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 extracts
Plant extracts are becoming more and more widespread as additives to replace synthetic
chemical products in flexible films and coating due to the consumer demand for more eco-
friendly ingredients and their antimicrobial capabilities. The antimicrobial activities of plant
extracts are possibly related to the functions of their constituents such as phenolic compounds,
terpenoids, essential oils, and weak acids (Hammer et al., 1999). The mechanism 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 membrane and results in the degradation of the cell wall
and damages 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 activity by damaging the cytoplasmic membrane and disturbing 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 effective against Listeria monocytogenes (L. monocytogenes).
Several studies 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 additives 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. Decades ago, the main goal
of using bio-based products was to reduce western dependence on imported oil. Nowadays, the
driving forces behind the use of bio-based products include the control of greenhouse gases, the
sustainability of our planet, and the current climate variation. It is estimated that more than 280
million tons of plastic were produced worldwide in 2012 (McCabe and Block, 2014). The
packaging sector uses about 39%, following by building construction with 20% and automotive
with 8% (Fuentes, 2014). Of the total volume of plastics, 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 extractives 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 regions 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, generating only a low income to
farmers. SBL 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 SBL 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 SBL. Commercial uses of SBL in the
manufacturing of high value products such as flexible packaging will help alleviate the burden
on the environment by replacing slow to non-degradable plastic based materials with degradable
and renewable materials. It will also help generate additional income for sugar beet farmers and
promote rural economics. 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 limits the stiffness
of resulting packaging. The lignin in the matrix resulted 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 structures for centuries (Moon et al., 2011). It can be carried out
in 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 structures (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 involving the use of oxygen and/or carbonate/sulfate are effective
methods due to their ability to produce important oxygen and peroxoacid oxidizing agents in
solution (Jääskeläinen et al., 2003). One of the advantages of peroxoacid is the use of
biodegradable and lower 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 et al., 2009; Zhao et
37
al., 2007). It is postulated that the use of lignin free SBL 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 tensile 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
absorption properties.
The specific objective of this paper is to evaluate the modifications generated by the pretreatment
of the sugar beet residue based lignocellulose by a combined acid and peroxoacid treatment.
Gravimetric analysis using established TAPPI methods was used to monitor and evaluate the
lignin, hemicellulose, and cellulose content before and after pretreatment. Nondestructive solid
state techniques including Fourier Transform Infrared (FTIR) and X-ray diffraction (XRD) were
used to identify and tentatively quantify the modifications of the SBL, such as lignin and
cellulose crystallinity after pretreatment.
3.2. Materials and methods
3.2.1. Materials
Sugar beet pellets were donated from the 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 SBL powder was defined as RSBL. The moisture
content of RSBL was measured by the oven drying method to be 7±1% according to ASTM
D442-07 (ASTM, 2007). Anhydrous calcium sulfate, calcium nitrate and potassium sulfate (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 peroxide (30%) were purchased
from Sigma Aldrich (St. Louis, MO, US) and used as received without further modifications.
3.2.2. Preparation of SBL
RSBL was Soxhlet extracted by following ASTM D1105 (ASTM, 1996) to remove the non-
structural components including waxes and oils. The extractive free sample was soaked in 10%
w/w sulphuric acid solution at 75 oC for 45 min, and then washed with distilled water until pH
neutral and labelled as ASBL. The ASBL was then delignificated during a 24 h treatment with
aqueous solution containing 40% v/v acetic acid, and 3% v/v of hydrogen peroxide at 75 oC. The
delignified pulp was washed with distilled water until a neutral pH and labelled as DSBL. All the
samples were air dried and ground to less than 250 μm for further use.
3.2.3. Gravimetric method to assess chemical composition of SBL
Chemical analysis followed 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 an autoclave tube, and 1ml of 72% sulphuric acid
was added. The mixture was stirred frequently for 1 h at 30 oC water bath, and 28 mL of distilled
water was added to the tube. Then the mixture was autoclaved for 1 hour at 121 oC. After cooling,
the lignin was transferred to the crucible and washed with distilled water repeatedly. The
collected lignin was dried at 105 oC 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 oC 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 filtered and washed in crucible with ethanol and water
respectively. After the washing, the sample was dried in oven at 105 oC for 24 h before weighing.
3.2.3.3. α-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 added to the beaker and stirred for 1 h.
The holocellulose residue was filtered and transferred to the crucible and washed with 8.3% of
sodium hydroxide, water and 10% of acetic acid. The crucible with α-celluloses was dried and
weighed.
3.2.3.4. Hemicellulose content
The hemicellulose content of SBL sample was determined by calculating the difference between
holocellulose and α-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 (RSBL, ASBL, and DSBL),
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. Samples were
scanned using an average of 64 scans over the range between 600 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. Prior to data analysis, all the FTIR spectra were
normalized by ratioing the intensity of the peaks to the intensity of the highest peak in the region
between 2000 and 800 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- Kα
radiation source (λ = 0.154 nm). Samples of particle size less than 250 μm were casted with
double sided tape on a quartz sample holder and scanned at a speed of 2o/min, range from 2θ =
10-40º, at room temperature. Biomass crystallinity as expressed by the crystallinity index (CrI)
was determined according to a method by Segal et al. (1959):
CrI =𝐼002 − 𝐼𝑎𝑚
𝐼002× 100
41
in which, I002 is the intensity for the crystalline portion of biomass (cellulose) at about 2θ = 22.4°,
and Iam is the peak for the amorphous portion (i.e., cellulose, hemicelluloses and lignin) at about
2θ = 16.6°.
3.2.6. Thermogravimetric analysis (TGA)
The thermal 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 controls. Five milligram for each sample was in sample cup
made of aluminium and performed in a nitrogen environment and at a heating rate of 10 oC/min
from 50 to 650 oC. 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 stages. The RSBL consists of
22.2% 𝛼-cellulose, 19.3% hemicellulose, and 5.9% lignin. This composition of hollocellulose
and lignin is similar to that found by Concha Olmos and Zúñiga Hansen (2012) in SBL from
Chile. The differences between our data and theirs may be attributed to various sample sources
and removal of some hemicellulose through the sodium chlorite and acetic acid method (Rowell,
1980). After acid pretreatment, the 𝛼-cellulose and hemicelluloses contents was increased by 115
and 43%, respectively, while Klason lignin was reduced to half of originals. The increased
cellulose and hemicellulose content was attributed to the removal of pectin from SBL after acid
extraction (Phatak et al., 1988). This result suggests that cellulose and hemicelluloses in SBL 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). Compared
with ASBL, the DSBL had more 𝛼-cellulose, less hemicellulose and Klason lignin; the contents
of 𝛼-cellulose, hemicelluloses, and Klason lignin were 80.4%, 10.5%, and 1.0%, respectively,
showing that the acetic acid and hydrogen peroxide pretreatment resulted in a significant level of
delignification. The higher cellulose content and lower hemicellulose content in DSBL compared
with that of ASBL revealed that bleaching removed hemicellulose from ASBL. This result was
similar to that of Kumar et al. (2013), which indicated that the peracetic acid delignification
procedure also result in hemicellulose removal.
Table 4 Chemical composition of SBL powders at different stages
RSBL ASBL DSBL
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
Figure 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 2θ = 22o. 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 crystallinity
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 of 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 crystallinity increased from 34.5% to 59.9%. This increase in crystallinity by means
10 15 20 25 30 35 40
Inte
nsi
ty
Diffraction Angle 2-theta, Degree
RSBL
ASBL
DSBL
Cellulose22.4
26.6
34.7
16.0
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 hemicellulose and lignin during delignification. This fact was confirmed by the
chemical characterization of our samples. In addition to the peak at 22o generally referenced in
the literature for cellulose, another peak appeared in the diffractograms of RSBL, ASBL, and
DSBL at 2θ values of 26.4o. This may be attributed to the pectin in SBL (Combo et al., 2013).
3.3.3. FTIR spectroscopy analysis
Figure 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 main 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 pretreatment. 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 pretreatment. 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-CH3 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-CH3 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. These
results were 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 intensities of 1157 and 1315 cm-1
are
sensitive to the amount of carbohydrate. The intensity ratios I1550/I1157 and I1550/I1315 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 methods and empirical lignin content
measured by these intensity ratios (Scholze and Meier, 2001; Schultz et al., 1985). Figure 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 (I1,550/I1,157 and I1,550/I1,315) ) corresponding to carbohydrate peaks. The coefficient
of 0.93 and 0.98 are shown for the correlation curve between I1,550/I1,157 and I1,550/I1,315.
80010001200140016001800
Ab
sorb
an
ce
Wavenumber (cm-1)
RSBLASBLDSBLLigninCellulose
1550 1315
1246
1203
1157
1103 1053 895 1735 1643
47
Figure 6 Correlation between the lignin content determined by gravimetric method and FTIR
peak intensity ratio (1) I1550/I1315, (2) I1550/1157
3.3.4. Thermal Degradation Behaviour
Figure 7 shows the thermogravimetric analysis of RSBL, ASBL, and DSBL. Cellulose, xylan,
and lignin were run as controls. The initial weight loss at 70 oC was attributed to the evaporation
of the free water in the samples. The curve of RSBL shows a wide decomposition temperature
between 210 and 400 oC, 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 oC,
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
Lig
nin
Con
ten
t (%
)
Intensity ratio
Intensity Ratio 1
Intensity Ratio 2
48
Figure 7 TG and DTG curves of RSBL, ASBL, and DSBL fibers. Cellulose whisker, xylan, and
lignin powder were running as control
0
20
40
60
80
100
50 150 250 350 450 550 650
Wei
ght
(%)
Temperature (oC)
RSBLASBLDSBLCelluloseXylanLignin
0
0.1
0.2
0.3
0.4
0.5
0.6
50 150 250 350 450 550 650
Der
iv .
Wei
ght
Ch
ange
(%
/oC
)
Temperature (oC)
RSBLASBLDSBLCelluloseXylanLignin
49
Compared with the curve of RSBL, the TG and DTG curve of ASBL shows a higher
decomposition temperature which starts at 230 oC and reached a dominant peak at 324
oC. The
increased decomposition temperature was attributed to the removal of pectin, some lignin, and
hemicellulose from SBL 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 pretreatment, therefore reducing the thermal stability of
cellulose as a result of the dehydration reaction (Kim et al., 2001). Another decomposition peak
was found at approximately 550 oC after acid pretreatment. This peak was ascribed to the
breakdown of sulphate 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 showed a higher thermal stability at temperature lower that 250 oC, 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 were found by
Sun et al. (2004b) for straw fibre, which implied that the unbleached cellulose 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 oC
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
samples revealed the partial removal of hemicelluloses and lignin. The crystallinity of the SBL
was increased by 48.7% after sulfuric acid pretreatment and subsequently 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 (Ghasemlou et al., 2011b). The 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
synthetic 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 matter equivalent was left over
by the sugar industry in 2011 in the United States (Finkenstadt, 2013). On a dry weight basis,
SBL contains 75%–80% polysaccharides, consisting roughly of 22%–24% cellulose, 30%
hemicelluloses (mainly arabinans and (arabino) galactans), and 25% pectin. Small amounts of fat,
protein, ash and lignin contents are also present in SBL, 1.4%, 10.3%, 3.7% and 5.9%,
respectively (Sun and Hughes, 1999). Of the compounds mentioned above, SBL 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 SBL is typical
primary wall cellulose (Sun and Hughes, 1999). SBL has been traditionally employed 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 SBL
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
SBL for possible applications as packaging materials.
Polyvinyl alcohol (PVOH), 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 with several natural renewable polymers (Chiellini
et al., 2003). Many researchers have studied various biodegradable packaging composite films
made from PVOH 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 properties of PVOH-SBL blend films.
Based on the considerations 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 SBL via a simple casting
method, 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 materials.
53
4.2. Materials and methods
4.2.1. Materials
Sugar beet pellets were donated by the Michigan Sugar Company (Bay City, USA). This SBL
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 defatted 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 H2SO4) 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 SBL (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 SBL was refrigerated at 4 °C in bottles covered with aluminum foil to prevent direct
exposure to light, until further analysis.
4.2.3. Chemical composition of SBL
The chemical composition of the SBL 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 content 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 hollocellulose
(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 hollocellulose using sodium hydroxide (NaOH) at
room temperature. The hemicellulose content was found by subtracting the cellulose content
from the hollocellulose 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
SBL/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. SBL/PVOH composite films were prepared by mixing different levels of
1% (w/w) purified SBL solutions provided from 4.4.2 section with various levels of PVOH
55
solution (denoted as SBL100, SBL75/PVOH25, SBL50/PVOH50 and SBL25/PVOH75). To
achieve complete dispersion, the mixture was stirred constantly for 40 min using a magnetic
stirrer at 500 rpm at 30 oC. The films prepared without plasticizer were brittle and cracked on the
casting plates during drying. Thus, plasticizer was incorporated into the FFD to achieve more
flexible films. Preliminary experiments were performed to compare the effectiveness of using
sorbitol or glycerol as a plasticizer. Sorbitol gave significantly better results than glycerol with
the latter producing wet films that were difficult to peel. 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 incorporated during stirring. The FFD were spread over polystyrene petri
dishes (15 cm diameter, 30 g FFD 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(NO3)2 solution) in a conditioning
desiccator 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 whole sample was used.
Replicates 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 × 1cm2 were maintained in a desiccator with calcium
sulphate desiccant (0% RH) for 20 days and weighed. Then, dry matter densities were calculated
by Eq. (1).
A
ms
where A is the film area (1 cm2), δ is the film thickness (cm), m is the film dry mass (g) and ρs is
the dry matter density of the film (g/cm3) (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 cup with
a circular opening of 0.000324 m2. 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 saturated 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 0.0001 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® Office Excel 2010 (the lines’ regression coefficients were > 0.998). The water vapor
transmission rate (WVTR) was obtained by dividing the slope (g/h) by the effective film area
(m2). 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:
𝑊𝑉𝑃 =∆𝑚
𝐴∆𝑡
𝑋
∆𝑝
Where ∆𝑚/∆𝑡 is the weight of moisture gain per unit of time (g/s), X is the average film
thickness (m), A is the area of the exposed film surface (m2), and ∆𝑝 is the water vapor pressure
difference between the two sides of the film (Pa). WVP was 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 Machine (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 Young’s modulus were calculated. 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 SBL100,
SBL75/PVOH25, SBL50/PVOH50 and SBL25/PVOH75 using a TGA 2950 with Universal
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 was 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 advanced X-ray
diffractometer (Bruker AXS GmbH, Karlsruhe, Germany) operated at 40 kV and 40 mA,
equipped with Cu- Kα radiation (λ = 1.5406 Å). Samples were scanned over a diffraction angle
(2θ) range of 10-40º, with a scanning rate of 2˚/min at room temperature. The d-spacing was
calculated using Bragg's diffraction equation, λ = 2d sinθ, where λ is the wavelength of the X-ray
radiation used (λ = 1.5406 Å), d is the spacing between diffractional lattice planes and θ is the
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).
Duncan’s multiple range tests were used to compare the differences 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 SBL
The chemical composition of SBL was described in Section 3.3.1.
4.3.2. Appearance and physical properties of the film
The SBL films were flexible and resistant when handled. The composite films formed from SBL
and PVOH were visually homogeneous, with no bubbles or cracks, as well as good handling
characteristics. 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 μm and
53 ± 2 μm and addition of PVOH did not significantly change (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 SBL 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 potentially increase the shelf-
life of the food product (Salarbashi et al., 2013). Figure 8 shows the WVP for different
composite films made with SBL and PVOH. The WVP was 1.78 × 10−10
g s−1
m−1
Pa−1
for the
plasticized SBL film sample. In the present study, the WVP of the SBL/PVOH composite films
was not
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
significant differences (p < 0.05) between the means obtained in Duncan’s test.
significantly (P > 0.05) affected by the inclusion of 25% of PVOH compared to SBL films. The
further addition of PVOH up to 50% to SBL 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
WV
P (
g S-1
m-1
Pa
-1 ×
10-1
0 )
Film type)
a
a
b
b
61
between SBL and PVOH molecules which have the effect of preventing water molecules from
diffusing through the films, 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
PVOH 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 relatively 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 oC 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). The WVP of SBL/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 applications.
4.3.4. Mechanical properties
Mechanical properties of films were characterized by measuring the tensile strength (TS)
elongation at break (EB) and Young’s Modulus, which are key elements of a film’s strength 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 Figure 9. Neat SBL 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). SBL/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 sorbitol 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% (SBL50/PVOH50) 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 contributed to increase in TS in which a higher force was required to
rupture those films but our study on the SBL/PVOH composite films, that is, replacing some
fractions of SBL by PVOH, did not give such a strengthening effect on the composite film. This
result 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 EB values. They suggested
that the formation of intermolecular hydrogen bonds between -OH groups of PVOH with the -
NH2 groups of chitosan is able to improve 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 SBL
films did not show 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% (12.45 ±
63
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 < 0.05) between the means
obtained in Duncan’s test.
b
c
a ab
0
10
20
30
40
50
60
70
SBL100 SBL75/PVA25 SBL50/PVA50 SBL25/PVA75
Ten
sile
str
engt
h (
MP
a)
Film type
b
d c
a
0
2
4
6
8
10
12
14
16
SBL100 SBL75/PVA25 SBL50/PVA50 SBL25/PVA75
Elo
nga
tio
n a
t b
reak
(%
)
Film type
b
a a
b
0
0.5
1
1.5
2
2.5
3
SBL100 SBL75/PVA25 SBL50/PVA50 SBL25/PVA75
Elas
tic
mo
du
lus
(GP
a)
Film type
(A)
(B)
(C)
64
Figure 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
Wei
ght
(%)
Temperature (oC)
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
De
riv.
Wei
ght
Ch
ange
(%
/oC
)
Temperature (oC)
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 SBL/PVOH films was evaluated by thermogravimetric analysis. Figure
10 shows the TGA weight loss and the derivative thermogravimetric (DTG) curves for the pure
films and SBL/PVOH composites in the temperature range from 50 °C to 500 °C. Previous
studies showed that the thermal degradation of SBL follows a two-step weight loss process
(Yılgın et al., 2010). 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 degradation because
PVOH also contains 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%, SBL/PVOH films started thermal degradation at lower temperatures. This
could be associated with the interaction between the SBL and PVOH matrix which might delay
the thermal degradation of the composite films. It can be seen from Figure 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
decomposition and carbon burning. Complete weight loss with a maximum at 333 and 278 °C
for pure SBL and PVOH, respectively, was detected. A similar behavior with SBL was observed
in SBL75/PVOH25, with a maximum at 331 °C, corresponding to the thermal decomposition of
66
the polymer. However, in the SBL25/PVOH75 composite films, a shift to lower temperatures of
about 40 °C was detected, indicating that the thermal degradation process of SBL25/PVOH75
happened at lower temperatures. It appeared that blending SBL 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 SBL and PVOH were subjected to X-ray diffraction (XRD)
analyses. Some typical examples of the results obtained from these analyses are shown in Figure
11. As can be seen from this figure, a very broad peak could be recognized at around 2θ=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 SBL fibers. PVOH showed an obvious
diffraction peak at 2θ=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 components, we expected that the composite
films made from SBL and PVOH would be partially crystalline materials, because the films
made with both pure SBL and pure PVOH, showed partially crystalline structures. The
diffraction peaks at 2θ = 22.27° of SBL crystal and 2θ = 19.58° of PVOH crystal were also
obviously shown in the XRD of composite SBL/PVOH film as shown in Figure 11. This shows
that the blend of SBL and PVOH cannot effectively break the crystals of SBL 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 SBL and PVOH,
became flatter and broader. It could be assumed that intermolecular interactions between SBL
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 reported that
films made with blends of PVOH and konjac glucomannan showed partially crystalline
structures.
Figure 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
Rel
ativ
e in
ten
sity
2 Theta (degrees)
(a)
(b)
(c)
(d)
(e)
68
4.3.7. Surface morphology of blend films
Figure 12 shows the representative electron scanning micrographs of the surfaces of SBL/PVOH
composite films plasticized with sorbitol. As can be seen, the surface of the SBL film was
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
SBL and composite films showed similar surface characteristics, addition of PVOH at higher
content brought out notable difference in the films’ surface microstructure. Insoluble SBL
blended with PVOH was visible in SBL75/PVOH25 films, indicating that SBL and PVOH did
not dissolve each other sufficiently. SBL50/PVOH50 (Figure 12(c)) was comparatively smooth
and the distribution was more uniform with some particles still existing, indicating that the
compatibility of the PVOH and SBL was good. An apparent phase separation was observed in
the SBL25/PVOH75 composite films as shown in Figure 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 samples’
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 compatibility 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 preparation of biodegradable films and coatings. The mechanical properties,
69
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/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 revealed that SBL and PVOH are compatible, and addition of PVOH
reduced 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 structure. While successful films were produced as part of this study, it
is clear that further studies are required to improve film formulations, composition and their
properties. Moreover, further studies need to be started using FT-IR spectroscopy to provide
evidence 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 on 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 sugar 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 cellulosic 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 limitations 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 nutrients.
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
(Fernández‐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 reported 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). Some 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 attributed to thujol, cedrol, α-, and β-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 hydrophobic 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, tensile, microbial growth, thermal properties and chemical interactions between oil and
SBL were monitored using TGA and FTIR.
5.2. Materials and methods
5.2.1. Materials
Dried and roughly ground sugar beet chip was obtained from Michigan Sugar Company (Bay
city, 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 were Soxhlet extracted for 24 h
with ethanol and water. Extractives free powder was treated with a 1 M H2SO4 solution at 75 oC
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 oC 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 rpm to particle size lower than 66 μm before
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 density of 0.94 g/cm3 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 oC in freezer. Subculture was carried out each 14
days to maintain bacterial viability. Bacteria were grown in Brain-heart infusion (BHI) broth at
37 oC at incubation chamber (Sheldon Manufacturing Inc, Cornelius, OR, USA). The bacterial
population in all the inoculated media was estimated to be more than 1 × 107 CFU/ml after 24 h
incubation.
5.2.2. Oil screening for antimicrobial 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. Films 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. About 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 oC for 24
75
h until visual evidence of formation of a film. Dried films were peeled from the Petri dish and
stored in a desiccator at 23 oC 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 cm2 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 oC. After 6 h, the undissolved specimens were dried at 105
oC 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:
𝐹𝑖𝑙𝑚 𝑆𝑜𝑙𝑢𝑏𝑖𝑙𝑖𝑡𝑦 𝑖𝑛 𝑤𝑎𝑡𝑒𝑟 (%) = (𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑂𝐷 𝑤𝑒𝑖𝑔ℎ𝑡 − 𝐹𝑖𝑛𝑎𝑙 𝑂𝐷 𝑤𝑒𝑖𝑔ℎ𝑡
𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑂𝐷 𝑤𝑒𝑖𝑔ℎ𝑡) × 100
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 density
Moisture content of the films was determined by measuring the weight loss of films upon drying
in an oven at 105 oC for 24 hours. To determine film density, samples of 2 × 2 cm were
maintained in a conditioned room at 50 % RH at 23 oC for 48 h and weighed. Densities were
calculated using the following equation:
76
𝜌𝑠 =𝑚
𝐴 × 𝛿
where A is the film area (4 cm2), 𝛿 is the film thickness (cm), 𝑚 and 𝜌𝑠 are the film mass (g) and
density of the film (g/cm3) at 50 % RH at 23
oC, 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 at 50% RH and
23 oC 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 the 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 oC 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
WVP =(WVTR)(thickness)
(pi − po)
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, pi and po the water vapor pressure inside (i) and outside (o) the
capsule. At 25 oC and 75% RH, the value of po is 2369 Pa while the value of pi is 0 Pa for an
absolute value of pi - po 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 width. The tensile properties were measured on specimens
equilibrated at 23 oC 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 tensile strength (TS), elongation at break (EB). The Young’s modulus was
estimated using Hooke’s law 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):
Young′s Modulus =Stress (MPa)
Elongation, %
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
elongation and therefore reduce the Young’s modulus. Four replicates were run for tensile testing
of each type of films.
5.2.4.6. FTIR spectroscopy
FTIR spectra of SBL films were recorded using a Shimadzu IR-Prestige 21 (Columbia, MD.,
USA) equipped with a Pike Technologies Miracle attenuated total reflectance (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 by 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). Samples 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. Differential scanning calorimetry (DSC)
The electrical and mechanical properties of polymers change significantly when the temperature
is close to or exceeds the glass transition temperature (Tg). Therefore, the Tg of SBL films
provides important information about their properties. In this study, Differential scanning
calorimetry (DSC) tests were conducted to determinate the Tg 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. Five milligram sample were placed in aluminum pans and heated up at a heating rate
of 10 oC/min from 0 to 400
oC. 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
drop of 1 μL distilled water and the surface of film. A syringe was used to deposit 1 μL DI water
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 different specimens per each
type of film. All measurements were taken at 50% RH and at a room temperature of 23 oC.
5.2.4.10. Antimicrobial activity of films
Three bacteria namely two Gram-negative, including Escherichia coli (E. coli) and Salmonella
enterica (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 (CFU) of
bacteria that appear on the countable agar plate (between 30 and 300) was counted. For the agar
diffusion methods, the 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 oC
for 24 h. The area of the inhibition zone defined as zone without apparent visual growth of
bacteria was measured with a caliper to the nearest 0.01 mm (Aa) The whole zone area that was
pre-inoculated with bacteria was calculated and used as the potential surface of bacteria growth
and corrected by subtracting the film surface (Ab). 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 antimicrobial 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 microbial 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 cm2 surface inhibition.
The antimicrobial index growth for bacteria used in this work was computed by using the
following equation (5):
Antimicrobial index (%) = Surface inhibited
Total potential surface× 100%
81
The zero or minimum surface inhibition of bacteria was obtained by using film with no oil
content while the maximum inhibition surface was estimated using films containing 2%
tetracycline as reference antibacterial (Bezić et al. 2003).
5.2.4.11. Statistical analysis
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 System Institute Inc., Cary, NC,
USA). Duncan’s multiple range tests were used to compare the differences among 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 plasticizer 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 ± 3 μm. 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/cm3, not 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/cm3 (Özdemir et al. 2013); a
82
density of 1.0 ± 0.1 g/cm3 will correspond to a porosity of about 33% following the equation
below (Twede et all 2015):
Pores , % =SGcell wall − SGfilm
SGcell wall× 100%
where Pores, % is the porosity or the percent volume of pores, SGcell wall the specific gravity of
cell wall close to 1.5 for lignocellulose and SGfilm, 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. Control 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 similar 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 interactions 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 permeability 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 oxidized 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 39.0o was obtained for control films indicating the hydrophilic nature of
lignocellulosic film surface from the numerous hydroxyl groups of SBL polysaccharides. The θ
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 acids 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 Young’s modulus of the
laboratory made films were evaluated and listed in Table 6 as a function of the type 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
elongation of 8% and a Young’s modulus of 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 oil. Films made with 0.1% CWO experienced an
elongation increase from 8.33% to 11.4%, representing an increase of almost 37% compared 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, facilitating the movement of the SBL polysaccharide
chains and improving the film flexibility. At a higher 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 film (Li and Larock, 2000).
The values of Young’s modulus confirm the high rigidity or lower flexibility of the films 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 Oil conc.
(% w/w)
Density
(g/cm3)
Moisture
content (%)
Soluble in
Water (%)
TS (MPa) EB (%) Young’s
modulus (GPa)
Contact angle
at 0 s (o)
Control 0 1.00±0.03a 12.35±0.46a 29.66±0.97a 54.33±4.34a 8.33±1.50bc 1.22±0.13a 38.98±0.30a
CWO 5 10.3±0.04a 12.06±0.39a 25.89±1.45b 47.41±5.01ab 11.37±1.55a 0.77±0.04b 57.73±0.43b
10 0.95±0.03a 10.81±0.27b 24.87±0.89b 36.08±5.13cd 9.85±1.52ab 0.81±0.03b 66.05±0.67c
15 1.00±0.08a 9.85 ±0.60c 21.75±1.22c 28.09±2.87e 6.37±0.47c 0.59±0.03c 75.35±0.28d
Tung oil 5 1.05±0.08a 11.41±0.42ab 25.76±0.86b 45.72±6.91bc 7.71±0.58c 1.23±0.06a 67.58±0.47e
10 0.99±0.02a 9.62±0.30c 22.02±1.04c 34.25±2.98de 3.21±0.54d 1.40±0.10a 76.97±1.16f
15 0.99±0.04a 8.62±0.21d 25.94±0.81b 32.77±1.84e 2.38±0.59d 1.31±0.14a 89.36±0.29g
Values represent the mean ± standard deviation of four replicates for TS, EB, and Young’s modulus; and three replicates for other test
Values in a column having different superscript letters are significantly different (P < 0.05).
86
Figure 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 SBL, SBL-span 80, and SBL containing 5, 10, and 15% CWO or tung oil are shown in
Fig. 14. The broad band at 3305 cm-1
in the SBL 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 SBL (Yang et al.,
2007). The peaks at 1440, 1365, 1311, and 1157 cm-1
are attributed to –O-CH3, CH stretching,
CH2 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 bonding forming from the
hydroxyl groups of Span 80.
1.9
2.1
2.3
2.5
2.7
2.9
0 5 10 15
WV
P (
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.
800130018002300280033003800
Ab
sorb
ance
Wavenumber (cm-1)
SBLSBL Span 80CWO 5% w/wCWO 10% w/wCWO 15% w/wCWO
800130018002300280033003800
Ab
sorb
ance
Wavenumber (cm-1)
SBLSBL Span 80Tung oil 5% w/wTung oil 10% w/wTung oil 15% w/wTung oil
2927
2866
1741 1246
1463
a
b
2927
2866
1741 1463
1236
88
The addition of CWO or tung oil into SBL film resulted in the appearance of two new bands at
1741 and 1643 cm-1
from the carbonyl (C=O) and unsaturated bond from oils, respectively.
They are associated with esters groups, carbonyl groups, acid groups, and double bonds from
poly unsaturated palmitic, stearic acid, linoleic chains of tung oil (Pereda et al., 2010; Trumbo
and Mote, 2001); and from the sesquiterpene alcohols, including cedrol, widdrol, sesquiterpenes,
such as cedrene, thujopsene from CWO (Kamatou et al., 2010; Panten et al., 2004). An 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 increase on the amount of
added oils.
5.3.5. Thermal properties of the films
TGA was performed to evaluate the thermal stability of the SBL film with and without oils. TGA
curves and their derivatives (DTG) are shown in Figure 15 and 16. The maximum
decomposition temperature (Td max) and the percentage of residues are listed in Table 7. The (Td
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 containing Tung oil at levels of 5, 10, and
15% w/w, and only four main stages in films containing CWO and films without oil. The 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 50 to 120 oC with Td max of about 82 ± 1
oC. 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
Figure 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
Wei
ght
(%)
Temperature ( oC)
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
Der
iv. W
eigh
t C
han
ge (
%/o
C)
Temperature (oC)
SBL
SBL Span 80
CWO 5% w/w
CWO 10% w/w
CWO 15% w/w
90
Figure 16 Typical results of TGA and DTG curves of SBL films including different
concentration of Tung oil
20
40
60
80
100
50 150 250 350 450 550 650
Wei
ght
(%)
Temperature ( oC)
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
Der
iv. W
eigh
t C
han
ge (
%/o
C)
Temperature (oC)
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)
Td max (oC) Residue
(%) 1o 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 oC 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, Td max at temperature ranging from 350 to 550 oC, is most likely due to the
degradation or decomposition of cellulose components and some oils components (Ahmad et al.
2012; Lin et al. 2008).
92
The fifth stage, Td max around 550 to 650 oC, is attributed 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 oC was considered as residues rich in calcium, charred
compounds impurities from SBL and 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 SBL films exhibited broad endothermic peaks at approximately 35-140
oC, mainly associated with the removal of moisture when the sample was heated up. When
temperature increased further ( > 200 oC), the DSC profile of films showed exothermic peaks at
234-350 oC, mostly attributed to the primary pyrolysis of cellulose components in SBL.
5.3.6. Antibacterial activity
Table 8 shows the inhibition zone in mm2 of the films after exposure to bacterium. Films
containing CWO inhibition all three tested bacteria, while control film without oil and films with
tung oil were not effective against any of the tested bacteria confirming and validating the
robustness of the bacteria growth in this study. Figure 17 shows the inhibitory effect of SBL
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
Figure 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 earlier on the performance of oils
from plant origin on better in inhibition 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 strips increased significantly for all tested bacteria.
0
5
10
15
20
25
0 5 10 15 20
Inh
ibit
ion
Ind
ex
(%)
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 (mm2) Inhibition Index (%)
L. innocua 0 0±0a 0
5 4.77±0.63a 0.82
10 42.17±1.27b 7.22
15 71.50±0.85c 12.24
20 136.01±0.93d 23.29
Tetracycline 584.06±7.65 100
S. enteria 0 0±0a 0
5 0±0a 0
10 32.88±0.80b 6.92
15 49.85±0.56c 10.49
20 94.81±0.81d 20.00
Tetracycline 475.34±8.45 100
E. coli O157:H7 0 0±0a 0
5 0±0a 0
10 36.12±0.71b 6.79
15 61.77±0.77c 11.61
20 107.45±0.97d 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 ofterpenes, α- and β-pinene, α- and β-
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 reaction with thiol groups of enzymes necessary
for DNA 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 spectra 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
effective 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 Work
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 acid 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 of SBL enhanced thermal stability, which makes them as a
promising candidate for use in thermoplastics.
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 were significantly influenced by added amount of PVOH. Introduction of PVOH into
SBL resulted in improvement in tensile properties and water vapor permeability of the film.
Antimicrobial activity is an important consideration for flexible films for active packaging.
Besides improving the water repellency of SBL film, some plant oils can be used as
antimicrobial agent. The SBL film incorporated with cedarwood oil (CWO) was effective against
both Gram-positive and Gram-negative pathogenic food bacteria. The films show better
inhibition of Gram-positive compared to Gram-negative bacteria.
This study indicated the possibility of application of SBL. Its workable mechanical and water
vapor barrier properties, combined 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. However, 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 combining biological, chemical, and physical needs to be considered to
increase the yield as well as economic efficiency of SBL.
In combination with PVOH, the SBL films showed excellent water vapor barrier properties
improvement, but did not contribute to improved tensile properties. In a future study, efforts
should be done to introduce crosslinkers to achieve better tensile properties.
SBL films enriched with plant oils showed 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
Figure 18 Typical DSC thermograms of SBL films incorporated with different concentration of
(a) CWO and (b) Tung oil
0 50 100 150 200 250 300 350 400
End
o
Temperature (oC)
SBLSBL Span 80CWO 5 % w/wCWO 10 % w/wCWO 15 % w/w
0 50 100 150 200 250 300 350 400
End
o
Temperature (oC)
SBLSBL Span80Tung oil 5 % w/wTung oil 10 % w/wTung oil 15 % w/w
a
b
101
Figure 19 Volume kinetics of water droplets deposited on surface of SBL films with different
levels of CWO and Tung oil
Control
CWO 5 % w/w
CWO 15 % w/w
Tung oil 5 % w/w
Tung oil 15 % w/w
t= 0 t= 15 t= 30 t= 60
102
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
L. innocua
S. enteria
E. coli
Control 5 % w/w 10 % w/w 15 % w/w 20 % w/w
103
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