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Louisiana State UniversityLSU Digital Commons
LSU Doctoral Dissertations Graduate School
2005
Development and characterization of antimicrobialedible films from crawfish chitosanKandasamy NadarajahLouisiana State University and Agricultural and Mechanical College
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Recommended CitationNadarajah, Kandasamy, "Development and characterization of antimicrobial edible films from crawfish chitosan" (2005). LSU DoctoralDissertations. 1630.https://digitalcommons.lsu.edu/gradschool_dissertations/1630
DEVELOPMENT AND CHARACTERIZATION OF ANTIMICROBIAL EDIBLE FILMS FROM CRAWFISH CHITOSAN
A Dissertation Submitted to the Graduate Faculty of the
Louisiana State University and Agricultural and Mechanical College
in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
in
The Department of Food Science
by Kandasamy Nadarajah
B.Sc., University of Peradeniya, 1994 M.Sc., University of Peradeniya, 1997
May 2005
ii
Dedicated to My parents
Mr. and Mrs. A. Kandasamy
iii
ACKNOWLEDGEMENTS
First, I would like to express my deepest gratitude to my academic advisor, Dr. Witoon
Prinyawiwatkul for his invaluable enthusiasm, guidance, and encouragement, and for giving me
the opportunity to complete my studies at my own pace.
I express my warmest gratitude to Professor Hong Kyoon No, for sharing his long
experience in the field of chitosan and for his suggestions and advice. I am especially thankful to
Dr. Marlene Janes for allowing me to use her Microbiology Lab and for guiding me with
microbiological techniques throughout this study.
I am thankful to Dr. Joan King, Dr. Frederick Shih, and Dr. Kevin Carman for their
constructive comments and suggestions for the improvement of this dissertation.
I am most grateful to Dr. Subramaniam Sathivel who has been a guide to me throughout
my career. Without his enthusiasm and help, this dissertation would not be materialized.
I am thankful to the whole staff of food science department providing me with the most
pleasant and convenient working environment.
Finally, my warmest thanks go to my parents and wife, Radha, for their everlasting
unconditional support.
Nada, February 2005
iv
TABLE OF CONTENTS
DEDICATION................................................................................................................................ ii
ACKNOWLEDGEMENTS........................................................................................................... iii
LIST OF TABLES........................................................................................................................ vii
LIST OF FIGURES ....................................................................................................................... ix
ABSTRACT.................................................................................................................................... x
CHAPTER 1. INTRODUCTION.................................................................................................. 1 1.1 Introduction............................................................................................................. 2 1.2 References............................................................................................................... 4
CHAPTER 2. LITERATURE REVIEW....................................................................................... 7 2.1 Chitosan .................................................................................................................. 8 2.2 Characterization of Chitosan................................................................................... 9
2.2.1 Degree of Deacetylation of Chitosan.......................................................... 9 2.2.2 Molecular Weight ..................................................................................... 11 2.2.3 Viscosity ................................................................................................... 11
2.3 Sources of Chitosan .............................................................................................. 12 2.3.1 Crawfish Shell Waste as a Source of Chitosan......................................... 13 2.3.2 Extraction of Chitosan from Crawfish...................................................... 13
2.4 Film-forming Ability of Chitosan......................................................................... 16 2.4.1 Film-forming Methods.............................................................................. 16 2.4.2 Film-forming Mechanisms........................................................................ 17 2.4.3 Film Morphology and Defects .................................................................. 17 2.4.4 Function of Plasticizers inFilm Formation ............................................... 18
2.5 Properties of Chitosan and Chitosan Films........................................................... 18 2.5.1 Safety of Chitosan Films........................................................................... 18 2.5.2 Biodegradation of Chitosan and Chitosan Films ...................................... 19 2.5.3 Mechanical Properties of Chitosan Films ................................................. 19 2.5.4 Transport Properties of Chitosan Films .................................................... 20 2.5.5 Antimicrobial Properties of Chitosan and Chitosan Films ....................... 22
2.6 Applications of Chitosan Films ............................................................................ 25 2.6.1 Applications of Chitosan and Chitosan Films in Foods ........................... 26
2.7 References............................................................................................................. 30
CHAPTER 3. PHYSICOCHEMICAL PROPERTIES OF EDIBLE CHITOSAN FILMS DEVELOPED FROM CRAWFISH SHELL WASTE......................................... 42
3.1 Introduction........................................................................................................... 43 3.2 Materials and Methods.......................................................................................... 44
3.2.1 Preparation of Chitosan from Crawfish Shell Waste................................ 44
v
3.2.2 Characterization of Crawfish Chitosan..................................................... 45 3.2.3 Preparation of Crawfish Chitosan Films................................................... 46 3.2.4 Thickness and Density of Crawfish Chitosan Films................................. 46 3.2.5 Color of Crawfish Chitosan Films ............................................................ 46 3.2.6 Transparency of Crawfish Chitosan Films ............................................... 47 3.2.7 Swelling of Crawfish Chitosan Films ....................................................... 47 3.2.8 Solubility of Crawfish Chitosan Films ..................................................... 48 3.2.9 Microstructure of Crawfish Chitosan Films ............................................. 48 3.2.10 Statistical Analysis.................................................................................... 48
3.3 Results and Discussions........................................................................................ 49 3.3.1 Characteristics of Crawfish Chitosan........................................................ 49 3.3.2 Film-forming Ability of Crawfish Chitosans............................................ 50 3.3.3 Thickness and Density of Crawfish Chitosan Films................................. 51 3.3.4 Color of Crawfish Chitosan Films ............................................................ 53 3.3.5 Transparency of Crawfish Chitosan Films ............................................... 55 3.3.6 Swelling of Crawfish Chitosan Films ....................................................... 56 3.3.6 Solubility of Crawfish Chitosan Films ..................................................... 58 3.3.7 Microstructure of Crawfish Chitosan Films ............................................. 59
3.4 Conclusions........................................................................................................... 61 3.5 References............................................................................................................. 62
CHAPTER 4. SORPTION AND WATER PERMEABILITY BEHAVIORS OF CRAWFISH CHITOSAN FILMS.............................................................................................. 65
4.1 Introduction........................................................................................................... 66 4.2 Materials and Methods.......................................................................................... 67
4.2.1 Preparation of Chitosan from Crawfish Shell Waste................................ 67 4.2.2 Characterization of Crawfish Chitosans ................................................... 68 4.2.3 Preparation of Crawfish Chitosan Films................................................... 69 4.2.4 Sorption Isotherm Experiments ................................................................ 69 4.2.5 Water Vapor Permeability of Crawfish Chitosan Films ........................... 71 4.2.6 Statistical Analysis.................................................................................... 71
4.3 Results and Discussion ......................................................................................... 71 4.3.1 Characteristics of Crawfish Chitosan........................................................ 71 4.3.2 Film-forming Ability and Film Characteristics ........................................ 72 4.3.3 Sorption Isotherms of Crawfish Chitosan Films....................................... 73 4.3.4 Sorption Model Analysis .......................................................................... 78 4.3.5 Water Vapor Transmission Rate............................................................... 83
4.4 Conclusions........................................................................................................... 88 4.5 References............................................................................................................. 89
CHAPTER 5. MECHANICAL PROPERTIES OF CRAWFISH CHITOSAN FILMS AS AFFECTED BY CHITOSAN PRODUCTION PROTOCOLS AND FILM CASTING SOLVENTS........................................................................................ 94
5.1 Introduction........................................................................................................... 95 5.1.1 Objective ................................................................................................... 96
5.2 Materials and Methods.......................................................................................... 97
vi
5.2.1 Materials ................................................................................................... 97 5.2.2 Characterization of Crawfish Chitosans ................................................... 97 5.2.3 Film Preparation........................................................................................ 98 5.2.4 Conditioning of Films ............................................................................... 98 5.2.5 Thickness of Films.................................................................................... 98 5.2.6 Tensile Test............................................................................................... 98 5.2.7 Puncture Strength...................................................................................... 99 5.2.8 Statistical Analysis.................................................................................... 99
5.3 Results and Discussions........................................................................................ 99 5.3.1 Tensile Strength ........................................................................................ 99 5.3.2 Effects of Acids and Chitosans on Tensile Properties ........................... 102 5.3.3 Comparison of Crawfish Chitosan Films with Selected Biopolymer .... 104 5.3.4 Comparison of Tensile Properties of Chitosan Films ............................ 105 5.3.5 Puncture Strengths of Crawfish Chitosan Films..................................... 107
5.4 Conclusions......................................................................................................... 109 5.5 References........................................................................................................... 110
CHAPTER 6. EFFICACY OF ANTIMICROBIAL EDIBLE FILMS MADE OF ORGANIC SALTS OF CRAWFISH CHITOSAN ............................................................... 113
6.1 Introduction......................................................................................................... 114 6.1.1 Objective ................................................................................................. 116
6.2 Materials and Methods........................................................................................ 116 6.2.1 Materials ................................................................................................. 116 6.2.2 Characterization of Crawfish Chitosan................................................... 117 6.2.3 Film Preparation...................................................................................... 117 6.2.4 Conditioning of Films ............................................................................. 118 6.2.5 Organisms and Culture Maintenance...................................................... 118 6.2.6 Zone Inhibition Assays ........................................................................... 119 6.2.7 Direct Inoculation Assay......................................................................... 120 6.2.8 Statistical Analysis.................................................................................. 120
6.3 Results and Discussions...................................................................................... 121 6.3.1 Zone Inhibition Test................................................................................ 121 6.3.2 Direct Inoculation Assay......................................................................... 126
6.4 Conclusions......................................................................................................... 133 6.5 References........................................................................................................... 135
CHAPTER 7. CONCLUSIONS................................................................................................. 138
VITA........................................................................................................................................... 142
vii
LIST OF TABLES
3.1 Physicochemical properties of chitosans extracted from crawfish shell .................... 49 3.2 Film-forming ability of unplasticized crawfish chitosan . .......................................... 52 3.3 Thickness and density of unplasticized crawfish chitosan films ................................ 53 3.4 Color attributes of crawfish chitosan films................................................................. 54 3.5 Solubility of unplasticized crawfish chitosan films .................................................... 59 4.1 Isotherm models used for fitting experimental data ................................................... 70 4.2 Constants of sorption models for chitosan acetate films ............................................ 79 4.3 Constants of sorption models for chitosan formate films ........................................... 80 4.4 Constants of sorption models for chitosan citrate films.............................................. 81 4.5 Water vapor transmission rate and water vapor permeability at 25oC and 50% RH
gradient. ...................................................................................................................... 85 4.6 Water vapor permeability of chitosan, edible and plastic films.................................. 87 5.1 Tensile measurements of crawfish chitosan films ................................................... 101 5.2 Comparison of tensile strength and percent elongation values for selected
biopolymer and synthetic polymer films .................................................................. 105 5.3 Comparison of tensile properties of chitosan films with organic acid...................... 106 5.4 Puncture strength of crawfish chitosan films............................................................ 108 6.1 Effect of chitosan films on selected food pathogenic bacteria. ................................ 122 6.2 Recovery of Listeria monocytogenes survivors from crawfish chitosan film with
different organic acids incubated at 37°C for 24 hours. ........................................... 127 6.3 Log reduction of Listeria monocytogenes survivors from crawfish chitosan film
with different organic acids incubated at 37°C for 24 hours. ................................... 128 6.4 Recovery of Staphylococcus aureus survivors from crawfish chitosan film with
different organic acids incubated at 37°C for 24 hours. ........................................... 130
viii
6.5 Log reduction of Staphylococcus aureus survivors from crawfish chitosan film with different organic acids incubated at 37°C for 24 hours. ................................... 130
6.6 Recovery of Salmonella typhimurium survivors from crawfish chitosan film with
different organic acids incubated at 37°C for 24 hours. ........................................... 131 6.7 Log reduction of Salmonella typhimurium survivors from crawfish chitosan film
with different organic acids incubated at 37°C for 24 hours. ................................... 132 6.8 Recovery of Shigella sonnei survivors from crawfish chitosan film with different
organic acids incubated at 37°C for 24 hours. .......................................................... 133 6.9 Log reduction of Shigella sonnei survivors from crawfish chitosan film with
different organic acids incubated at 37°C for 24 hours. ........................................... 134
ix
LIST OF FIGURES 2.1 Chemical structure of cellulose, chitin and chitosan .................................................... 8 2.2 Conversion of chitin into chitosan .............................................................................. 10 2.3 Scheme for chitosan production (Modified from No and Meyers 1995).................... 15 3.1 Transparency of unplasticized crawfish chitosan films .............................................. 56 3.2 The % swelling of crawfish chitosan films formed with acetic and formic acids.. .... 57 3.3 The unplasticized DPMA chitosan film formed with formic acid.............................. 57 3.4 The SEM of crawfish chitosan films. ......................................................................... 60 4.1 Moisture sorption isotherms of representative crawfish chitosan films at 25oC. ....... 74 4.2 Moisture sorption isotherms of chitosan acetate films at 25oC................................... 75 4.3 Moisture sorption isotherms of chitosan formate films at 25oC. ................................ 76 4.4 Effect of water activity on clustering of water molecules in chitosan acetate films... 84 5.1 Stress-strain curves of crawfish chitosan films with organic acid solvents.............. 100 5.2 Tensile strength of films made with different crawfish chitosans and acids.. .......... 102 5.3 Young's modulus of films made with different crawfish chitosans and acids.......... 103 6.1 Inhibition of food pathogenic bacteria by chitosan citrate films.. ............................ 124 6.2 Inhibition zones produced by crawfish chitosan films.. ........................................... 125
x
ABSTRACT Inherent antibacterial/antifungal properties and film-forming ability of chitosan make it
ideal for use as a biodegradable antimicrobial packaging material. This study was attempted to
develop antimicrobial films from crawfish chitosan. Traditional chitosan production involves:
deproteinization (DP), demineralization (DM), decolorization (DC), and deacetylation (DA).
Modification of chitosan production affects film properties. Effects of chitosan production
protocols, film-casting solvents, and plasticizer contents on physicochemical, mechanical and
antibacterial properties were investigated. Four chitosans were prepared from traditional
(DPMCA) and modified processes [excluding either DP, DC or both DP and DC]. Chitosan
(1%w/v) was dissolved in 1% acetic, ascorbic, citric, formic, lactic and/or malic acid, and cast
with and without glycerol (a plasticizer) at a ratio of 1:0.1, 1:0.2, 1:0.3, 1:0.4 and 1:0.5
(chitosan:glycerol, w/w) to form films.
Flexible and transparent films could be prepared from chitosans with acetic, formic or
citric acid without a plasticizer. DMCA acetate films showed higher tensile strength (135.8
MPa), but poor antibacterial properties. DPMCA formate films with tensile strength of 76.8 MPa
reduced microbial loads of Staphylococcus aureus, Salmonella typhimurium, and Shigella
sonnei by more than 2.5 log CFU/mL in 24 hours. DMA citrate films showed tensile strength of
29.3 MPa and reduced Listeria monocytogenes, Staphylococcus aureus, Salmonella
typhimurium, and Shigella sonnei by more than 4.4 log CFU/mL in 24 hours. This study
demonstrated the feasibility of developing antimicrobial edible films from crawfish chitosans.
Some critical factors required for desirable film properties were identified.
1
CHAPTER 1
INTRODUCTION
2
1.1 INTRODUCTION
Chitosan is a carbohydrate polymer that can be derived from crustacean seafood wastes
such as shells of crabs, shrimps and crawfish. Chitosan has a wide range of applications in
diverse fields ranging from medical sutures and seed coatings to dietary supplements and
coagulants for waste treatment. Physicochemical properties of chitosans and their functionalities
are affected by their sources (Rhazi and others 2004) and the methods employed to extract them
(Brine and Austin 1981). Hence, different physicochemical properties and functionalities can be
expected from chitosans derived from crawfish shell by different extraction protocols.
Physicochemical properties of different chitosans derived from crawfish shell waste were
reported earlier (Rout 2001). However, their application potentials in food and other fields are
yet to be investigated. Recently, the potential of developing flexible and transparent edible films
and packaging materials from crawfish chitosan was reported by Nadarajah and Prinyawiwatkul
(2002). Development of antimicrobial edible films and packaging materials from crawfish
chitosans may expand their applications in food systems. The possibility of producing crawfish
chitosans with varying physicochemical properties presents a niche for selecting the most
suitable chitosans for the development of antimicrobial films and packaging materials.
Contamination of food products by pathogenic bacteria has emerged as a serious public
concern. Bacteria such as Listeria monocytogenes, Bacillus cereus, Escherichia coli (O157:H7),
Staphylococus aureus, Salmonella typhimurium, and Shigella sonnei are identified as the most
potent pathogens associated with food born illnesses in U.S.A (Mead 1999). Antimicrobial
packaging is one promising approach to prevent both contamination of pathogens and growth of
spoilage microorganisms on the surface of food. Direct addition of antimicrobial substances into
food formulations or onto food surfaces may not be sufficient to prevent the growth of
3
pathogenic and spoilage microorganisms as antimicrobial substances applied could be partially
inactivated or absorbed by the food systems (Ouattara and others 2000). Antimicrobial films
render sustained release of antimicrobial substances onto the food surface and compensate for
the partial inactivation or absorption of them by food systems (Siragusa and Dickson 1992).
Chitosan is an ideal biopolymer for developing such antimicrobial films due to their non-
toxicity (Hirano and others 1990), biocompatibility (Muzzarelli 1993), biodegradability
(Shigemasa and others 1994), film-forming ability (Averbach 1978) and inherent antimicrobial
properties (Sudarshan and others 1992). Moreover, antimicrobial properties of chitosan can be
enhanced by irradiation (Matsuhashi and Kume 1997), ultraviolet radiation treatment, partial
hydrolyzation (Davydova and others 2000), chemical modifications (Nishimura and others
1984), synergistic enhancement with preservatives (Roller and others 2002), synergistic
enhancement with antimicrobial agents (Lee and others 2003), or in combination with other
hurdle technologies.
One of the simplest and most economical ways of producing antimicrobial films is to
incorporate antimicrobial substances into films (Weng and Hotchkiss 1993). Various organic
acids that naturally occur in fruit and vegetables and possess general antimicrobial activity such
as acetic, lactic, malic, and citric, sorbic, benzoic and succinic acids can be used for this purpose
(Beuchat 1998). Further, since chitosan needs to be dissolved in slightly acidic solutions, the
production of antimicrobial films from chitosan with organic acids is straightforward (Begin and
Calsteren 1999). However, the interaction between the preservatives and the film-forming
material may affect film casting, release of preservative and mechanical properties (Chen and
others 1996).
4
Antimicrobial edible films developed with several organic acids have demonstrated their
effectiveness in reducing bacterial levels on meat products. Baron (1993) showed that edible
corn starch films with potassium sorbate and lactic acid inhibited S. typhimurium and E. coli
(O157:H7) on poultry. Siragusa and Dickson (1992) reported that organic acids were more
effective against L. monocytogenes, S. typhimurium and E. coli (O157:H7) on beef carcass when
immobilized in edible film than when applied directly. Recently, chitosan films containing
acetic and propionic acids controlling Enterobacteriaceae and Serratia liquefaciens on bologna,
regular cooked ham, and pastrami were reported (Ouattara and others 2000). These results
indicate potentials for developing antimicrobial edible films from crawfish chitosan.
This dissertation study was intended to investigate the possibility of producing
antibacterial edible films from crawfish chitosans by (1) extracting various chitosans from
crawfish shell waste by employing different extraction protocols, (2) formulating films with
different organic acids, and (3) screening (i) physicochemical properties, (ii) sorption behaviors,
(iii) water permeability characteristics, (iv) mechanical properties, and (v) antibacterial activities
of the chitosan films to identify properties best suited to develop antimicrobial films from
crawfish chitosan.
1.2 REFERENCES Averbach BL. 1978. Film-forming capability of chitosan. In: Muzzarelli RAA, Pariser ER.
editors. Proceedings of the First International conference on Chitin/Chitosan. MIT: Cambridge, MA. p 199-209.
Baron JK. 1993. Inhibition of S. typhimurium and E. coli O157:H7 by an antimicrobial
containing film [MSc thesis]. Lincoln, Nebr.: University of Nebraska. Begin A, Calsteren MRV. 1999. Antimicrobial films produced from chitosan. Int J Biol
Macromol 26:63-7. Beuchat LR. 1998. Surface decontamination of fruits and vegetables eaten raw: a review. Food
safety issues. Geneva, Switzerland: Food Safety Unit/World Health Organization. 42 p.
5
Brine CJ, Austin PR. 1981. Chitin variability with species and method of preparation. Comp
Biochem Physiol 69B:283-86. Chen M, Yeh GH, Chiang B. 1996. Antimicrobial and physicochemical properties of
methylcellulose and chitosan films containing a preservative. J Food Process Preserv 20:379-90.
Davydova VN, Yermak IM, Gorbach VI, Krasikova IN, Solov’eva TF. 2000. Interaction of
bacterial endotoxins with chitosan: Effect of endotoxin structure, chitosan molecular mass and ionic strength of the solution on the formation of the complex. Biochemistry 65(9):1082-90.
Hirano S, Itakura C, Seino H, Akiyama Y, Nonaka I, Kanbara N, Kanakami T, Arai K and
Kinumaki T. 1990. Chitosan as an ingredient for domestic animal feeds. J Agric Food Chem 38:1214-7.
Lee CH, An DS, Park HF, Lee DS. 2003. Wide-spectrum antimicrobial packaging materials
incorporating nisin and chitosan in the coating. Pack Technol Sci 16:99-106. Matsuhashi S, Kume T. 1997. Enhancement of antmicrobial activity of chitosan by irradiation. J
Sci Food Agric 73:237-41. Mead PS, Slutsker L, Dietz V, McCaig LF, Bresee JS, Shapiro C, Griffin PM, Tauxe RV. 1999.
Food-Related Illness and Death in the United States. Emerging Infectious Diseases 5(5):607-25.
Muzzarelli RAA.1993. Biochemical significance at exogenous chitins and chitosans in animals
and patients. Biomaterials 20:7-16. Nadarajah K. and Prinyawiwatkul W. 2002. Filmogenic properties of crawfish chitosan
[abstract]. In: 54th Pacific Fisheries Technologists Annual Meeting Book of Abstracts; 2002 February 24-27; Reno, NV. Abstract nr 37.
Nishimura K, Nishimura S, Nishi N, Saiki I, Tokura S, Azuma I. 1984. Immunological activity
of chitin and its derivatives. Vaccine 2(1):93-9. Ouattara B, Simard RE, Piette G, Bégin A,. Holley RA. 2000. Inhibition of surface spoilage
bacteria in processed meats by application of antimicrobial films prepared with chitosan. Internat J Food Microbiol 62:139-48.
Rhazi M, Desbrieres J, Tolaimate A, Alagui A, Vottero P. 2004. Investigation of different natural
sources of chitin: influence of the source and deacetylation process on the physicochemical characteristics of chitosan. Polymer International 49(4):337-44.
6
Roller S, Sagoo S, Board R, O’Mahony T, Caplice E, Fitzgerald G, Fogden M, Owen M, Fletcher H. 2002. Novel combination of chitosan, carnocin and sulphite for the preservation of chilled pork sausages. Meet Sci 62:165-77.
Rout SK, Prinyawiwatkul W. 2001. Process simplification and modification affecting physico-
chemical properties of chitosan and conversion efficiency of chitin [abstract]. In: IFT Annual Meeting Book of Abstracts; 2001 June 23-27; New Orleans, LA. Chicago, Ill.: Institute of Food Technologists. Abstract nr 15D-25.
Shigemasa Y, Saito K, Sashiwa H, Saimoto H. 1994. Enzymatic degradation of chitins and partially deacetylated chitins. International Journal of Biological Macromolecules 16(1):43-9.
Siragusa GA, Dickson JS. 1992. Inhibition of Listeria monocytogenes on beef tissue by
application of organic acids immobilized in a calcium alginate gel. J Food Sci 57:293- 96. Sudarshan NR, Hoover DG, Knorr D. 1992. Antibacterial action of chitosan. Food Biotechnol
6(3):257-72. Weng YM, Hotchkiss JH. 1993. Anhydrides as antimycotic agents added to polyethylene films
for food packaging. Packaging Technol Sci 6:123-8.
7
CHAPTER 2
LITERATURE REVIEW
8
2.1 CHITOSAN
Chitosan is a modified natural carbohydrate polymer derived from chitin which has been
found in a wide range of natural sources such as crustaceans, fungi, insects and some algae
(Tolaimate and others 2000). The primary unit in the chitin polymer is 2-acetamido-2-deoxy-β-
D-glucose. These units are combined by 1-4 glycosidic linkages, forming a long chain linear
polymer without side chains. Chitin is chemically identical to cellulose, except that the
secondary hydroxyl group on the alpha carbon atom of the cellulose molecule is substituted with
acetoamide groups (Figure 2.1).
Removal of most of the acetyl groups of chitin by treatment with strong alkali yields
chitosan (Peniston and Johnson, 1980) which is 2-amino-2-deoxy-β-D-glucose. A sharp
nomenclature with respect to the degree of N-deacetylation has not been defined between chitin
and chitosan (Muzzarelli 1977). In general, chitin with a degree of deacetylation of above 70% is
considered as chitosan (Li and others 1997a).
Figure 2.1: Chemical structure of cellulose, chitin and chitosan
Chitosan is insoluble in water but soluble in acidic solvents below pH 6. Organic acids
such as acetic, formic and lactic acids are used for dissolving chitosan, and the most commonly
used solvent is 1% acetic acid solution. Solubility of chitosan in inorganic acid solvent is quite
limited. Chitosan is soluble in 1 % hydrochloric acid but insoluble in sulfuric and phosphoric
acids. Chitosan solution's stability is poor above pH 7 due to precipitation or gelation that takes
9
place in alkali pH range. Chitosan solution forms a poly-ion complex with anionic hydrocolloid
and provides gel.
2.2 CHARACTERIZATION OF CHITOSAN
Chitosan can be characterized in terms of its quality, intrinsic properties such as purity,
molecular weight, viscosity, and degree of deacetylation and physical forms (Sanford 1989). The
quality and properties of chitosan product may vary widely because many factors in the
manufacturing process can influence the characteristics of the final chitosan product (Li and
others 1992).
2.2.1 Degree of Deacetylation of Chitosan
Among many characteristics, the degree of deacetylation is one of the more important
chemical characteristics, which influences the performance of chitosan in many of its
applications (Muzzarelli 1977; Li and others 1992; Baxter and others 1992). In addition, the
degree of deacetylation, which reveals the content of free amino groups in the polysaccharides
(Li and others 1992), can be used to differentiate between chitin and chitosan. In general, chitin
with a degree of deacetylation of above 70% is considered as chitosan (Li and others 1997a). In
the process of deacetylation, acetyl groups from the molecular chain of chitin are removed to
form amino groups (Figure 2.2).
Variables such as temperature or concentration of sodium hydroxide solution affect the
removal of acetyl groups from chitin, resulting in a range of chitosan molecules with different
properties and hence its applications (Baxter and others 1992; Mima and others 1983). Since the
degree of deacetylation depends mainly on the method of purification and reaction conditions
(Baxter and others 1992; Li and others 1997), it is essential to characterize chitosan by
determining its degree of deacetylation prior to its utilization.
10
Figure 2.2: Conversion of chitin into chitosan
A number of methods have been used to determine the degree of deacetylation, such as
linear potentiometric titration (Ke and Chen 1990), infrared spectroscopy (Baxter and others
1992), nuclear magnetic resonance spectroscopy (Hirai and others 1991), pyrolysis-mass
spectrometry (Nieto and others 1991), first derivative UV-spectrophotometry (Muzzarelli and
Rocchetti 1985), and titrimetry (Raymond and others 1993). Some of the methods are either too
tedious, too costly for routine analysis (e.g., nuclear magnetic resonance spectroscopy), or
destructive to the sample (Khan and others 2002). From the literature, the degree of deacetylation
values of chitosan appear to be highly associated with the analytical methods employed (Khan
and others 2002). However, one of the most frequently used methods is infrared spectroscopy
because of its simplicity.
11
2.2.2 Molecular Weight The molecular weight of chitosan varies depending on the raw material sources and
preparation methods (Li and others 1992). Most commercial chitosans have a degree of
deacetylation that is greater than 70% and a molecular weight ranging between 100,000 Da and
1.2 million Da (Li and others 1997; Onsoyen and Skaugrud 1990). Various factors, such as
temperature, dissolved oxygen concentration, and shear stress can cause degradation of chitosan.
The molecular weight of chitosan can be determined by methods such as chromatography
(Bough and others 1978), light scattering (Muzzarelli 1977), and viscometry (Maghami and
Roberts, 1988). Among many methods, viscometry is a simple and rapid method for the
determination of molecular weight. The intrinsic viscosity of a polymer solution is related to the
polymer molecular weight according to the Mark-Houwink equation:
[η]=KMa
where [η] is the intrinsic viscosity, M the viscosity-average molecular weight, and K and a are
constants for a given solute-solvent system and temperature.
2.2.3 Viscosity
As with molecular weight and degree of deacetylation, viscosity is an important
characteristic of chitosan. Viscosity of chitosan is highly dependent on the degree of
deacetylation, molecular weight, concentration of solution, ionic strength, pH, and temperature.
The processes involved in the extraction of chitosan also affect the viscosity of chitosan. For
instance, chitosan viscosity decreases with an increased time of demineralization (Moorjani and
others 1975). Bough and others (1978) found that elimination of the demineralization step in the
chitin preparation decreased the viscosity of the final chitosan products. Moorjani and others
(1975) reported that bleaching chitosan with acetone or sodium hypochlorite at any stage of the
12
extraction process leads to considerable reduction in viscosity. No and others (1999)
demonstrated that chitosan viscosity is considerably affected by physical (grinding, heating,
autoclaving, ultrasonication) and chemical (ozone) treatments, except for freezing, and decreases
with an increase in treatment time and temperature.
2.3 SOURCES OF CHITOSAN Chitosan is converted from chitin, which is a structural polysaccharide found in the
skeleton of marine invertebrates, insects and some algae. Chitin is perhaps the second most
important polysaccharide after cellulose and is an abundantly available renewable natural
resource. The aquatic species that are rich in chitinous material (10-55 % on a dry weight basis)
include squids, crabs, shrimps, cuttlefish and oysters. Mucoraceous fungi, which are known to
contain chitin and the deacetylated derivate, chitosan, in cell walls (22 to 44%), have been used
for commercial chitin production (Muzzarelli 1977; Muzzarelli and others 1994). However, in
comparison with marine sources, which yield more than 80,000 metric tons of chitin per year
(Muzzarelli 1977; Subasingle 1995), chitin production from fungal waste is negligible.
Depending on the sources, the physicochemical properties and functionalities of chitosan
differ (Rhazi and others 2004). For example, chitosan prepared from squid contains β-chitin
(amine group aligned with the OH and CH2OH groups) and those prepared from crustaceans
contain α-chitin (anti-parallel chain alignment) (Shepherd and others 1997; Felt and others
1999). Despite a wide range of available sources, chitosan is commercially manufactured only
from crustaceans (crab, shrimp, krill, and crayfish) primarily because a large amount of
crustacean exoskeleton is available as a byproduct of food processing. Disposal of crustacean
shell waste has been a challenge for seafood processors. Therefore, production of value-added
products, such as chitin, chitosan and their derivatives, and utilization of these value added
13
products in different fields are of utmost interest to food industries. Continual use of new raw
materials as a source of chitin would enable production to be significantly increased. Major
progress is being made in the development of profitable technology for isolation of chitin and its
derivatives (Rashidova and others 2004). However, commercial extraction has been hampered by
the corrosive nature of the strong acids and bases used in the manufacture of chitosan, which
destroys equipment, requires careful handling by workers, and presents potential environmental
hazards (Peniston and Johnson 1980; Leffler 1997).
2.3.1 Crawfish Shell Waste as a Source of Chitosan Louisiana is the world’s largest producer of crawfish. Crawfish consumed in Louisiana
belongs to two species, the red swamp crawfish and the white river crawfish. The red swamp
crawfish, Procambarus clarkii, is most common, accounting for 60% of the catch in the
Atchafalaya river basin (No and Meyers 1995). Louisiana produces crawfish with an average
annual harvest exceeding 100 million pounds. Since the edible portion of crawfish accounts for
only 15%, about 85 million pounds of crawfish shell waste is produced in Louisiana annually.
The crawfish shell waste contains 23.5% chitin, which can be converted into chitosan (No and
Meyers 1992). The peeling waste has been used as animal feed with low economic value,
although it is an inexpensive source of the biopolymer chitosan (Prinyawiwatkul and others
2002). Various applications of crawfish chitosan have been reported. However,
commercialization of such findings is yet to be realized.
2.3.2 Extraction of Chitosan from Crawfish
Chitosan can be extracted from chitin sources by conventional chemical extraction or
using enzymes. Depending on procedures and sources, resultant chitosans differ in their
chemical and physical properties and functionality (Rhazi and others 2004). The conventional
14
chitosan extraction method involves, deproteinization to remove proteinous materials by an
alkali treatment, demineralization to remove calcium carbonate and calcium phosphate by an
acid treatment, decoloration to remove pigments by solvent extraction and bleaching, and
deacetylation to convert chitin into chitosan by an alkali treatment.
No and Meyers (1995) established a procedure to extract chitin from crawfish shell
waste and to convert chitin into chitosan by an alkali treatment with 50% NaOH (solid:
solvent, 1:10, w/v) for 30 minutes at 100oC in air or 121oC at 115 psi (Figure 2.3). Rout and
Prinyawiwatkul (2001) investigated properties of chitosan extracted from crawfish shell using
different extraction processes and reported that the deacetylation time in air has to be increased
by another 30 minutes (a total of 60 minutes) to obtain chitosan with film-forming ability.
They also suggested that deprotenization and decoloration steps could be excluded since the
harsh deacetylation process denatures any protein and removes most of pigments from
crawfish shell. Nadarajah and Prinyawiwatkul (2002) investigated the degree of deacetylation
of crawfish chitosan versus film- forming ability and reported that crawfish chitosan obtained
by 60 minutes deacetylation led to a lower degree of deacetylation and poor film formation.
Further, they suggested that a 90 minutes of the deacetylation process yielded chitosan with a
higher degree of deacetylation which serve as good film formers. However, chitosan with
similar film-forming ability can be obtained with an autoclaving process at a shorter time of 30
minutes (No and others 2000) (Figure 2.3). Apart from these studies, information on film-
forming ability of crawfish chitosan and their functionalities is lacking. It is envisaged that
chitosan obtained from modified or simplified extraction processes and film formation with
different solvents could yield a film with elicited antimicrobial activity while bringing down
the production cost.
15
Wet crawfish shell
Washing and drying
Grinding and sieving
Deproteinization
Deproteinized shell
Washing
Demineralization
Washing
Extraction with Acetone
Drying
Bleaching
Washing and Drying
Deacetylation
Washing and Drying
Chitosan
Figure 2.3: Scheme for chitosan production (Modified from No and Meyers 1995).
3.55 % NaOH (w/v) for 2 h at 65oC solid: solvent (1:10, w/v)
1 N HCl for 30 min at room temp. solid: solvent (1:15, w/v)
0.315% NaOCl (w/v) for 5 min at room temp. solid:solvent (1:10, w/v)
50% NaOH for 30 min at 115 psi / 121oC, solid:solvent (1:10, w/v)
16
2.4 FILM-FORMING ABILITY OF CHITOSAN Chitosans with higher molecular weight have been reported to have good
film-forming properties as a result of intra- and intermolecular hydrogen bonding (Muzzarelli
1977). A patent was granted to G.W. Rigby in 1936 for the earliest attempt to form films from
chitosan. These films were described as flexible, tough, transparent, and colorless with a tensile
strength of about 9,000 psi and prepared by a solvent casting method. Chitosan films prepared
by similar methods were reported later by Muzzarelli and others (1974), Averbach (1978),
Butler and others (1996), Caner and others (1998) and Wiles and others (2000). These films
were described to have good gas barrier and mechanical properties. The chitosan film
characteristics, however, varied from one report to another. Differences in the sources of chitin
used to produce chitosan, chitosan properties, solvents used, methods of film preparation, and
types and amounts of plasticizers used affect the quality of the chitosan films (Lim and Wan
1995; Remuñán-López and Bodmeier 1996; Begin and Calsteren 1999; Nunthanid 2001). The
film-forming ability of chitosan extracted from crawfish has been reported by Nadarajah and
Prinyawiwakul (2002).
2.4.1 Film-forming Methods
Edible films are formed by either a wet- or dry-process mechanism. The wet-process-
mechanism is based on a film-forming dispersion or solution in which polymers are first
dispersed or solubilized into a liquid phase, and then dried. Freeze drying is employed to obtain
sponge-type scaffolds used in tissue engineering. The wet process is often preferred as it permits
the application of films as coatings in a liquid form directly onto food products by dipping,
brushing or spraying (Peressini and others 2004). Some edible films, such as starch films, can be
prepared using a dry-process, such as thermoplastic extrusion. This extrusion process is based on
17
the thermoplastic properties of polymers when plasticized and heated above their glass transition
temperature under low water content-conditions (Warburton and others 1993; Psomiadou and
others 1997; Arvanitoyannis and Billiaderis 1998).
2.4.2 Film-forming Mechanisms
Polymeric solutions form films through a series of phases. When the polymer solution is
cast on a surface, cohesion forces form a bond between the polymer molecules (Banker 1996).
When the cohesive strength of the polymer molecules is relatively high, continuous surfaces of
the polymer material coalesce. Coalescence of an adjacent polymer molecule layer occurs
through diffusion. Upon evaporation of water, gelation progresses and allows the polymer chains
to align in close proximity to each other and to get deposited over a previous polymer layer
(Harris and Ghebre-Sellassie, 1997). When there is adequate cohesive attraction between the
molecules, sufficient diffusion, and complete evaporation of water, polymer chains align
themselves to form films (Harris and Ghebre-Sellassie 1997).
2.4.3 Film Morphology and Defects
Polymeric films should be uniform and free from defects for their applications.
Uniformity of the films is critical for their functionalities. The processing variables involved in
conversion of chitin into chitosan, especially the uniformity of particle size of shells used as a
starting material, greatly influence the properties of chitosan (No and others 1999), and hence the
uniformity of films produced. During the film-forming process, shrinkage of the films due to
evaporation of water or rapid drying often causes defects such as cracks or curling in the films
(Obara and McGinity 1995). Addition of plasticizers such as glycerol or sorbitol is often used to
reduce such defects.
18
2.4.4 Function of Plasticizers in Film Formation
Films prepared from pure polymers tend to be brittle and often crack upon drying.
Addition of food-grade plasticizers to film-forming solution alleviates this problem (McHugh
and Krochta, 1994). When a plasticizer is added, the molecular rigidity of a polymer is relieved
by reducing the intermolecular forces along the polymer chain. Plasticizer molecules interpose
themselves between the individual polymer chains, thus breaking down polymer-polymer
interactions, making it easier for the polymer chains to move past each other. The plasticizer
improves flexibility and reduces brittleness of the film. Polyethylene glycol, glycerol, propylene
glycol, and sorbitol are the most commonly used plasticizers in edible film production (Aydinli
and Tutas 2000).
The amount of plasticizer added can cause adverse effects on film properties such as
increasing mass transfer through the films. Hence, plasticizers must be used with caution. When
the plasticizer concentration exceeds its compatibility limit in the polymer, it causes phase
separation and physical exclusion of the plasticizer (Aulton and others 1981). This leads to
development of a white residue on edible films which has been referred to as “blooming”
(Aulton and others 1981) or “blushing” (Sakellariou and others 1986). The amount of plasticizer
used in film formation should also be small enough to avoid probable toxic effects (Nisperos-
Carriedo 1994).
2.5 PROPERTIES OF CHITOSAN AND CHITOSAN FILMS
2.5.1 Safety of Chitosan Films
Chitosan is non-toxic and safe to domestic animals (Hirano and others 1990). According
to Rao and Sharma (1997), chitosan films were non toxic and free from pyrogens. Many medical
and pharmaceutical applications of chitosan films require sterility of films. Chitosan films can be
19
sterilized by irradiation (Lim and others 1998) and autoclaving (Rao and Sharma 1995), although
these processes lead to some degradation of the films.
2.5.2 Biodegradation of Chitosan and Chitosan Films
Many studies have shown that chitin and chitosan are biodegradable polymers. Davies
and others (1969) reported that chitosan is most susceptible to hydrolysis by lysozyme at pH 5.2,
and the optimum range of pH value is between pH 5.2 and 8.0 (Davies and others 1969;
Shigemasa and others 1994).
Pangburn and others (1982) studied the effect of deacetylation on susceptibility of chitin
and chitosan to lysozyme and found that pure chitin (0% deacetylation) was most susceptible to
lysozyme, while pure chitosan (100% deacetylation) was not degraded by lysozyme. Sashiwa
and others (1990) studied the relative rates of degradation of six chitosans varying in degree of
deacetylation (45%, 66%, 70%, 84%, 91%, and 95%), and reported that 70% deacetylated
chitosan degraded most quickly.
Shigemasa and others (1994) investigated the effects of preparation methods on chitosan
degradation. They found that for the same molecular weight and degree of deacetylation,
homogeneously prepared chitosans were more susceptible to hydrolysis by lysozyme than those
heterogeneously prepared.
2.5.3 Mechanical Properties of Chitosan Films
For edible films to be employed as a food packaging material, they should satisfy the
requirement of being durable, stress resistant, flexible, pliable, and elastic. Thus, they should
possess desirable tensile properties which could bear stresses exerted during various handling
processes. Only limited literature is available on mechanical properties of chitosan films. There
are variations of physical property values of chitosan films reported in the literature due to
20
different chitosans and testing conditions used. Films produced with low molecular weight
chitosan at 3% w/w in 1% acetic acid, with glycerol as a plasticizer at 0.25 and 0.50 mL/g of
chitosan, were reported to have tensile strength (TS) of 15 to 35 MPa and percent elongation at
break (%E) of 17 to 76 (Butler and others 1996). Caner and others (1998) reported that films
produced by a similar method but with different solvents (acetic, formic, lactic and propionic
acid) at 1% and 7.5% concentrations exhibited the TS value range of 12 to 32 Mpa and %E value
range of 14 to 70 with an exception of the film made with 7.5% lactic acid having the lowest TS
value of 6.85 MPa and the highest %E value of 51. They also reported that increasing the
plasticizer content decreased TS and increased %E. Kittur and others (1998) reported a much
higher TS value of 70.3 MPa and a lower %E value of 6.2 for a film made of 2% w/w chitosan
in 1 % acetic acid. Variations in mechanical strength of chitosan films are due to the type of
chitosan and concentration used, the type of plasticizer and its content, and solvent. The TS
values of chitosan films are comparable to those of commercial DDPE and LDPE films but, their
%E values are significantly lower than the commercial films (Briston 1988). However, compared
to films made of other biopolymers (wheat gluten, corn zein protein and soy protein isolate),
chitosan films exhibited significantly higher TS values (Cunningham and others 2000).
2.5.4 Transport Properties of Chitosan Films In general, edible films and coatings provide the potential to control transport of
moisture, oxygen, aroma, oil, and flavor compounds in food systems, depending on the nature of
the edible film-forming materials (Donhowe and Fennema 1993; Krochta 1997; Krochta and De
Mulder-Johnston 1997). However, when films are formed using biopolymers alone, they are very
brittle. To lessen brittleness and to make flexible films, plasticizers are used. However,
plasticizers increase the film permeability (Gontard and others 1993), especially for plasticized
21
hydrophilic films. Increased permeability of edible films is undesirable for food applications, so
there is a need to minimize the use of plasticizers. Another potential approach to increase film
flexibility is reduce polymer molecular weight, thus reducing intermolecular forces along
polymer chains and increasing polymer chain end groups and polymer free volume (Sears and
Darby, 1982). This approach may permit a decrease in the required amount of added plasticizer
in films; consequently, it may minimize permeability of films while producing needed film
flexibility (Sothornvit and Krochta 2000).
Chitosan films exhibit gas barrier properties. Oxygen permeability of chitosan is as low
as many conventional plastic films such as poly vinylidene dichloride (PVdC) and ethyl vinyl
alcohol (EVOH) (Webber 2000). Since chitosans obtained from various sources and methods
vary in their characteristics, barrier properties of film made of various chitosans also vary.
Muzzarelli and others (1974) reported a water vapor transmission rate of 1200 g/m2/d measured
at 100 °F and 90% relative humidity for chitosan membranes with 20 µm thickness. Wong and
others (1992) reported a water vapor permeability (WVP) value of 0.41 g mm/m2/d/mmHg for
chitosan and chitosan-lipid films cast from 1% chitosan solution using formic acid. Butler and
others (1996) reported that chitosan films made with plasticizer (glycerol) levels of 0.25 and 0.50
ml/g had a mean WVP of 2.89 × 10–4 g/m/d/mmHg at 25 °C between 0% to 11% RH.
Manufacturing biopolymer based films with adequate water barrier properties is a major
challenge as many of the bioplymers are hydrophilic by nature (Webber 2000). Butler and others
(1996) stated that their chitosan films were extremely good barriers to oxygen, while having
higher water vapor barrier properties because of their hydrophilic nature. They also reported that
increasing plasticizer concentrations negatively affected barrier properties but improved
formation, mechanical, and handling properties. Caner and others (1998) prepared chitosan films
22
using various acid and plasticizer concentrations and reported water vapor permeability
coefficients ranging from 1.74 × 10–5 to 7.04 × 10–4 g/m/d/mmHg at 25°C between 50% to 100%
RH. They also suggested that storage time had no effect on barrier properties of chitosan films.
Attempts to improve vapor barrier properties of chitosan films yielded only limited
success. Wong and others (1992) used lipid to form chitosan-lipid composite films to improve
moisture barrier properties. Hoagland and Parris (1996) developed a chitosan/pectin-laminated
film to alter water vapor permeability and water solubility. Tual and others (2000) produced
chitosan films with improved barrier properties by crosslinking chitosan with glutaraldehyde.
However, these films were reported to be brittle due to formation of chemical junctions.
2.5.5 Antimicrobial Properties of Chitosan and Chitosan Films
Microbial growth on the surface of food is a major cause of food spoilage and food-borne
illness. Therefore, the concept of using edible active coating to inhibit spoilage and pathogenic
microorganism has received considerable interest (Rico-Pena and Torres 1991; Weng and
Hotchkiss 1992, 1993; Ouattara and others 2000; Coma and others 2001). Development of
antimicrobial plastics with added antimicrobial agents is less preferred as their releasing rate is
unsatisfactory and there is growing environmental concern. Direct application of antimicrobial
agents onto food surfaces by spraying, dipping or coating has proven to be less effective as there
is loss of activity because of leaching onto the food, enzymatic activity, and reaction with other
food components (Jung and others 1992; Ray 1992; Ouattara and others 2000). Hence, use of
packaging films or coating as a matrix to deliver antimicrobial agents may provide an alternative
approach to prevent food spoilage and food-borne illness. Such packaging or coating can
maintain a high concentration of antimicrobial agents on the food surface and allow low
migration into food (Torres and others 1985; Siragusa and Dickson 1992; Ouattara and others
23
2000). Chitosan films having the ability for controlled release of added substances would help in
this context.
Chitosan possesses unique properties that make it an ideal ingredient for development of
antimicrobial edible film. Chitosan possesses film-forming properties (Averbach 1978), greater
and broader spectra of antibacterial activity compared to disinfectants, a higher bacterial/fungal
killing rate, and lower toxicity toward mammalian cells (Franklin and Snow, 1981; Takemono
and others 1989). Further, Rhoades and Roller (2000) reported that the interaction (binding or
chelation) of chitosan with endotoxins of gram-negative bacteria decreased their acute toxicity.
Because of the strong chelating ability of chitosan, external chelating agents such as EDTA may
not be required, when antimicrobial agents such as nisin are added to chitosan to control gram-
negative bacteria.
Antimicrobial properties of chitosan have been reported by many investigators.
Chitosan's ability to inhibit a wide variety of bacteria (Sudarshan and others 1992; Yalpani and
others 1992), fungi (Allan and Hadwiger 1979; Stossel and Leuba 1984; Kendra and others
1989; Fang and others 1994), yeasts (Ralston and others 1964), and viruses (Kochkina and
others; 1995; Pospieszny 1997; Chirkov 2002) make it a broad spectrum antimicrobial agent. A
variety of research has been conducted to assess inhibitory effects of chitosan in a solution state
or its oligosaccharides in terms of minimum inhibitory concentration (MIC). Chitosan is more
effective in inhibiting bacteria than chitosan oligomers (Jeon and others 2001). Antimicrobial
activity of chitosan is influenced by its molecular weight, degree of deacetylation, concentration
in solution, and pH of the medium (Lim and Hudson 2003). No and others (2002b), reported that
chitosan with different organic acid solvents exhibited varying inhibitory effects on bacteria. In
general, acetic acid, lactic acid, and formic acids were more effective in inhibiting bacterial
24
growth than propionic and ascorbic acids. Chitosan shows stronger antimicrobial activity for
gram-positive than gram-negative bacteria (Jeon and others 2001). Chitosan has been observed
to act more quickly on fungi and algae than on bacteria (Cuero, 1999); however, like other
properties of chitosan, this activity may be dependent on the type of chitosan, chitosan molecular
weight, and degree of deacetylation, among other factors influencing the environment in which
the chitosan is stored.
Antimicrobial property of chitosan can be enhanced by irradiation (Matsuhashi and
Kume 1997), ultra violet radiation treatment, partial hydrolyzation (Davydova and others 2000),
using different organic solvents (No and others 2002a, 2002b), chemical modifications
(Nishimura and others 1984; Tanigawa and others 1992), synergistic enhancement with
preservatives (Chen and others 1996; Roller and others 2002), synergistic enhancement with
antimicrobial agents (Lee and others 2003; Song and others 2002), or in combination with other
hurdle technologies.
Several mechanisms were proposed for the antimicrobial activity of chitosan. One
mechanism is that the polycationic nature of chitosan interferes with the negatively charged
residues of macromolecules at the cell surface, presumably by competing with Ca2+ for
electronegative sites on the membrane without conferring dimensional stability, rendering
membrane leakage (Young and Kauss 1983). The other mechanism is that oligomeric chitosan
penetrates into the cells of the microorganism and prevents the growth of cells by prohibiting the
transformation of DNA into RNA (Hadwinger and others 1986). Tokura and others (1997)
suggested that antimicrobial activity is related to the suppression of the metabolic activity of the
bacteria by blocking nutrient penetration through the cell wall rather than the inhibition of the
transcription from DNA.
25
Although several papers on the antimicrobial properties of chitosan have been published,
relatively little work has been reported on the antimicrobial properties of chitosan films. Coma
and others (2002) investigated antibacterial properties of chitosan acetate films on Listeria
monocytogenes and Listeria innocua using Emmental cheese as a model system. The authors
reported 100% inhibition of Listeria monocytogenes for 8 days but decreased antibacterial
activity with time which was attributed to decreased availability of amino groups. Chen and
others (1996) incorporated food preservatives, such as potassium sorbate and sodium benzoate,
into a chitosan film matrix and compared their inhibitory effects on microbial growth. They
reported that chitosan films made in dilute acetic acid solutions were able to inhibit the growth of
Rhodotorula rubra and Penicillium notatum by direct application of the film on the colony-
forming organism. Lee and others (2003) reported enhanced microbial stability of milk and
orange juice that were exposed to paperboard coated with chitosan and nisin. Coma and others
(2003) assessed antimicrobial activity of chitosan coating on the growth of Staphylococcus
aureus, Pseudomonas aeruginosa, and Listeria monocytogenes. They reported that chitosan
coating could be used to increase the microbial lag phase while decreasing the maximum density
of selected microorganisms, and could have potential applications for dairy product preservation.
Rodriguez and others (2003) reported that the use of chitosan in acetic acid as edible coating for
precooked pizza (0.079 g/100 g pizza) delayed the growth of Alternaria sp, Penicillium sp, and
Cladosporium sp (Deuteromycetes).
2.6 APPLICATIONS OF CHITOSAN FILMS
Chitosan can be used in vastly diverse fields ranging from flocculant for seed coating,
toiletry components, controlled drug delivery systems (Graham 1990), membrane based
transdermal drug delivery systems (Thacharodi and Rao 1993, 1995), eye contact lens (Felt and
26
others 1999), wound-healing, dressing material and artificial skin (Biagini and others 1992; Ueno
and others 2001), various medical supplies including surgical dressing, sanitary cottons, gauzes,
bandages, plasters, and sanitary pads, separation membranes, matrix for immobilization of
biomolecules such as peptides (Bernkop-Schnurch and Kast 2001) and genes (Borchard 2001),
bioseparation, support for bio sensors (Ng and others 2001), and bioadhesive to increase
retention at the site of application (He and others 1998; Calvo and others 1997).
2.6.1 Applications of Chitosan and Chitosan Films in Foods
Chitosan possesses many desirable properties for use in food systems. The film-forming
ability of chitosan and gas-barrier properties of chitosan film favor its use as an edible food
packaging material. Their inherent antimicrobial properties along with non-toxicity and
biocompatibility offer their use as antimicrobial additives. Further, as an additive chitosan can
offer a variety of functionalities.
To date, only a few attempts have been made to assess the effects of chitosan films and
coatings in real foods. Most of the work has been centered on the antimicrobial properties of
chitosan. Agulló and others (1998) evaluated the capacity of chitosan films to extend the shelf-
life of precooked pizzas. The study showed that increased shelf-life was mainly due to antifungal
properties of chitosan instead of its action as a water vapor barrier. Rodriguez and others (2003)
demonstrated that chitosan in acetic acid as an edible coating (0.079 g/100 g pizza) delayed the
growth of Alternaria sp, Penicillium sp, and Cladosporium sp (Deuteromycetes) in precooked
pizza. They also demonstrated that the use of chitosan in the dough is not effective because the
biopolymer loses its antimicrobial capacity due to the Maillard reaction. Skonberg and Gillman
(2000) reported extension of shelf life of fresh salmon and haddock fillets by chitosan coating.
Nadarajah and others (2003) reported the use of chitosan coating on catfish fillets for retaining
27
color, inhibiting lipid oxidation, and retarding microbial spoilage. They demonstrated that the
shelf life of refrigerated catfish fillets could be prolonged up to 8 days with high molecular
weight (1,100 kDa) chitosan at 1% concentration. Srinivasa and others (2002) investigated the
effect of modified atmosphere packaging by chitosan film on the quality of mango fruits and
reported that mangos stored in chitosan-covered boxes showed an extension of shelf-life of up to
18 days without any microbial growth and off flavor.
Only limited literature is available on direct application of chitosan as an antimicrobial
additive in foods. Darmadji and Izumimoto (1994) investigated the effect of chitosan on
development of spoilage in minced beef patties stored at 30oC for 2 days and at 4oC for 10 days.
At higher storage temperature, a reduction of one to two log cycles of total bacteria,
pseudomonads, Staphylococci, coliforms, Gram-negative bacteria and Micrococci was observed
in the presence of 1% chitosan; at lower storage temperature, similar reductions in spoilage flora
were reported after 10 days. Fang and others (1994) investigated the use of chitosan as an
antimicrobial agent against mold spoilage in candied kumquat. The authors reported that a
concentration of 6 g/L of chitosan was required to maintain a mold-free shelf life of 65 days
when the sugar concentration in the syrup was reduced from the traditional 65o Brix to 61.9o Brix
at pH 4. No and others (2002a) reported inhibition of growth of bacteria isolated from spoiled
tofu by chitosan and the possibility of using chitosan to extend the shelf life of tofu. They also
showed that antibacterial activity of chitosan was higher than chitosan oligomers. Oh and others
(2001) used mayonnaise as a complex food model and demonstrated that addition of chitosan can
inhibit the growth of spoilage microorganisms Lactobacillus fructivorans and
Zygosaccharomyces bailii when stored at 25oC. Roller and Covill (1999) found that chitosan at a
rate of 1-5 g/L on apple juice reduced the growth rate of Mucor racemosus and Byssochlamys
28
spp. Coma and others (2002) reported use of chitosan film to inhibit growth of Listeria
monocytogenes on Emmental cheese. Antimicrobial activity of chitosan on bacterial strains
isolated from fish meat paste (Cho and others 1998) was also reported. Chitosan is frequently
used as a preservative in solid foods in Japan in products such as kamaboko, noodles, soy sauce.
However, as reported by Roller and Covill (1999), these reports are lacking in details so that the
condions/formulations would be difficult to replicate and verify (Li and others 1997a; Hirano
1997).
Chitosan offers various functionalities to foods. It has been considered as dietary fiber,
which is recognized to reduce apparent fat digestibility (Deuchi and others 1994; Kanauchi and
others 1995) and, therefore, it would be a promising approach for dietary supplementation when
applied to foods. Kim and others (2000) inferred that absorption of fat in the human body from
high fat foods such as whipped cream can be reduced by addition of chitosan without
compromising sensory qualities. Austin (1982) reported that chitosan can be used to reduce
lactose intolerance. Kataoka and others (1998) found that addition of 1.5% chitosan to walleye
pollock surimi in combination with setting at 20oC resulted in a twofold increase in gel strength.
Similarly, Benjakul and others (2000) reported that incorporation of chitosan, particularly in the
presence of CaCl2 greatly improved the gelling properties of surimi from barred garfish without
changes in color.
Chitosan is an ideal preservative coating for fresh fruit and vegetables because of its film-
forming and biochemical properties. Chitosan has a particular adhesiveness towards biological
surfaces because of its positive charge and the negative charge of biological membranes
(Henriksen and others 1996) and, therefore, is capable of forming stable films (Romanazzi and
others 2002).
29
Chitosan has been shown to regulate gas exchange, decrease transpiration losses and
maintain the quality of harvested fruits. Chitosan coating prolongs storage life and controls decay
of strawberries (El Ghaouth and others 1991; Zhang and Quantick 1998), litchi (Zhang and
Quantick 1997), apples (Du and others 1998), peach (Li and Yu 2000), mango (Srinivasa and
others 2002), and table grapes (Romanazzi and others 2002).
It was reported that chitosan, as a semi-permeable coating, could delay the ripening of
strawberries (El Ghaouth and others 1991), tomatoes (El Ghaouth and others 1992a), cucumbers
and bell peppers (El Ghaouth and others 1992b), apples (Hu and Zou 1998; Zheng and others
1996), and pears (Zheng and others 1996; Li and Yu 2000) by slowing down the production of
anthocyanin and ethylene. Chitosan helps retain the fruits’ firmness, fresh weight, titratable
acidity, soluble carbohydrates and vitamin C. A polymeric coatings made of chitosan (Nutri-
Save composed of N, O-carboxymethyl chitosan) has also shown promise in delaying ripening of
pears (Meheriuk and Lau 1998).
Chitosan reduces the growth of many phytopathogenic bacteria and fungi (Allan and
Hadwiger 1979). Moreover, it elicits phytoalexin formation (Reddy and others 1999), and
induces the production of antifungal hydrolases (Zhang and Quantick 1998; Hirano 1999) and an
increase of phenylalanine ammonia-lyase (PAL) activity (Romanazzi and others 2002). Chitosan
has generally been applied in postharvest treatments (Baldwin and others 1997), and there are
very few examples of preharvest application (Reddy and others 2000; Romanazzi and others
2000, 2002). Romanazzi and others (2002) suggested that pre-harvest spray of chitosan is best
for commodities such as grapes since exposure to postharvest liquid-based treatments is not
advisable for commodities as it could cause damage. However, since the effect of preharvest
fungicide spraying is often inefficient because of the heavy foliage which obstructs full coverage
30
(Sholberg and Gaunce 1996) and the development of fungicide resistant isolates of postharvest
pathogens (Hong and Michailides 1996), postharvest spraying of chitosan may be preferable and
more advantageous.
2.7 REFERENCES Agulló E, Gschaider ME, Rodríguez MS, Ramos VM, Pedroni V. 1998. Efecto antifúngico de
películas de quitosano sobre prepizzas. Inform Tecnol 9(3):123-8. Allan CR, Hadwiger LA.1979. The fungicidal effect of chitosan on fungi of varying cell wall
composition. Exp Mycol 3:285-7. Arvanitoyannis I, Biliaderis CG. 1998. Physical properties of polyol-plasticized edible films
made from sodium caseinate and soluble starch blend. Food Chem 62:333-44. Aulton ME, Abdul-Razzak MH, Hogan JE. 1981. The mechanical properties of
hydroxypropylmethylcellulose films derived from aqueous systems. Part 1: The influence of plasticizers. Drug Dev Ind Pharm 7(6):649-68.
Austin PR. 1982. Lactose-rich animal feed formulations and method of feeding animals. U.S.
Patent 4,320,150. Averbach BL. 1978. Film-forming capability of chitosan. In: Muzzarelli RAA, Pariser ER.
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CHAPTER 3
PHYSICOCHEMICAL PROPERTIES OF EDIBLE CHITOSAN FILMS
DEVELOPED FROM CRAWFISH SHELL WASTE
43
3.1 INTRODUCTION
Naturally renewable biopolymers have attracted much research interest in recent years
because of their potential use as edible and biodegradable films and coatings for food packaging.
Chitosan, which can be derived from abundantly available chitin sources such as crustacean shell
wastes, has excellent film-forming ability and inherent antimicrobial properties suitable for to
development of edible antimicrobial films (Ralston and others 1964; Allan and Hadwiger, 1979;
Stossel and Leuba, 1984; Kendra and others 1989; Sudarshan and others 1992; Yalpani and
others 1992; Fang and others 1994; No and others 2002). Moreover, the possibility of enhancing
antimicrobial properties of chitosans by irradiation (Matsuhashi and Kume 1997), partial
hydrolyzation (Davydova and others 2000), using different organic solvents (No and others
2002), chemical modifications (Nishimura and others 1984; Tanigawa and others 1992),
synergistic enhancement with preservatives (Chen and others 1996; Roller and others 2002), or
by combing with other hurdle technologies, make them ideal for use as antimicrobial edible
packaging materials.
Very limited literature is available on physicochemical properties of chitosan films
derived from crawfish shell waste. Chitosan derived from crawfish shell wastes was used to form
various flexible and transparent films resembling plastic packaging materials (Nadarajah and
Prinyawiwatkul 2002). Properties of chitosan films are reported to be affected by the chitosan
extraction process (Rout 2001; Nadarajah and Prinyawiwatkul 2002). Furthermore, various
organic acids used to dissolve chitosan affect resultant film properties (Kienzle-Sterzer and
others 1982; Caner and others 1998; Rhim and others 1998; Park and others 1999; Park and
others 2002). Therefore, a variety of crawfish chitosan films can be developed using various
chitosan extraction methods and organic acids. The knowledge of physicochemical properties of
44
such films would allow more efficient selection of crawfish chitosan films for their specific and
suitable applications.
Therefore, the primary objectives of this study were to develop a variety of crawfish
chitosan based films and to identify their physicochemical and functional properties. Proper
understanding of the physical and chemical fundamentals underlying the functional properties of
crawfish chitosan films would help us develop process protocols suitable to prepare
antimicrobial edible films. Variations in physicochemical properties and functionalities were
intended to be achieved by using (1) different crawfish chitosans based on their extraction
method, (2) different film casting (organic acid) solvents, and (3) different amounts of added
plasticizer (glycerol).
3.2 MATERIALS AND METHODS
3.2.1 Preparation of Chitosan from Crawfish Shell Waste
All chemicals used in this study were analytical grade and purchased from Sigma
Chemical Co. (St. Louis, MO). Crawfish shell waste collected from a local seafood processing
facility was washed with warm tap water to remove foreign materials and remaining muscle
particles. The shells were then dried at 60oC overnight, ground with a centrifugal grinding mill
(Retsch/Brinkmann ZM-1, Westbury, NY), and shell particles between a mesh size of 20 (0.841
mm) and 40 (0.420 mm) were used as starting material. The methods established to extract chitin
from crawfish shell waste by No and Meyers (1995) and further processing of chitin into
chitosan through autoclaving (No and others 2000) were used to prepare different chitosan
samples for this study.
Dried crawfish shell particles were treated with 1 N NaOH at 65oC for 1 hour at a solid:
solvent ratio of 1:10, w/v for deproteinization (DP). Following a washing step, deproteinized
45
shell particles were treated with 1 N HCl at room temperature for 30 minutes at a solid: solvent
ratio of 1:1.5, w/v for demineralization (DM). Particles were then treated with acetone at 1: 10
w/v concentration, washed, and bleached with 0.315% NaOCl at a 1:10 w/v ratio for 5 minutes
for decoloration (DC). Resultant chitin was treated with 50% NaOH at a 1:10 w/v ratio at
121oC/15 psi for 30 minutes for deacetylation (DA). Subsequent washing and drying steps
yielded chitosan. Four different chitosan samples were prepared using the traditional method
involving all above steps (DPMCA), the traditional method excluding deproteinization (DMCA),
the traditional method excluding decoloration (DPMA), and the traditional method excluding
both deproteinization and decoloration (DMA).
3.2.2 Characterization of Crawfish Chitosan
The viscosity average molecular weight of crawfish chitosans was determined by the
intrinsic viscosity using the Mark-Houwink equation ([η] = KMa) where [η] is the intrinsic
viscosity, M is the molecular weight, K=1.81 x 10-3 and a=0.93 at 25oC (Maghami and Roberts
1988). Crawfish chitosan films deprotonated by methanolic ammonia was used to determine the
degree of deacetylation with IR spectroscopy (M2000 FTIR spectrophotometer, Midac Corp.,
Irvine, CA, USA) and the equation proposed by Sabnis and Block (1997) was used to calculate
degree of deacetylation values. Viscosity was determined for 1% chitosan in 1% acetic acid
(w/v) using the Brookfield viscometer at 50 rpm and 25oC with a spindle number RV5.
The moisture content (%) of the chitosans was determined gravimetrically in triplicate by
drying samples at 100oC for 48 hours. Nitrogen (%) was determined by the combustion method
using the Leco CHN analyzer (Model # FP-428, Leco Corporation, St. Joseph, Michigan, USA).
The ash content (%) was determined according to the standard method # 923.03 (AOAC 1990).
46
3.2.3 Preparation of Crawfish Chitosan Films
Four types of crawfish chitosans (DPMCA, DPMA, DMCA, and DMA) were separately
dissolved with 1% of film casting solvents (acetic, ascorbic, lactic, formic, citric or malic acids).
Each solution was vigorously agitated for 30 minutes, immersed in boiling water for 10 minutes,
cooled down to room temperature, and filtered through glass-wool to remove undissolved
particles. Resultant film casting solution was divided into two batches. One batch contained
glycerol as a plasticizer at a ratio of 0.10, 0.20, 0.30, 0.40, and 0.50 (chitosan: glycerol, w/w),
and the other batch was without a plasticizer. The film casting solution (300 mL) was cast onto a
31 × 31 cm Teflon coated plate and allowed to dry at room temperature (23oC) for 48 hours.
Dried films were carefully peeled out and stored in desiccators containing saturated NaBr until
further tested.
3.2.4 Thickness and Density of Crawfish Chitosan Films
A micrometer (Model 293-766; Mitutoyo, Tokyo, Japan) was used to measure the film
thickness to the nearest 0.001 mm. Thickness of each film (mm) was measured at room
temperature (23oC and 45% RH) and expressed as an average of 10 random measurements and
standard deviation. Weight of a 25 × 25 mm sample was taken to determine a density of each
film (g/cm3), expressed as an average of 3 measurements and standard deviation.
3.2.5 Color of Crawfish Chitosan Films
Color of the film samples was measured using a portable Minolta spectrophotometer
(Model CM-508d, Minolta Camera Co. Ltd., Osaka Japan) with 2o standard observer and D65
illuminant, and expressed as CIE color characteristics; L*, a* and b* values, where L* describes
lightness (ranging from black to white), a* and b* describe the chromatic coordinates (ranging
from –a: greenness, –b: blueness, +a: redness, +b: yellowness). Film specimens were placed on
47
the surface of a white standard plate (L*=92.91, a*=0.92, b*=0.03). A mean value of 10
measurements was reported for each color attribute. The total color difference (∆E) was
calculated by the following equation.
( ) ( ) ( )2*2*2* baLE ∆+∆+∆=∆
The results were expressed as ∆E values with the acetate film made with DPMCA
chitosan serving as a reference.
3.2.6 Transparency of Crawfish Chitosan Films Transparency of crawfish chitosan films was measured according to the procedure of Han
and Floros (1997) using a spectrophotometer (Spectronic Genesis 2, Thermo Spectronic,
Rochester, USA). This method was modified from the ASTM method D1746-92, which is the
standard test method for transparency of plastic sheeting (ASTM 1987). The transparency of the
plastic film was determined from the following equation, based on film thickness and %
transmittance (or absorbance) of the light at 600 nm (T600 or A600).
bT
orb
AcyTransparen 600600 log
=
where T600 is transmittance at 600 nm, A600 is absorbance, and b is the length of the light path
through the medium (i.e., film thickness).
3.2.7 Swelling of Crawfish Chitosan Films
Water sorption capacities of chitosan films were determined by soaking them in
phosphate buffered saline (PBS at pH 7.4) at room temperature. A known weight of chitosan
film was placed in the PBS media for 30 minutes. The wet weight of the film was determined by
first blotting the surface of the chitosan film with filter paper to remove excess water, and the
48
film weighed immediately. The percentage of water adsorption in the medium (Wsw) was
calculated from the equation:
1000
030 ×−
=W
WWWsw
where W30 represents the weight of the chitosan film after 30 minutes of sorption and W0 is the
initial weight of the chitosan film. The Wsw values were expressed as an average of 3
measurements and standard deviation.
3.2.8 Solubility of Crawfish Chitosan Films
The film solubility (S%) expresses the percentage of film's dry matter solubilized after
immersion in water for 24 hours at 25oC, according to Gontard and others (1992). The
experiment was performed under slow agitation. Film solubility (S%) was calculated from the
following equation:
⎟⎟⎠
⎞⎜⎜⎝
⎛ −=
initial
finalinitial
wtwtwt
S%
where Wtinitial is the initial weight of chitosan film and Wtfinal is the weight of chitosan film
after immersion.
3.2.9 Microstructure of Crawfish Chitosan Films
Scanning electron microscopy (SEM) was used to examine representative film surfaces.
All films were equilibrated at 53% relative humidity prior to preparation for scanning. Film
samples were sputter coated with silver and scanned using a FEI Quanta 200 ESEM (FEI
Company, Hillsboro, Oregon, USA) with an accelerating beam voltage of 3.9 kV.
3.2.10 Statistical Analysis
All data from the physicochemical properties were analyzed using SAS (SAS 2002). The
PROC GLM procedure with orthogonal contrasts was used to test the effect of film casting
49
solvents and chitosan extraction methods on physicochemical properties. Means were compared
by the Tukey's studentized range test. Significance of differences was defined at p < 0.05.
3.3 RESULTS AND DISCUSSIONS
3.3.1 Characteristics of Crawfish Chitosan
Chitosan extracted from crawfish shell was white or light pink in color depending on the
extraction methods. Chitosans extracted without the decoloration process (DPMA and DMA
chitosans) exhibited more pinkish color. This indicates that the harsh deacetylation process
applied to convert chitin into chitosan was not sufficient to remove all pigments from crawfish
shells.
Table 3.1 describes the general characteristics of chitosans extracted from crawfish shell
waste. Molecular weight is one of the important factors governing the functional properties of
chitosan. The viscometric method is the simplest and the most effective method for determining
molecular weight of polymers (Muzzarelli 1985). Crawfish chitosans had molecular weight in
the range of 3584-10168 Da. The traditional extraction method of chitosan (DPMCA) resulted in
Table 3.1: Physicochemical properties of chitosans extracted from crawfish shell
Chitosan Typea
Molecular Weight (Da)
Viscosity (cP)
Deacetylation (%)
Moisture (%)
Ashb (%)
Nitrogenb (%)
DPMCA 3584 8 83.06 2.19 0.35 7.35
DPMA 10168 48 82.43 2.74 0.25 7.20
DMCA 7269 16 81.87 2.16 0.31 7.22
DMA 7839 40 81.78 4.77 0.33 7.36
a DPMCA = Deproteinized + demineralized + decolorized + deacetylated, DPMA = Deproteinized + demineralized + deacetylated, DMCA = Demineralized + decolorized + deacetylated, DMA = Demineralized + deacetylated. b Dry weight basis.
50
the lowest molecular weight of 3584 Da. The lowest molecular weight of the DPMCA crawfish
chitosan can be attributed to the harsh autoclaving deacetylation procedure, which caused
depolymerization. The viscosity measures correlated well with the molecular weight of crawfish
chitosan. All crawfish chitosans showed similar physiochemical properties in terms of degree of
deacetylation, ash, and nitrogen content.
3.3.2 Film-forming Ability of Crawfish Chitosans
Chitosan is known for its film-forming ability. Chitosan filmed with various organic
acids vary in their physiochemical properties. Flexible and transparent films prepared from
crawfish chitosan, resembling plastic films, were reported earlier (Nadarajah and Prinyawiwatkul
2002). The solvent casting method of film formation, in which chitosan is first dissolved in
acidic solvent, cast on plates, and air dried to form films, is often used owing to its simplicity. In
this study, all chitosans extracted from crawfish shell waste exhibited good film-forming ability.
There was no apparent visual difference among films prepared from different crawfish chitosans,
provided that films were cast with the same acid solvent. However, the film-forming ability of
crawfish chitosan was greatly influenced by the casting solvents. The crawfish chitosan films
formed with lactic or malic acids were highly sticky and hygroscopic. These films also exhibited
a high degree of shrinking and deformed instantly upon peeling. Although the films made with
lactic or malic acid were less sticky when a plasticizer was avoided, they still exhibited shrinkage
and deformation upon peeling. All crawfish chitosans formed films with ascorbic acid; however,
resultant films were very brittle. They became far too fragile to handle when no plasticizer was
added. Furthermore, all the ascorbate films developed deep brown coloration, presumably due to
browning reactions, and thus became unappealing for packaging purposes.
51
The film-forming ability of crawfish chitosans was desirable when acetic, formic, or
citric acid was used, resulting in highly flexible and transparent films. However, all plasticized
films with acetic, formic and citric acids exhibited high stickiness and hygroscopic nature, even
in the presence of the lowest plasticizer content. The high degree of hydrophilicity of chitosan is
attributed to the deacetylated amino groups present in the polymeric chain, favoring considerable
migration of water molecules to these sites. Nadarajah and Prinyawiwatkul (2002) reported that
plasticized crawfish chitosan films with acetic acid exhibited more hygroscopic nature compared
to those films prepared with commercial chitosans. Addition of plasticizers minimizes or
eliminates brittleness of the films. However, they also adversely affect film properties by making
them more hygroscopic and contributing to greater absorption of moisture (Lawton 1992).
Lawton (2004) demonstrated that addition of hydrophobic plasticizers can yield edible films that
are minimally hydrophilic. This indicates the possibility of producing a variety of plasticized
crawfish chitosan films if compatible hydrophobic plasticizers are identified.
Films made without plasticizers using acetic, formic and citric acids exhibited neither
stickiness nor hygroscopic nature, and they resembled plastic films. However, the unplasticized
films formed with citric acids were slightly brittle in nature. This suggests that flexible and
transparent films that resemble plastic films can be produced from crawfish chitosans using
acetic or formic acid without any plasticizers. The film-forming ability of unplasticized crawfish
chitosans with different organic acids and their film properties are described in Table 3.2.
3.3.3 Thickness and Density of Crawfish Chitosan Films
We observed that the thickness of chitosan films was highly influenced by the type of
film casting solvent used. Crawfish chitosan formed thicker films with citric acid compared to all
other solvents used in the present study. This is in agreement with Begin and Calsteren (1999)
52
Table 3.2: Film-forming ability of unplasticized crawfish chitosan with different organic acids†.
Film casting solvent†† Film properties
Acetic acid A yellow tinted, flexible, transparent, non-sticky film with smooth shiny surface and slight acidic odor
Ascorbic acid A brown colored, highly brittle film
Citric acid A yellowish, flexible, transparent, non-sticky film with slight brittle and grainier surface without any acidic odor
Formic acid A yellow tinted, flexible, transparent, non-sticky film with smooth shiny surface without any acidic odor
Lactic acid Highly sticky films which shrink upon peeling, becoming a sticky mass/clump
Malic acid Highly sticky films which shrink upon peeling, becoming a sticky mass/clump
† 1% chitosan in film casting solvent by w/v. †† 1% acid by w/v.
who reported that pronounced increase in thickness of film cast with lactic or citric acid.
According to Begin and Calsteren (1999), the citric acid chitosan-film solution gelled before the
molecules could align and pack, which resulted in a thicker film. They also stated that the ability
of citric acid to form multiple salt bridges with amino groups could act as a reticulating agent to
promote gel formation.
The thickness of films varied from 0.02 mm for acetate and formate films to 0.06 mm for
citrate films (Table 3.3). There was no difference in thickness between acetic and formic acid
films. Citrate films showed significantly (p<0.05) greater thickness than acetate and formate
films. The density of films also varied significantly (p<0.05) depending on the acid used. The
formate films had relatively higher density than citrate and acetate films. However, no
correlation between thickness and density of chitosan films was evident.
53
3.3.4 Color of Crawfish Chitosan Films
Color of films is an important attribute which influences its appearance, marketability,
and their suitability for various applications. Clear edible films are typically desirable. The color
attributes of unplasticized crawfish chitosan films are described in Table 3.4. All crawfish
chitosans formed transparent films with slightly yellowish tint. The yellowish tint was more
Table 3.3: Thickness and density of unplasticized crawfish chitosan films
Solvent Chitosan Type* Thickness† (mm)
Density†† (g/cm3)
Acetic acid DPMCA 0.021d 1.47 ± 0.11bcd
DPMA 0.020d 1.76 ± 0.07ab
DMCA 0.024cd 1.19 ± 0.01d
DMA 0.026c 1.34 ± 0.03cd
Formic acid DPMCA 0.027c 1.52 ± 0.12bc
DPMA 0.020d 1.65 ± 0.07abc
DMCA 0.020d 1.88 ± 0.14a
DMA 0.021d 1.64 ± 0.15abc
Citric acid DPMCA 0.059a 1.37 ± 0.05cd
DPMA 0.050b 1.36 ± 0.018cd
DMCA 0.050b 1.42 ± 0.08cd
DMA 0.052b 1.18 ± 0.01d
* DPMCA = Deproteinized + demineralized + decolorized + deacetylated, DPMA = Deproteinized + demineralized + deacetylated, DMCA = Demineralized + decolorized + deacetylated, DMA = Demineralized + deacetylated. Means within the same column sharing same letters are not significantly different at p≥ 0.05. † Means of 10 random measurements and standard deviation. †† Means of 3 measurements and standard deviation.
54
prominent in the citric acid films as shown by greater b* values (3.69 - 4.87). Chitosan films
with similar yellow coloration were reported by Caner and others (1998) and Wiles and others
(2000). The a* values obtained for all films were very low and close to 0 indicating that the films
were more neutral in terms of red or green chromaticity. No difference in color lightness was
visually evident among the films, although statistical analysis revealed some significant (p<0.05)
Table 3.4: Color attributes of crawfish chitosan films†
Solvent Chitosan Type†† L* a* b* ∆E
Acetic acid DPMCA 88.05 ± 0.44abcd -0.54 ± 0.24g 3.70 ± 0.90bc (Reference)
DPMA 87.54 ± 0.38e 0.75 ± 0.12ab 4.37 ± 1.22ab 2.01 ± 0.55a
DMCA 87.69 ± 0.30cde -0.01 ± 0.15de 3.28 ± 0.86bc 1.04 ± 0.47b
DMA 88.23 ± 0.13ab -0.04 ± 0.05de 3.16 ± 0.16c 1.02 ± 0.69b
Formic acid DPMCA 88.16 ± 0.33ab -0.35 ± 0.22fg 3.31 ± 0.85bc 1.20 ± 0.78ab
DPMA 88.33 ± 0.21a 0.58 ± 0.07bc 3.26 ± 0.67bc 1.53 ± 0.67ab
DMCA 88.18 ± 0.40ab -0.21 ± 0.18ef 3.15 ± 0.85c 1.55 ± 1.06ab
DMA 88.10 ± 0.35abc -0.01 ± 0.12de 3.27 ± 0.80bc 1.20 ± 0.83ab
Citric acid DPMCA 87.85 ± 0.21bcde 0.08 ± 0.18d 4.87 ± 0.93a 1.79 ± 0.61ab
DPMA 87.91 ± 0.18abcde 0.82 ± 0.02a 3.69 ± 0.39bc 1.58 ± 0.36ab
DMCA 87.69 ± 0.17cde 0.15 ± 0.11d 4.26 ± 0.52abc 1.27 ± 0.42ab
DMA 87.61 ± 0.38de 0.48 ± 0.04c 3.84 ± 0.22abc 1.46 ± 0.45ab
† Mean of 10 measurements and standard deviation. †† DPMCA = Deproteinized + demineralized + decolorized + deacetylated, DPMA = Deproteinized + demineralized + deacetylated, DMCA = Demineralized + decolorized + deacetylated, DMA = Demineralized + deacetylated. Means within the same column sharing same letters are not significantly different at p≥0.05.
55
difference in L* values. Compared to acetic and citric acid films, the formic acid films exhibited
slightly lighter color (higher L* values), especially the DPMA formate films. The total color
difference (∆E) gives better differentiation of color for comparison purposes (Francis 1983). The
∆E obtained for the chitosan films falling in a narrow range of 1.02 to 2.01 indicates that almost
all the films look alike, in terms of color. It is generally known when ∆E values are less than 3.0,
color differences cannot be easily detected by naked eyes (Francis 1983). Therefore, it can be
inferred that crawfish chitosan type and organic solvents have minimal effect on the color
attributes of films.
3.3.5 Transparency of Crawfish Chitosan Films
Transparency is one of the common optical properties of light permeable materials.
Spectrophotometry is used to measure the transparency of a material by light-transmittance or
absorbance using the Beer-Lambert’s law relating the amount of light absorbed or transmitted by
a material to the nature of the light absorbing material (Chang 1981; Han and Floros 1997).
Development of transparent packaging materials which allow product visibility is a general trend
and requirement in packaging films.
The type of chitosan and acid used to form the film significantly (p<0.05) affected
transparency of crawfish chitosan films (Figure 3.1). In general, the formate films were more
transparent with a mean transparency value (absorbance/mm) of 197.7, followed by acetate films
with 167.6 and citrate films with 84.1. The lowest transparency values obtained for the citric acid
films can be attributed to the higher tint of yellowness (b* values) observed. However, compared
to conventional low density polyethylene films with the transparency values of 20 - 30 (Han and
Floros 1997), all crawfish films were exceptionally more transparent, thus may be used as see-
through packaging or coating materials.
56
0
50
100
150
200
250
300
DPMCA DPMA DMCA DMA
Crawfish chitosan films
Tran
spar
ency
(Abs
orba
nce/
mm
)
Acetate acidFormic acidCitric acid
Figure 3.1: Transparency of unplasticized crawfish chitosan films. Black bars indicate standard deviation. Refer Table 3.1 for abbreviations.
3.3.6 Swelling of Crawfish Chitosan Films
Chitosan, being a hydrophilic polymer, shows high affinity towards water. Hence, upon
hydration, chitosan films absorb water and swell. The swelling behaviors of unplasticized
crawfish chitosan film formed with acetic or formic acid are presented in Figure 3.2. The citrate
films formed with all crawfish chitosans exhibited complete solubility in water and hence
demonstrated no swelling effect. Figure 3.3 shows the formic acid film made with DPMA
chitosan before and after swelling.
Crawfish chitosan films formed with acetic and formic acids showed higher degree of
swelling ranging from 2797.68 to 18785.85 %. Chitosan films exhibiting such extreme swelling
0
50
100
150
200
250
300
DPMCA DPMA DMCA DMA
Crawfish chitosan films
Tran
spar
ency
(Abs
orba
nce/
mm
)
Acetic acidFormic acidCitric acid
57
0
5000
10000
15000
20000
25000
DPMCA DPMA DMCA DMA
Crawfish chitosan films
% S
wel
ling
Acetic acidFormic acidCitric acid
Figure 3.2: The % swelling of crawfish chitosan films formed with acetic and formic acids. Black bars indicate standard deviation. The citric acid film which dissolved upon hydration is indicated as 0 swelling.
(a)
(b)
Figure 3.3: The unplasticized DPMA chitosan film formed with formic acid; (a) before swelling and (b) after swelling.
58
was reported earlier (Bonina and others 2004: Bergera and others 2004). According to Bergera
and others (2004), chitosan films exhibiting higher swelling are beneficial in biomedical
applications since higher swelling allows absorption of water and/or bioactive compounds
without dissolution and permits drug release by diffusion.
The formate film formed from DPMA and DMA chitosans had significantly (p<0.05)
greater swelling than the acetic film. Despite their extreme swelling behavior, all acetate and
formate films retained their integrity and showed no trend of dissolving in water, unlike the
citrate films. Therefore, the crawfish chitosan films which showed lowest swelling (acetic and
formic films with DPMCA or DMCA chitosan and acetic acid film with DPMA chitosan), are
recommended for packaging purposes. The citrate films which dissolve in water may be a good
candidate to develop edible films and packaging materials which require complete solubility
upon hydration.
The crawfish chitosan films showing extremely high swelling (formic acid films with
DPMA or DMA chitosan) may be desirable for development of absorbent pads such as those
used in meat packages to absorb exudates from meat. In such case, the chitosan films can be
directly used as absorbent pads without any plastic lining materials that are normally used to
prevent desorption. The inherent antimicrobial property of chitosan films can also be beneficially
utilized to improve shelf life of package contents. This would also help to replace currently used
plastic wrapped absorbent pads.
3.3.6 Solubility of Crawfish Chitosan Films
Solubility of crawfish chitosan films is presented in Table 3.5. All citrate films were
completely soluble and, hence, accounted for highest solubility. Formate films were more
soluble than acetate films. Data showed that solubility of crawfish chitosan was influenced by
59
the type of acid used and not by the type of chitosan. An acetate film having lower solubility is
favorable for packaging purpose.
3.3.7 Microstructure of Crawfish Chitosan Films
The scanning electron micrographs of crawfish chitosan films are given in Figure 3.4.
The SEM revealed that the structure of crawfish chitosan films vary with the organic solvents
used. Films formed with formic and citric acid were more homogeneous and continuous
Table 3.5: Solubility of unplasticized crawfish chitosan films
Solvent Chitosan Type* Solubility† (S%)
Acetic acid DPMCA 0.22 ± 0.03cd
DPMA 0.15 ± 0.02d
DMCA 0.14 ± 0.01d
DMA 0.16 ± 0.01d
Formic acid DPMCA 0.35 ± 0.01bcd
DPMA 0.55 ± 0.17bc
DMCA 0.58 ± 0.28b
DMA 0.59 ± 0.03b
Citric acid DPMCA 1.00a
DPMA 1.00a
DMCA 1.00a
DMA 1.00a
* DPMCA = Deproteinized + demineralized + decolorized + deacetylated, DPMA = Deproteinized + demineralized + deacetylated, DMCA = Demineralized + decolorized + deacetylated, DMA = Demineralized + deacetylated. Means within the same column sharing same letters are not significantly different at p≥ 0.05. † Means of 3 measurements and standard deviation.
60
(a) (b) (c)
Figure 3.4: The SEM of crawfish chitosan films: (a) acetic acid film, (b) formic acid film, and (c) citric acid film
61
compared to those formed with acetic acid. The lamellar structure found in the acetate and
formate films was not evident with citric acid films. Further research is required to deduce
structure of citric acid films.
3.4 CONCLUSIONS
This study indicates that chitosans derived from crawfish shell waste by different
methods can be used to formulate edible films with selected film casting solvents. All crawfish
chitosans exhibited good film-forming ability. The film-forming ability was significantly
affected more by the type of organic solvents and addition of a plasticizer than the type of
chitosan used. Addition of plasticizers made the films more hydrophilic, sticky, and difficult to
handle. The films formed with lactic and malic acids were highly hygroscopic with high
shrinkage upon peeling. Films formed with ascorbic acids were highly brittle regardless whether
plasticizer was added or not. Therefore, selection of an organic solvent and omission of
plasticizers are important to obtain desirable films from crawfish chitosans.
The films formed with acetic, formic or citric acids without plasticizers were highly
flexible and transparent, and resembled plastic films. No visual color difference was evident
among these films and their transparency far exceeded that of conventional low density
polyethylene. All citrate films exhibited high solubility in water and, hence, have potential for
developing edible packaging material intended for easy solubility. The crawfish chitosan films
(DPMA, DMA formic acid films and DMA acetic films) with higher swelling can be utilized to
form absorbent pads. The acetate and formate films with DPMCA or DMCA chitosan and acetic
acid film with DPMA chitosan exhibited lower swelling upon hydration and, therefore, are
recommended for further research work on developing edible/biodegradable packaging
materials.
62
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films: Effect of solvent acid. Makromol Chem 183: 1353-9. Lawton JW. 1992. Viscoelasticity of zein-starch doughs. J Cereal Chem 69:351-5. Lawton JW. 2004. Plasticizers for zein: their effect on tensile properties and water absorption of
zein films. J Cereal Chem 81(1):1-5. Maghami GG, Roberts GAF. 1988. Evaluation of the viscometric constants for chitosan.
Makromol Chem 189:195-200. Matsuhashi S, Kume T. 1997. Enhancement of antimicrobial activity of chitosan by irradiation. J
Sci Food Agric 73:237-41. Muzzarelli RAA. 1985. Chitin. In: Aspinall ed. Polysaccharides. London, Academic Press. p
417-50. Nadarajah K. and Prinyawiwatkul W. 2002. Filmogenic properties of crawfish chitosan
[abstract]. In: 54th Pacific Fisheries Technologists Annual Meeting Book of Abstracts; 2002 February 24-27; Reno, NV. Abstract nr 37.
Nishimura K, Nishimura S, Nishi N, Saiki I, Tokura S, Azuma I. 1984. Immunological activity
of chitin and its derivatives. Vaccine 2(1):93-9. No HK, ChoYI, Kim HR, Mayers SP. 2000. Effective deacetylation of chitin under conditions of
15 psi/121oC. J Agric Food Chem 48(6):2625-7. No HK, Meyers SP. 1995. Preparation and characterization of chitin and chitosan – a review.
Journal of Aqua Food Pro Techno. 4:27-52. No HK., Park NY, Lee SH, Meyers SP. 2002. Antimicrobial activity of chitosans and chitosan
oligomers with different molecular weights. J Food Microbiol 74:65-72. Park HJ, Jung ST, Song JJ, Kang SG, Vergano PJ, Testin RF. 1999. Mechanical and barrier
properties of chitosan-based biopolymer film. Chitin and Chitosan Research, 5(1):19-26.
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Park SY, Marsh KS, Rhim JW. 2002. Characteristics of different molecular weight chitosan films affected by the type of organic solvents. J Food Sci 67(1) 194-7.
Ralston GB, Tracey MV, Wrench PM. 1964. Inhibition of fermentation in bakers' yeast by
chitosan. Biochem Biophys Acta. 93(3):652-5. Rhim JW, Weller CL, Ham KS. 1998. Characteristics of chitosan films as affected by type of
solvent acid. Food Sci Biotech 7(4):263-8. Roller S, Sagoo S, Board R, O’Mahony T, Caplice E, Fitzgerald G, Fogden M, Owen M,
Fletcher H. 2002. Novel combination of chitosan, carnocin and sulphite for the preservation of chilled pork sausages. Meat Sci 62:165-77.
Rout SK, Prinyawiwatkul W. 2001. Process simplification and modification affecting physico-
chemical properties of chitosan and conversion efficiency of chitin [abstract]. In: IFT Annual Meeting Book of Abstracts; 2001 June 23-27; New Orleans, LA. Chicago, Ill.: Institute of Food Technologists. Abstract nr 15D-25.
Sabnis S, Block LH. 1997. Improved infrared spectroscopic method for the analysis of degree of
N-deacetylation of chitosan. Polym Bull 39:67-71. SAS, Release 9.0 (TS MO), 2002. SAS Institute, Inc. Cary, NC, USA. Stossel P, Leuba J.L. 1984. Effect of chitosan, chitin and some amino-sugars on growth of
various soilborne phytopathogenic fungi. Phytopath. Z. 111: 82–90. Sudarshan NR, Hoover DG, Knorr D. 1992. Antibacterial action of chitosan. Food Biotechnol
6(3):257-72. Tanigawa T, Tanaka Y, Sashiwa H, Saimoto H, Shigemasa Y. 1992. In: Advanced in Chitin and
Chitosan. Brine J, Sandford PA, Zikakis JP. editors. London and New York: Elsevir Applied Science. p 206-15.
Wiles JL, Vergano PJ, Barron FH, Bunn JM, Testin RF. 2000. Water vapor transmission rates
and sorption behavior of chitosan films. J Food Sci 65(7):1175-9 Yalpani M, Johnson F, Robinson LE. 1992. Advances in Chitin and Chitosan. London. Elsevier
Applied Science. 543 p.
65
CHAPTER 4
SORPTION AND WATER PERMEABILITY BEHAVIORS OF CRAWFISH CHITOSAN FILMS
66
4.1 INTRODUCTION
In recent years, there has been an increasing interest in the development of antimicrobial
packaging materials. The use of biodegradable and renewable polymers in an attempt to replace
plastic packaging materials would reduce environmental pollution. With potent antibacterial
(Sudarshan and others 1992; Yalpani and others 1992; No and others 2002a,b), and antifungal
(Allan and Hadwiger, 1979; Stossel and Leuba, 1984; Kendra et al., 1989; Fang et al., 1994,
Ralston et al., 1964) properties, chitosan is one of the most promising and abundant resources for
this purpose. Chitosan possesses a greater and broader spectra of antibacterial activity compared
to disinfectants, a greater microbial killing rate, and lower toxicity toward mammalian cells
(Franklin and Snow, 1981; Takemono and others 1989).
Crawfish shell waste is an abundant, inexpensive and relatively unexploited resource for
chitosan extraction. Since physicochemical properties of chitosan are greatly affected by the
sources (Rhazi and others 2004) and the extraction methods (Rout 2001; Nadarajah and
Prinyawiwatkul 2002), chitosans derived from crawfish shell waste by different extraction
methods may differ in their functionalities. Chitosan dissolves in dilute organic acids.
Consequently, the use of acids having antimicrobial properties such as acetic, lactic, formic,
malic, and citric may offer a simple method to produce antimicrobial films from crawfish
chitosan. Chitosan films formed with various organic acids such as acetic, formic, lactic, citric,
propionic and malic acids have varying physicochemical properties (Butler and others 1996;
Caner and others 1998; Rhim and others 1998; Park and others 1999; Park and others 2002).
Chitosan films exhibited selective permeabilities to gases (Wong and others 1992; Butler
and others 1996) and possessed stronger texture. However, they lacked resistance to water
transmission because of their hydrophilic nature. Hence, there is a major drawback in using
67
chitosans as packaging materials as they are far too humidity sensitive to be used in direct
contact with food or for direct handling (Olbarrieta and others 2001). Hydrophilic films also lose
their mechanical and barrier properties in high moisture conditions (Gontard and others 1994).
Thorough understanding of sorption and water transmission behavior of chitosan film is
necessary to formulate films that are less sensitive to humidity changes.
This study was conducted to determine the sorption and water transmission properties of
crawfish chitosan films, in an attempt to select the films that are less sensitive to humidity and
have better water barrier properties. Effects of chitosan types and film casting organic solvents
on sorption and water transmission behaviors were studied. Models capable of describing
moisture sorption behavior of crawfish chitosan films were identified.
4.2 MATERIALS AND METHODS
4.2.1 Preparation of Chitosan from Crawfish Shell Waste
All chemicals used in this study were analytical grade and purchased from Sigma
Chemical Co. (St. Louis, MO). Crawfish shell waste collected from a local seafood processing
facility was washed with warm tap water to remove foreign materials and remaining muscle
particles. The shells were then dried at 60oC overnight, ground with a centrifugal grinding mill
(Retsch/Brinkmann ZM-1, Westbury, NY), and shell particles between a mesh size of 20 (0.841
mm) and 40 (0.420 mm) were used as starting material. The method established to extract chitin
from crawfish shell waste by No and Meyers (1995) and further processing of chitin into
chitosan through autoclaving (No and others 2000) were used to prepare different chitosan
samples for this study.
Dried crawfish shell particles were treated with 1 N NaOH at 65oC for 1 hour at a solid:
solvent ratio of 1:10, w/v for deproteinization (DP). Following a washing step, deproteinized
68
shell particles were treated with 1 N HCl at room temperature for 30 minutes at a solid: solvent
ratio of 1:1.5, w/v for demineralization (DM). Particles were then treated with acetone at 1: 10
w/v concentration, washed, and bleached with 0.315% NaOCl at a 1:10 w/v ratio for 5 minutes
for decoloration (DC). Resultant chitin was treated with 50% NaOH at a 1:10 w/v ratio at
121oC/15 psi for 30 minutes for deacetylation (DA). Subsequent washing and drying steps
yielded chitosan. Four different chitosan samples were prepared using the traditional method
involving all above steps (DPMCA), the traditional method excluding deproteinization (DMCA),
the traditional method excluding decoloration (DPMA), and the traditional method excluding
both deproteinization and decoloration (DMA).
4.2.2 Characterization of Crawfish Chitosans
The viscosity average molecular weight of crawfish chitosans was determined by the
intrinsic viscosity using the Mark-Houwink equation ([η] = KMa) where [η] is the intrinsic
viscosity, M is the molecular weight, K=1.81 x 10-3 and a=0.93 at 25oC (Maghami and Roberts
1988). Crawfish chitosan films deprotonated by methanolic ammonia was used to determine the
degree of deacetylation with IR spectroscopy (M2000 FTIR spectrophotometer, Midac Corp.,
Irvine, CA, USA) and the equation proposed by Sabnis and Block (1997) was used to calculate
degree of deacetylation values. Viscosity was determined for 1% chitosan in 1% acetic acid
(w/v) using the Brookfield viscometer at 50 rpm and 25oC with a spindle number RV5.
The moisture content (%) of the chitosans was determined gravimetrically in triplicate by
drying samples at 100oC for 48 hours. Nitrogen (%) was determined by the combustion method
using the Leco CHN analyzer (Model # FP-428, Leco Corporation, St. Joseph, Michigan, USA).
The ash content (%) was determined according to the standard method # 923.03 (AOAC 1990).
69
4.2.3 Preparation of Crawfish Chitosan Films
Four types of crawfish chitosans (DPMCA, DPMA, DMCA, and DMA) were separately
dissolved with 1% of film casting solvents (acetic, ascorbic, lactic, formic, citric or malic acids).
Each solution was vigorously agitated for 30 minutes, immersed in boiling water for 10 minutes,
cooled down to room temperature, and filtered through glass-wool to remove undissolved
particles. The film casting solution (300 mL) was cast onto a 31 × 31 cm Teflon coated plate and
allowed to dry at room temperature (23oC) for 48 hours. Dried films were carefully peeled out
and stored in desiccators containing saturated NaBr until further tested.
A micrometer (Model 293-766; Mitutoyo, Tokyo, Japan) was used to measure the film
thickness to the nearest 0.001 mm. Thickness of each film (mm) was measured at room
temperature (23oC and 45% RH) and expressed as an average of 10 random measurements and
standard deviation.
4.2.4 Sorption Isotherm Experiments
Moisture isotherms of chitosan films were determined in triplicate gravimetrically
(Hatakcyama and Hatakcyama, 1998) at 25oC. In order to avoid possible curing effects that may
arise due to heating, film samples were dried at 25oC for 3 weeks in hermetically sealed
desiccators containing Drierite desiccant (W.A. Hammond Drierite Co. Ltd., Xena, OH).
Film samples were weighed in glass containers and placed inside a vacuum desiccator
containing a saturated salt of known water activity (Robinson and Stokes, 1959). The range of
water activities from 0.112 to 0.927 was studied using saturated salt solutions of lithium chloride
(aw = 0.113), magnesium chloride (aw = 0.327), potassium carbonate (aw = 0.431), sodium
bromide (aw = 0.576), potassium iodide (aw = 0.688), sodium nitrate (aw = 0.742), potassium
chloride (aw = 0.843), and potassium nitrate (aw = 0.935). All chemicals were analytical grade
70
purchased from Sigma Chemical Co. (St. Louis, Mo., U.S.A.). The desiccators with the samples
were kept in a temperature controlled environment at 25oC. Equilibrium was considered to be
reached after three weeks as our initial experiments revealed that sample weight did not change
by more than 2 mg of water/g of chitosan film by dry weight. The amount of water adsorbed on
the film sample was determined by re-weighing the containers and the contents.
Six sorption isotherm models: Guggenheim-Anderson-De Boer (GAB model) (Anderson
1946; De Boer 1953; Guggenheim 1966), Smith (1947), Halsey (1948), Caurie (1970), Oswin
(1946), Henderson (1952) were used to fit the experimental sorption isotherm data (Table 4.1).
GAB model parameters were calculated using a non-linear regression program developed by
Professor T.P. Labuza, University of Minnesota, MN, USA.
Table 4.1: Isotherm models used for fitting experimental data
Isotherm Model Equation No.
Oswin (1946) n
w
w
aaam
⎭⎬⎫
⎩⎨⎧
⎥⎦
⎤⎢⎣
⎡−
=1
(1)
Smith (1947) ( )[ ]wa-1ln ×−= ab mmm (2)
Halsey (1948) ( ) ( )[ ]{ }wabam lnlnln −×+= (3)
Henderson (1952) ( )
( ) ( )BA
Ba
m w ln11lnln
ln −⎭⎬⎫
⎩⎨⎧
⎥⎦
⎤⎢⎣
⎡−
= (4)
Caurie (1970) ( ) ⎥⎦
⎤⎢⎣
⎡ −+−=⎥⎦
⎤⎢⎣⎡
w
w
oo a
am
cmcm
1ln2.ln1ln (5)
GAB ( )( )
woow
w aCm
CCkmmka
a⎥⎦
⎤⎢⎣
⎡ −+=
−11
1 (6)
Nomenclature: m = moisture content aw= water activity a, b, ma, mb, A, B, c, C, k are constants mo = monolayer moisture content
71
4.2.5 Water Vapor Permeability of Crawfish Chitosan Films
Water vapor permeability of crawfish chitosan films was measured in triplicate using the
Permatran W3/31 modular system (Modern Controls, Inc., Minneapolis, MN) according to the
American Society of Testing and Materials Standard Method E 96-95 (ASTM, 1995). All films
were placed on a stainless steel mask with an open testing area of 5 cm2. One side of the film
was exposed to flowing nitrogen gas, and the other side was exposed to flowing water vapor at
25°C and 50 % RH.
The water vapor permeability, WVP, (ng·m/m2·s·Pa) of film was calculated as follows:
( ) LP
WVTRWVP ×∆
=
Where WVTR is the measured water vapor transmission rate (ng/m2·s) through a film, L
is the mean film thickness (m), and ∆P was the partial water vapor pressure difference (Pa)
across the 2 sides of the film.
4.2.6 Statistical Analysis
All experiments were carried out in triplicate and average values were reported. Linear
regression analysis (PROC REG) was performed to derive regression models and to calculate the
best fitted values of constants in the sorption isotherm equations using SAS (SAS, 2002). The
suitability of the equations was evaluated and compared using the coefficient of regression (R2).
PROC ANOVA with a nested factorial model was applied to test the differences among
isotherms. Significance of differences was defined at P < 0.05.
4.3 RESULTS AND DISCUSSION
4.3.1 Characteristics of Crawfish Chitosan
The physicochemical properties of all chitosans used in this study are shown in Table 3.1.
The chitosan extracted by the traditional method (DPMCA) had a lowest molecular weight due
72
to depolymerization of chitosan. The DPMCA chitosans also showed the lowest viscosity and
highest degree of deacetylation. The DMA chitosan was subjected to least chemical treatments
but had lower molecular weight than that of DPMA chitosan. However, considering the general
range of molecular weight of commercial chitosans falling between 100,000 and 1,200,000
Daltons (Li and others, 1992), all crawfish chitosans produced in this study can be convincingly
categorized as low molecular weight chitosans. Viscometric measurements agreed with the
magnitude of molecular weight accounting lowest viscosity for DPMCA chitosan and highest
viscosity for DPMA chitosan. The DMA chitosan contained about twofold moisture content of
that of other chitosans.
4.3.2 Film-forming Ability and Film Characteristics
The film-forming ability of plasticized crawfish chitosans can be considered poor owing
to the highly hygroscopic nature of the films, which makes them adsorb moisture from the
atmosphere and makes the films sticky. Further, plasticized films formed with malic and lactic
acids undergo shrinkage and deformation immediately after being peeled out from casting plates.
The sticky and shrunk chitosan films are not desirable for handling and, therefore, not suitable
for packaging applications. Nadarajah and Prinyawiwatkul (2002) reported that chitosan acetate
films made from crawfish chitosan (deacetylated by a distillation process) with molecular weight
46,000 Da showed higher hygroscopic nature compared to the films made from commercially
available shrimp chitosan. Although many have reported film-forming ability of chitosan
(Muzzarelli and others, 1974; Averbach, 1978; Butler and others, 1996; Caner and others, 1998),
the hygroscopic nature of plasticized chitosan films have not been reported and presumably not
encountered with the chitosans used.
73
Among the unplasticized films, those made with malic and lactic acid showed stickiness
similarly observed with plasticized films, although shrinkage of unplasticized films was not
prominent. Regardless of the types of chitosans used (DPMCA, DMPA, DMCA, DMA), the
films made with acetic or formic acid were highly transparent and flexible with slight yellow tint.
All citrate films were yellowish and brittle in nature compared to acetate and formate films.
Chitosan films possessing slight yellow tint was reported by Caner and others (1998) and Wiles
and others (2000). All crawfish chitosan films showed the tendency of becoming yellowish
during prolonged storage at higher humidity conditions. Similar observations were also reported
by Wiles and others (2000) for chitosan acetate films. Apart from that, our crawfish chitosan
films showed no apparent visual difference among them. Interestingly, the flexibility of chitosan
acetate and formate films was not impaired at low water activity levels (aw=0.113) despite the
fact that unplasticized films tend to become brittle and lose their integrity at low water activity
levels. However, the citrate films became more brittle at lower water activity levels and broken
apart when handled. This reveals that flexible crawfish chitosan films can be formulated without
added plasticizers and prepared with acetic and formic acids. Plasticizers are used to reduce
brittleness of polysaccharide or protein-based films but their use entails decreased efficiency of
barrier properties of edible packaging (Butler and others 1996; Shaw and others 2002). Further,
the film-forming ability of crawfish DMA chitosan indicates the possibility of formulating more
economical chitosan films because DMA chitosans undergo the least number of processing steps,
chemicals, and time to produce.
4.3.3 Sorption Isotherms of Crawfish Chitosan Films
The moisture sorption characteristics of crawfish chitosan films are critical for
ascertaining their use as a packaging material at varying humidity conditions. The sorption
74
isotherms of all (DPMCA, DPMA, DMCA, DMA) chitosan films showed a classical sigmoid
curve described as the type II isotherm in BET classification (Brunanuer 1945) (Figure 4.1).
Most biological products follow a sigmoid curve representing the type II isotherm BET
classification (Labuza 1984).
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0 0.2 0.4 0.6 0.8 1
Water activity (aw)
g w
ater
/g c
hito
san
DPMCA acetate filmDPMCA formate filmDPMCA citrate film
Figure 4.1: Moisture sorption isotherms of representative crawfish chitosan films at 25oC.
The acetate and formate films exibited similar adsorption curves. However, the
adsorption curve of citrate films deviated from both acetate and formate films (Figure 4.1). The
citrate films exhibited stickiness above water activity level of 0.74 and brittleness at below water
75
activity level of 0.33. However, the acetate and formate films retained their flexibility and non-
sticking behavior throughout the water activity continuum.
The chitosan acetate films made from different crawfish chitosans exhibited identical
sorption isotherms, despite the differences in physicochemical properties of chitosans (Figure
4.2). The PROC ANOVA procedure performed with nested water activity levels revealed that
there was no significant difference (p>0.05) among the isotherms obtained from different types
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0 0.2 0.4 0.6 0.8 1
Water activity (aw)
g w
ater
/g c
hito
san
DPMCA acetate filmDPMA acetate filmDMCA acetate filmDMA acetate film
Figure 4.2: Moisture sorption isotherms of chitosan acetate films at 25oC.
76
of crawfish chitosans. Thus, we can deduce that the extraction process of chitosan and their
physicochemical properties have negligible influence on the sorption behavior of chitosan
acetate films evaluated in this study.
Unlike chitosan acetate films, chitosan formate films made from different types of
crawfish chitosans showed significantly different (p<0.05) isotherms (Figure 4.3). The PROC
ANOVA with nested water activity levels revealed that the isotherm of DPMCA chitosan
formate film was significantly different (p<0.05) from that of DMCA and DMA formate films.
0
0.1
0.2
0.3
0.4
0.5
0.6
0 0.2 0.4 0.6 0.8 1
Water activity (aw)
g w
ater
/g c
hito
san
DPMCA formate filmDPMA formate filmDMCA formate filmDMA formate film
Figure 4.3: Moisture sorption isotherms of chitosan formate films at 25oC.
77
No significant difference (p>0.05) in isotherms was found between DPMCA and DPMA
chitosan formate films as well as between DPMA and DMCA chitosan formate films.
Comparison of isotherms of chitosan acetate with chitosan formate films indicated that
the formate films were significantly different (p<0.05) from the acetate films, except for
DPMCA acetate and DPMCA formate films (Figures 4.2 and 4.3). Chitosan citrate films were
significantly (p<0.05) different from both acetate and formate films (Figure 4.1). Statistical
analysis revealed that both the type of acids and chitosans used for film formation influenced the
sorption behavior of crawfish chitosan films. However, the effect of chitosan type was less
prominent.
At elevated water activity levels, a pronounced increase in sorption uptake was observed
(Figures 4.2 and 4.3). Similar observations were also reported by Despond and others (2001).
The pronounced increase in the equilibrium moisture content at elevated water activity levels
indicates possible swelling effects in the films. Swelling would cause changes in the
microstructure of the film and increase moisture sorption, and water vapor would act as a
plasticizer inside the chitosan matrix (Wiles and others 2000; Rogers 1985). At water activity
levels of 0.743 to 0.936, the amount of water absorbed by chitosan films increased by 138% (dry
basis) for acetate films, by 192% (dry basis) for formate films, and by 285 % (dry basis) for
citrate films. Since chitosan citrate film changes its nature from very brittle to very soggy at
elevated water activity levels, it cannot be a good candidate for packaging purposes. Though
both chitosan acetate and formate films retained their flexibility and integrity along the water
activity continuum, the chitosan acetate films, which absorb less water and produce lower lying
adsorption isotherm, are of better qualify for packaging purposes. However, apart from
adsorption, the absorption and swelling behaviors of chitosan films have to be considered to
78
select more appropriate films. Accordingly, the chitosan acetate film with DMCA chitosan and
the formate film with DPMCA chitosan, having lower swelling, qualify for packaging purposes.
4.3.4 Sorption Model Analysis
The isotherm models used in this study and the calculated constant values are
summarized in Table 4.2, 4.3, and 4.4 for acetate, formate and citrate films, respectively. A good
agreement between experimental and predicted data was found with the GAB models with the
coefficient of determination (R2) of 0.97 to 0.99 for all the films tested, regardless of the type of
acids and chitosans used. Therefore, the GAB model is most appropriate for the prediction of
equilibrium moisture content of crawfish chitosan acetate, formate, and citrate films. The GAB
equation has been described to predict the moisture sorption of many foods and natural polymers
with adequate accuracy (Joncquières and Fane 1998; Taoukis 1988). Despond and others (2001)
reported the adequacy of the GAB model for water sorption of chitosan films.
Among other models tested, the Oswin and Caurie models can be used to predict sorption
behavior of acetate and formate films with R2 values of 0.93 to 0.98. However, except for the
GAB model, most of the models tested did not accurately describe chitosan citrate films. This
can be attributed to the deviation found in the sorption behavior of chitosan citrate films in which
they adsorbed much lower and much higher moisture, respectively, at below and above the water
activity level of 0.84.
The GAB model and the water clustering theory of Zimm and Lundberg (1956) were
utilized to infer the water clustering phenomenon in chitosan films (Despond and other 2001).
Zimm and Lundberg (1956) developed an equation to calculate a cluster integral from the
sorption isotherm. The clustering function (G11/v1) is a characteristic quantity that enables the
79
Table 4.2: Constants of sorption models for chitosan acetate films
Isotherm Chitosan Type* Constantsb R2
GAB k C mo DPMCA 0.90 16.63 0.062 0.98 DPMA 0.93 27.77 0.069 0.98 DMCA 0.87 9.61 0.069 0.98 DMA 0.88 13.25 0.068 0.98 Smith Mb Ma DPMCA 0.021 0.134 0.95 DPMA 0.036 0.111 0.91 DMCA 0.021 0.125 0.96 DMA 0.025 0.118 0.95 Oswin a n DPMCA 0.115 0.459 0.97 DPMA 0.116 0.393 0.93 DMCA 0.115 0.459 0.97 DMA 0.107 0.450 0.94 Halsey a b DPMCA -2.46 -0.60 0.95 DPMA -2.41 -0.52 0.93 DMCA -2.45 -0.59 0.93 DMA -2.52 -0.59 0.92 Caurie C mo DPMCA 0.162 0.709 0.97 DPMA 0.151 0.771 0.93 DMCA 0.162 0.709 0.97 DMA 0.155 0.691 0.94 Henderson A B DPMCA 14.41 1.47 0.93 DPMA 24.18 1.73 0.89 DMCA 109.86 2.17 0.97 DMA 14.02 2.21 0.94
*DPMCA = Deproteinized + demineralized + decolorized + deacetylated, DPMA = Deproteinized + demineralized + deacetylated, DMCA = Demineralized + decolorized + deacetylated, DMA = Demineralized + deacetylated. b Refer to Nomenclature for constants
80
Table 4.3: Constants of sorption models for chitosan formate films
Isotherm Chitosan Type* Constants byb R2
GAB k C mo DPMCA 0.92 9.53 0.060 0.98 DPMA 0.92 14.08 0.066 0.97 DMCA 0.93 15.15 0.063 0.98 DMA 0.92 14.36 0.065 0.98 Smith Mb Ma DPMCA 0.010 0.146 0.94 DPMA 0.016 0.159 0.94 DMCA 0.009 0.169 0.93 DMA 0.0131 0.161 0.93 Oswin a n DPMCA 0.111 0.501 0.95 DPMA 0.127 0.471 0.96 DMCA 0.125 0.483 0.94 DMA 0.127 0.460 0.94 Halsey a b DPMCA -2.52 -0.65 0.93 DPMA -2.37 -0.62 0.95 DMCA -2.40 -0.64 0.94 DMA -2.36 -0.61 0.95 Caurie C mo DPMCA 0.167 0.667 0.95 DPMA 0.173 0.734 0.96 DMCA 0.174 0.730 0.95 DMA 0.171 0.746 0.94 Henderson A B DPMCA 79.00 1.99 0.95 DPMA 79.05 2.11 0.96 DMCA 73.67 2.06 0.94 DMA 86.20 2.17 0.94
*DPMCA = Deproteinized + demineralized + decolorized + deacetylated, DPMA = Deproteinized + demineralized + deacetylated, DMCA = Demineralized + decolorized + deacetylated, DMA = Demineralized + deacetylated. b Refer to Nomenclature for constants
81
Table 4.4: Constants of sorption models for chitosan citrate films
Isotherm Chitosan Type* Constants byb R2
GAB k C mo DPMCA 0.96 1.31 0.030 0.97 DPMA 0.89 0.38 0.083 0.99 DMCA 0.95 0.77 0.045 0.99 DMA 0.96 0.73 0.042 0.99 Smith Mb Ma DPMCA -0.008 0.080 0.82 DPMA -0.035 0.124 0.88 DMCA -0.046 0.138 0.91 DMA -0.052 0.143 0.91 Oswin a n DPMCA 0.035 0.807 0.83 DPMA 0.036 0.912 0.91 DMCA 0.043 0.806 0.96 DMA 0.036 0.909 0.95 Halsey a b DPMCA -3.85 -1.02 0.76 DPMA -3.78 -1.11 0.85 DMCA -3.52 -0.95 0.94 DMA -3.49 -1.04 0.87 Caurie C mo DPMCA 0.119 0.294 0.83 DPMA 0.129 0.284 0.91 DMCA 0.131 0.326 0.96 DMA 0.128 0.283 0.95 Henderson A B DPMCA 63.21 1.23 0.83 DPMA 37.08 1.09 0.91 DMCA 49.38 1.23 0.96 DMA 38.09 1.09 0.95
*DPMCA = Deproteinized + demineralized + decolorized + deacetylated, DPMA = Deproteinized + demineralized + deacetylated, DMCA = Demineralized + decolorized + deacetylated, DMA = Demineralized + deacetylated. b Refer to Nomenclature for constants
82
calculation of tendency of the (water) molecules to cluster in the given polymer matrix. The
clustering function is defined as the ratio:
1)1(
,
1
1
1
11
11 −
⎥⎥⎥⎥
⎦
⎤
⎢⎢⎢⎢
⎣
⎡
∂
⎟⎟⎠
⎞⎜⎜⎝
⎛∂
−−=
Tp
vG
αφα
φ (7)
where G11 is the cluster integral, calculated from the molecular pair distribution, 1v is the partial
molar volume of the penetrant (e.g., water), φ 1 is the volume fraction, and a1 is the activity of
component 1. For an ideal solution, the activity coefficient (a1/φ 1) does not vary with
concentration, i.e., the activity is proportional to the volume fraction, and , therefore, eq. (7)
becomes:
11
11 −=v
G (8)
For nonrandom mixing solutions, however, the activity coefficient decreases with
increasing φ 1 so that G11/v1 is greater than -1. The extent of clustering in the solution is indicated
by the extent to which G11/v1 exceeds -1. The quantity φ 1G11/v1 is the mean number of excess
water molecules in the neighborhood of a given water molecule. The average number of solvent
molecules in a cluster can be described by the following equation:
11
111 +=
vGNc φ (9)
where Nc is the cluster number. For an ideal solution where there is no clustering of water, Nc is
equal to 1. The Nc values greater than 1 represent clustering of water molecules. Positive Nc
values indicate that the solute increases the free volume of the polymer matrix, increasing the
83
sorption capacity, diffusivity, and permeability. By combining eqs. (6, Table 4.1), (7), and (9), Nc
can be expressed as (Zhang and others 1999):
⎟⎟⎠
⎞⎜⎜⎝
⎛−−++−×−−= 1)222()1(
0
11 CkaCka
CmN wwc
φφ (10)
Where mo is the monolayer moisture content, and k and C are the GAB constants. To
deduce this equation, the variable m in eq. (6) (Table 4.1) was transformed from gravimetric to
volumetric fraction φ 1 using densities of crawfish chitosan films.
The Nc values deduced from the equation 10 indicate that when water activity exceeds
0.57, clustering of water molecules takes place (Figure 4.4). At lower water activity levels, water
was distributed mainly through the polymer matrix, probably absorbed on the active sites of
hydrogen bonds. As water activity increased, water molecules predominately clustered on these
hydrogen bonding sites, likely resulting in plasticization of the polymer. Water molecules that
initially entered the polymer structure may have opened the structure to make it easier for
subsequent water molecules to absorb in the neighborhood of the initially absorbed molecules
(Despond and others 2001).
4.3.5 Water Vapor Transmission Rate
The water vapor transmission rates of crawfish chitosan films are shown in Table 4.5.
Thickness of crawfish chitosan films was dependent of the type of chitosans and film casting
solvents used. Chitosan citrate films had significantly (p<0.05) higher thicknesses than chitosan
acetate and formate films. In general, chitosan acetate and formate films had similar film
thickness. The variation in the film thickness observed in this study is in agreement with those
described earlier by Caner and others (1998) and Begin and Calsteren (1999). Ideal polymeric
films should not exhibit thickness effect on water vapor permeability. However, hydrophilic
84
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0.0 0.2 0.4 0.6 0.8 1.0
Water activity (aw)
Nc
DPMCA acetate film
DPMA acetate filmDMCA acetate film
DMA acetate film
Figure 4.4: Effect of water activity on clustering of water molecules (Nc values greater than 1) in chitosan acetate films.
films often, but not always, exhibit a positive slope relationship between thickness and water
vapor permeability (Miranda and others 2004).
All citrate films showed significantly (p<0.05) lower WVTR compared to acetate and
formate films. However, there was no significant difference in WVTR among all chitosan citrate
films. The lower WVTR obtained by the citrate films can be attributed to the greater thickness
85
compared to that of other films. In general, the chitosan formate films had higher WVTR than
the acetate films. This observation is in agreement with that described by Caner and others
(1998). The highest WVTR was obtained for DPMA formate films.
Table 4.5: Water vapor transmission rate and water vapor permeability at 25oC and 50% RH gradient.
Solvent Chitosan Type*1 Thickness*2 (mm)
WVTR*3 (g/m2.day)
WVP (ng.m/m2.s.Pa)
Acetic acid DPMCA 0.021d 244.35 ± 5.34c 0.038f
DPMA 0.020d 217.12 ± 5.81d 0.032g
DMCA 0.024cd 273.69 ± 2.58b 0.048c
DMA 0.026c 298.45 ± 0.63a 0.057b
Formic acid DPMCA 0.027c 310.53 ± 2.71a 0.061a
DPMA 0.020d 311.10 ±1.35a 0.045cd
DMCA 0.020d 272.87 ± 8.05b 0.040ef
DMA 0.021d 277.94 ± 11.91b 0.043de
Citric acid DPMCA 0.059a 44.59 ± 3.12e 0.019h
DPMA 0.050b 40.73 ± 4.36e 0.015hi
DMCA 0.050b 38.36 ± 1.77e 0.014hi
DMA 0.052b 44.52 ± 5.23e 0.017hi
*1DPMCA = Deproteinized + demineralized + decolorized + deacetylated, DPMA = Deproteinized + demineralized + deacetylated, DMCA = Demineralized + decolorized + deacetylated, DMA = Demineralized + deacetylated. Means within the same column sharing same letters are not significantly different at p > 0.05. *2 Means of 10 random measurements and standard deviation. *3 Means of 3 measurement and standard deviation.
86
Water vapor permeability of a film is constant and independent of the driving force on
water vapor transmission. However, in hydrophilic films, water molecules interact with polar
groups in the film structure, causing plasticization or swelling, which, in term results in varying
permeability (Miranda and others 2004).
Statistical analysis showed that both WVTR and WVP were significantly (p<0.05)
influenced by the film casting solvents. Chitosan types showed significant (p<0.05) influence on
WVTR of films. Hence, it can be deduced that both chitosan types and film casting solvent are
critical in water transmission.
Prediction of water transport through chitosan films becomes complex due to films’
hydrophilic nature. The complexity is due to their nonlinear water sorption isotherms and water
content dependent diffusivity. Chitosan, possessing high cationic and being hydrophilic in
nature, leads to higher interaction with water molecules, which increases the permeation of
water vapor. With increasing moisture, the films start swelling. Swelling would cause
conformational changes in the microstructure of the film that not only increase moisture sorption,
but also increases channels in the polymeric structure to allow the increase in permeant flow.
Swelling results in deviation from Fickian behavior (Miranda and others 2004).
Table 4.6 shows a comparison of results obtained from our study with past literatures on
chitosan films, common edible films and some plastic films. The WVP values obtained in the
present study are in agreement with those obtained by Butler and others (1996) and Wong and
others (1992). However, much higher WVR values were reported by Miranda and others (2004)
and Caner and others (1998). The WVP of crawfish chitosan films is lower than that of many
edible starch or protein based films, but is much higher than those of plastic films.
87
1Table 4.6: Water vapor permeability of chitosan, edible and plastic films.
Film Water vapor permeability (ng.m/m2.s.Pa) Reference
Crawfish chitosan filmsa
Acetate films 0.032 - 0.057 Present study
Formate films 0.040 - 0.061 Present study
Citrate films 0.014 - 0.019 Present study
Chitosan film from the literature
Chitosan acetateb 0.025 Butler and others (1996)
Chitosan acetatec 0.078 - 0.125 Caner and others (1998)
Chitosan formatec 0.107 - 0.135 Caner and others (1998)
Chitosan formate 0.0365 Wong and others (1992)
Chitosan acetated 0.345 Miranda and others (2004)
Edible films
Carboxymethylated starch 1.2 - 2.1 Kim and others (2002)
Soybean protein 0.17 Ghorpade and others (1995)
Wheat gluten 0.14 Aydt and others (1991)
Rice bran 9.20 Gnanasambandam and others (1997)
Corn zein 0.12 - 0.33 Park and Chinnan (1995)
Plastic films
Low density polyethylene 0.00055 Krochta and Johnson (1997)
High density polyethylene 0.00020 Krochta and Johnson (1997)
Polyvinyl chloride 0.00071 Park and Chinnan (1995)
aFilms made with 1% chitosan in 1% acetic acid (w/v). Test condition: 25oC and 50% RH gradient. bFilms made with 3% chitosan in 1% acetic acid (w/v). Test condition: 25oC and 50% RH gradient. cFilms made with 3% chitosan in 1% acetic acid (w/w). Test condition: 25oC and 50% RH gradient. dFilms made with 2% chitosan in 1% acetic acid (v/v). Test condition: 28 - 32oC and 95 - 16% RH gradient.
88
4.4 CONCLUSIONS
Crawfish chitosan forms flexible and transparent films when acetic or formic acid was
used as a film casting solvent without a plasticizer. Chitosan extraction methods and film casting
solvents significantly affected the sorption behavior of crawfish chitosan films although the
effect was not apparent for those films made with acetic acid. The unplasticized crawfish
chitosan acetate films, which are flexible, transparent and reaching lower moisture content at any
water activity level compared to chitosan formate films, are good candidates for developing
packaging. On average, chitosan formate films adsorbed more moisture than chitosan acetate or
citrate films. While acetate and formate films maintained their flexibility, the citrate films
exhibited brittleness to soggy nature along the water activity continuum and, are therefore, not
suitable for packaging applications.
The sorption behavior of crawfish chitosan acetate and formate films can be best
predicted by the GAB model (R2 = 0.97-0.98) followed by the Oswin and Caurie models (R2 =
0.93-0.97). The sorption behavior of chitosan citrate films, which showed much deviation from
those of acetate and formate films, can only be best predicted by the GAB model (R2 = 0.98-
0.99). Therefore, the GAB model can be appropriately used to predict sorption behavior of all
crawfish chitosan films. Furthermore, the GAB model provided information on clustering of
water molecules at water activity exceeding 0.57.
The water vapor permeability of crawfish chitosan films was significantly influenced by
the chitosan types and film casting solvents used. The chitosan citrate films showed the lowest
WVP followed by chitosan acetate films. The highest WVP was obtained for DPMCA chitosan
formate films.
89
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94
CHAPTER 5
MECHANICAL PROPERTIES OF CRAWFISH CHITOSAN FILMS AS AFFECTED BY CHITOSAN PRODUCTION PROTOCOLS AND
FILM CASTING SOLVENTS
95
5.1 INTRODUCTION
Knowledge of mechanical properties is necessary for any packaging material to ensure
integrity of it and its content. Developing edible films with mechanical properties comparable or
superior to those of synthetic packaging materials has been a challenge. Most edible films lack
mechanical properties appropriate for packaging purposes.
Chitosan is a natural polymer that is readily biodegradable by many chitosanase-
producing bacteria. Chitosan possesses excellent film-forming properties (Muzzarelli and others
1974; Averbach 1978; Butler and others 1996; Caner and others 1998; Wiles and others 2000).
Chitosan films are described as being tough, long lasting, flexible, and very difficult to tear
(Butler and others 1996). Thus, most of their mechanical properties are comparable to many
medium-strength commercial polymers (Butler and others 1996). However, the pursuit of
making even better chitosan films with more strength and functionalities is in progress. Cross-
linking chitosan with glutaraldehyde, divalent metal ions, polyeletrolytes (Dutkiewiez and
others 1992), and anionic polysaccharide (Hoagland and Parris 1996) or laminating chitosan with
other polysaccharides such as pectin (Hoagland and Parris 1996) and methylcellulose (Chen and
others 1996) are some of such attempts.
Chitosan films formed with various organic acids such as acetic, formic, lactic, citric or
and malic acid have been reported to have varying physicochemical and mechanical properties
(Kienzle-Sterzer and others 1982; Butler and others 1996; Caner and others 1998; Rhim and
others 1998; Park and others 1999; Park and others 2002). Selection of organic acids that give
maximum mechanical strengths to films is one straightforward approach to obtain high strength
chitosan films. Various organic solvents (acetic, lactic, formic, citric, malic, and propionic acids)
have been used for preparing chitosan films (Park and others 2002; Kienzle-Sterzer and others
96
1982; Butler and others 1996; Rhim and others 1998; Park and others 1999). The chitosan films
formed without plasticizers were brittle films and not suitable for packaging applications.
Nadarajah and Prinyawiwatkul (2002) stated that crawfish chitosans extracted by
different extraction methods affected film-forming ability of resultant chitosans and their film
properties. They further reported that crawfish chitosans had excellent film-forming properties
and can be used to produce flexible and transparent films, resembling plastic films. However,
these films were so humidity sensitive, that makes them inappropriate for packaging purposes.
We discovered that crawfish chitosan films that are less humidity sensitive can be formulated
using different combinations of organic acids without any plasticizers and different types of
chitosan obtained through modified chitosan extraction protocols. This study was attempted to
investigate the effects of different film casting solvents and chitosan extraction protocols on
mechanical properties of crawfish chitosan films.
5.1.1 Objective
The overall research objective was to formulate crawfish chitosan films with maximum
mechanical properties by selecting appropriate casting solvent and chitosan extraction protocol.
The specific objectives were;
1. to determine the effects of different organic acids on tensile and puncture
strengths of crawfish chitosan films,
2. to determine the effects of crawfish chitosan extraction protocols on tensile and
puncture strengths of crawfish chitosan films,
3. to establish an optimum combination of film casting solvent and chitosan
extraction protocol to achieve maximum mechanical strength for crawfish
chitosan films.
97
5.2 MATERIALS AND METHODS
5.2.1 Materials
Crawfish chitosans were derived from crawfish shell waste using the traditional and/or
modified methods. The traditional method involves deproteinization (DP), demineralization
(DM), decoloration (DC) and deacetylation (DA) (No and Meyers 1995; No and others 2000).
Four different chitosan samples were prepared using the traditional method involving all above
steps (DPMCA), the traditional method excluding deprotienization (DMCA), the traditional
method excluding decoloration (DPMA), and the traditional method excluding both
deproteinization and decoloration (DMA).
5.2.2 Characterization of Crawfish Chitosans
The viscosity average molecular weight of crawfish chitosans was determined by the
intrinsic viscosity using the Mark-Houwink equation ([η] = KMa) where [η] is the intrinsic
viscosity, M is the molecular weight, K=1.81 x 10-3 and a=0.93 at 25oC (Maghami and Roberts
1988). Crawfish chitosan films deprotonated by methanolic ammonia was used to determine the
degree of deacetylation with IR spectroscopy (M2000 FTIR spectrophotometer, Midac Corp.,
Irvine, CA, USA) and the equation proposed by Sabnis and Block (1997) was used to calculate
degree of deacetylation values. Viscosity was determined for 1% chitosan in 1% acetic acid
(w/v) using the Brookfield viscometer at 50 rpm and 25oC with a spindle number RV5.
The moisture content (%) s of the chitosans was determined gravimetrically in triplicate
by drying samples at 100oC for 48 hours. Nitrogen (%) was determined by the combustion
method using the Leco CHN analyzer (Model # FP-428, Leco Corporation, St. Joseph, Michigan,
USA). The ash content (%) was determined according to the standard method # 923.03 (AOAC
1990).
98
5.2.3 Film Preparation
Four types of crawfish chitosans (DPMCA, DPMA, DMCA, and DMA) were separately
dissolved with 1% of film casting solvents (acetic, ascorbic, lactic, formic, citric or malic acids).
Each solution was vigorously agitated for 30 minutes, immersed in boiling water for 10 minutes,
cooled down to room temperature, and filtered through glass-wool to remove undissolved
particles. The film casting solution (300 mL) was cast onto a 31 × 31 cm Teflon coated plate and
allowed to dry at room temperature (23oC) for 48 hours. Dried films were carefully peeled out
and stored.
5.2.4 Conditioning of Films
All film specimens used for the mechanical tests were preconditioned for 48 hr at 25°C
and 52.8% relative humidity inside desiccators containing saturated solutions of magnesium
nitrate. To avoid post aging impact on films, films of 3 days old were used (Caner and others
1998).
5.2.5 Thickness of Films
A micrometer (Model 293-766; Mitutoyo, Tokyo, Japan) was used to measure the film
thickness to the nearest 0.001 mm prior all tests. The film thickness was used for calculation of
tensile and puncture strengths. For tensile tests, a mean of five measurements across each film
specimen was used. For puncture tests, a mean was calculated from 10 random measurements
made across each film.
5.2.6 Tensile Test
Tensile measurements for tensile strength, yield strength, elongation, and Young’s
Modulus were performed with the Instron testing system (model 4411, Instron Engineering
Corp., Canton, MA) following the ASTM standard D882 (ASTM 1998b). Uniform film
99
specimens of a 100 mm × 5 mm size were prepared from chitosan film samples. Film strips were
placed in the pneumatic grips of the testing machine, which were set at an initial separation of 50
mm. The crosshead speed was set at 50 mm/min. At least 12 specimens for each film type were
tested and an average of 8 measurements was reported.
5.2.7 Puncture Strength
Puncture strength (PS) of the chitosan film samples was determined, according to the
ASTM standard D3763-97a (ASTM 1998a), using the texture analyzer system (TA.XT plus,
Stable Micro System, Haslemere, Surrey, UK) equipped with a 5kg load cell and a film testing
rig (TA-108). A square film sample of 100 mm × 100 mm was used. The puncture head was a
cylindrical rod of 3 mm diameter (TA-53) and the puncture speed was set at 1 mm/s. The PS was
measured 6 times for each sample and reported as N/mm (the peak force divided by the thickness
of the film).
5.2.8 Statistical Analysis
Data from tensile, tear, and puncture tests were analyzed using SAS (SAS 2002). The
PROC GLM procedure with orthogonal contrasts was used to test the effect of film casting
solvents and chitosan extraction methods on tensile, tear, and puncture strengths. Significance of
differences was defined at P < 0.05.
5.3 RESULTS AND DISCUSSIONS
5.3.1 Tensile Strength
The tensile strength curves of crawfish chitosan acetate and formate films followed
typical stress-strain curve of LDPE films, except for the short elongation phase. However, the
citrate films behaved differently and failed to exhibit any Hookian behavior (reversible
100
deformation zone) or true yield point before break (Figure 5.1). Similar observation was also
reported by Begin and Calsteren (1999) for chitosan citrate films.
Figure 5.1: Stress-strain curves of crawfish chitosan films with organic acid solvents.
The tensile strength values of crawfish chitosan films prepared with different organic
acids are shown in Table 5.1. Depending on the type of chitosan and film casting solvent, tensile
strength of crawfish chitosan films varied widely ranging from 18.9 to 135.8 MPa. Among the
acids used, acetic acid resulted in significantly (p<0.05) higher tensile strength, followed by
formic and citric acids. This result agrees with that of Rhim and others (1998), Begin and
Calsteren (1999), Khan and others (2000), and Park and others (2002), who found acetic acid
formed films with highest tensile strength values compared to those formed with formic, malic,
lactic, and citric acids. As per Billmeyer (1984), the acetic acid and formic acid films accounting
101
for tensile strength of 76.8 to 135.8 MPa and Young’s modulus in the range of 2400.3 to 4120.5
Mpa, can be considered as hard. The citric acid films, accounting for lower tensile strength and
%E values, can be considered soft and brittle. According to Park and others (2000), in acetic acid
solution, chitosan forms dimers indicating that the intermolecular interaction is relatively strong,
which leads to tighter structure than those prepared with other acid solutions.
Table 5.1: Tensile measurements of crawfish chitosan films
Solvent Chitosan Type*
Tensile Strength (MPa)
Yield Strength (MPa)
% Elongation
Modulus (MPa)
Acetic acid DPMCA 82.0 ± 8.7c 72.4 ± 4.2b 25.0 ± 8.9e 3309.5abc
DPMA 129.5 ± 4.2ab 73.7 ± 5.6b 45.0 ± 6.7ab 3120.6bc
DMCA 135.8 ± 9.8a 92.1 ± 5.9a 37.1 ± 3.1bcd 3384.8ab
DMA 132.9 ± 9.6a 77.3 ± 7.2b 43.2 ± 3.6 b 4120.5a
Formic acid DPMCA 76.8 ± 4.8c 61.5 ± 5.0c 31.5 ± 6.6cde 2848.4bc
DPMA 114.8 ± 16.1b 52.8 ± 7.1d 54.9 ± 8.3a 2400.3c
DMCA 90.1 ± 4.7c 71.0 ± 3.2b 31.2 ± 3.9cde 3180.8bc
DMA 91.5 ± 12.9c 56.0 ± 1.6cd 40.0 ± 7.0bc 2858.4bc
Citric acid DPMCA 18.9 ± 4.2d NTYP 5.9 ± 1.1f 504.3d
DPMA 24.4 ± 5.9d NTYP 24.2 ± 8.4e 666.8d
DMCA 26.9 ± 3.5d NTYP 13.0 ± 4.2f 514.1d
DMA 29.3 ± 7.5d NTYP 26.7 ± 8.5de 285.6d
*DPMCA = Deproteinized + demineralized + decolorized + deacetylated, DPMA = Deproteinized + demineralized + deacetylated, DMCA = Demineralized + decolorized + deacetylated, DMA = Demineralized + deacetylated. NTYP = No true yield point. Means within the same column sharing same letters are not significantly different at p> 0.05.
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5.3.2 Effects of Acids and Chitosans on Tensile Properties
Results (Table 5.1) indicate that both the type of chitosans and acids used influence the
tensile properties of crawfish chitosan films. However, effects of acids on tensile properties were
more prominent. The influences of acids and chitosan type on tensile strength and Young's
modulus are easily observed in Figure 5.2 and Figure 5.3, respectively.
DPM
CA
DPM
A
DM
CA
DM
A
Citri
cFo
rmic
Ace
tic
0
20
40
60
80
100
120
140
Tens
ile S
tren
gth
(MPa
)
Chitosan Type Acid
Figure 5.2: Tensile strength of films made with different crawfish chitosans and acids. Refer to Table 5.1 for chitosan abbreviations.
Films made with different acids showed significant (p<0.05) difference in tensile strength
values with few exceptions. The acetate films generally had significantly (p<0.05) higher tensile
strength values than the formate and citrate films. The acetate and formate films possessed
comparable %E while the citrate films possessed significantly lower %E than both films. The
Young's modulus values of films formed with different acids were also significantly (p<0.05)
103
different. The acetate films also possessed relatively higher Young's modulus than formate films
indicating that acetic acid films are tougher than formic acid films. Films formed with citric acid
were weakest with significantly (p<0.05) lower tensile strength, %E and Young's modulus
compared to acetate and formate films. This suggests that acetic and formic acid films are
suitable to form flexible and tough films with crawfish chitosans without any plasticizers.
DPM
CA
DPM
A
DM
CA
DM
A
Citri
cFo
rmic
Ace
tic
0500
10001500200025003000350040004500
Mod
ulus
(MPa
)
Chitosan Type Acid
Figure 5.3: Young's modulus of films made with different crawfish chitosans and acids. Refer to Table 5.1 for chitosan abbreviations.
The type of chitosans used to form films also significantly influenced tensile strength and
%E of the films. The Young's modulus was not significantly influenced by the type of chitosans
used for a given acid. There was significant difference (p<0.05) between tensile strength values
of the films obtained from the traditional method (DPMCA) and the non-traditional methods
(DPMA, DMCA, and DMA). The chitosan obtained by the traditional extraction (DPMCA)
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formed significantly (p<0.05) weaker films in terms of tensile strength when acetic acid was
used. The traditional extraction method yielded chitosan (DPMCA) with lower molecular weight
of 3584 Da (refer to Table 3.1); this may contribute to the weaker films. Increasing tensile
strength values with increasing Mw of chitosans was observed by Muzzarelli and others (1977)
and Park and others (2002). Higher molecular weight chitosans offer large numbers of amino and
hydroxyl groups, resulting in increased numbers of hydrogen bonding which is attributed for
formation of higher tensile strength films (Park and others 2002). Chitosans produced by non-
traditional methods (DPMA, DMCA, and DMA) showing no significant (p<0.05) difference in
their tensile strength values can be attributed to their close molecular weight (Table 3.1). The
type of chitosans used also significantly (p<0.05) affected the elongation of the films, similarly
observed for tensile strength for acetate and formate films.
5.3.3 Comparison of Crawfish Chitosan Films with Selected Biopolymer
The tensile strength, %E, and thickness for selected biopolymer and synthetic polymer
films are presented in Table 5.2. Most biopolymers are soft and brittle in nature with lower
tensile strength, %E, and Young’s modulus values. Addition of plasticizers to biopolymer
relieves their brittleness and makes them tougher. The films made in the present study, especially
those made with acetic acid, without a plasticizer possess much higher tensile strength values
than most biopolymers (Table 5.2). However, their %E is lower then some biopolymers,
indicating that they are not tougher. This can be attributed to added plasticizers with the
biopolymers which make them tougher. Compared to the synthetic films such as LDPE, HDPE,
Saran®, and cellophane, the chitosan acetate and formate acid films are harder with higher
tensile strength values. However, their %E is significantly lower than that of synthetic polymers,
105
except for that of cellophane. Nevertheless, the tensile strength and %E for crawfish chitosan
films are adequate enough to form hard yet flexible packaging materials.
5.3.4 Comparison of Tensile Properties of Chitosan Films
Chitosan films formed with various organic acids with or without plasticizers are
presented in the Table 5.3. The variations found in the tensile strength values can be attributed to
differences in the chitosan type, concentrations of chitosan and acids, plasticizer type and
content, and various testing conditions adopted in their studies. The chitosan films made without
Table 5.2: Comparison of tensile strength and percent elongation values for selected biopolymer and synthetic polymer films
Film Type Tensile strength (MPa)
%E Reference
Wheat Gluten:Gly 2.6 276.2 Gennadios and others (1993)
Whey Protein Isolate: Gly 13.9 30.8 McHugh and Krochta (1994)
Zein:Oleic acid 6.81 3.18 Lai and Padua (1997)
Soy Protein Isolate: Gly 5.23 90.27 Brandenburg and others (1993)
Sodium caseinate: Gly 36.9 18.0 Chen (1995)
Calcium caseinate: Gly 4.25 1.4 Benerjee and others (1994)
Cellophane 114.0 20.0 Aydt and others (1991)
Saran® 27.6 – 62.1 20 - 150 Karel (1975)
HDPE 17.3 - 34.6 300 Brinston (1988)
LDPE 8.6 - 17.3 500 Brinston (1988)
Chitosan 82.0-135.8 25.0 - 45.0 Present study
Gly = Glycerol plasticizer
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Table 5.3: Comparison of tensile properties of chitosan films with organic acid. Film casting solvent
Tensile strength (MPa) %E Modulus
(Mpa) Reference
Acetic acid:PLa 22.67 32.19 NA Caner and others (1998)
Acetic acid:PLb 7.23 - 48.37 22.98-167.2 NA Miranda and others (2004)
Acetic acid:PLc 14.6-18.7 45.9-76.0 NA Butler and others (1996)
Acetic acid:UPd 39.47 37.44 NA Miranda and others (2004)
Acetic acid:UPe 67.6 – 70.3 6.2 – 7.1 NA Kittur and others (1998)
Acetic acid:UPf 57.1 6.7 1741 Begin and others (1999)
Acetic acid:UPg 67.1 21.35 NA Khan and others (2000)
Acetic acid:UPh 68.8-150.2 4.1-7.6 NA Park and others (2004)
Formic acid:PLa 22.86 27.99 NA Caner and others (1998)
Formic acid:UPf 74.3 6.4 1741 Begin and others (1999)
Citric acid:UPf 2.9 37.9 183 Begin and others (1999)
Citric acid:UPh 6.7-17.4 41.9-117 NA Park and others (2004)
Lactic acid:UPh 17.1-62.6 19.6-31.1 NA Park and others (2004)
Lactic acid:UPf 22.2* 26.2 683 Begin and others (1999)
Lactic acid:UPg 59.8 67.1 NA Khan and others (2000)
Malic acid:UPh 27.4-62.4 17.8-29.9 NA Park and others (2004)
Propionic acid:PLa 18.56 24.57 NA Caner and others (1998) PL a
b
c
d
e
f
g
h
= Plasticized, UP = Unplasticized, * = Yield strength, NA=Not available 3% chitosan in 1% acid with polyethylene glycol at 0.25 or 0.50 mL/g chitosan as plasticizer. 2% chitosan in 1% acetic acid (v/v) with glycerol, sorbitol, polyethylene glycol, Tween 60 and Tween 80 at 0.3% and 0.6% w/v concentrations as plasticizers. Tensile strength variations due to plasticizer contents. 3% chitosan in 1% acetic acid (w/v) with glycerol at 0.25 or 0.50 g/g chitosan as plasticizer. 2% chitosan in 1% acetic acid (v/v) without any plasticizers. 2% chitosan in 1% acetic acid (v/v) without any plasticizers. 1% chitosan in 2% acid. 1.4% chitosan (w/v) in 2% acid (w/v). 2% chitosan (w/v) in 2% acid. Tensile strength variations due to different molecular weight of chitosans.
107
a plasticizer show higher tensile strength and lower %E compared to those plasticized films.
Plasticizers are known to reduce tensile strength and increase %E and make the films more
flexible with reduced toughness (Butler and others 1996; Banker 1966).
Kittur and others (1998) and Park and others (2004) reported unplasticized acetic acid
film with higher tensile strength values ranging from 67.6 - 70.3 and 68.8 - 150.2 MPa,
respectively. Although they used double concentration of chitosan in their studies, their tensile
strength values are comparable to those obtained for our present study (82.0 – 135.8 MPa).
However, their films were brittle and not suitable for packaging applications owing to lower %E
values (<8.0%). The acetic acid film formed with crawfish chitosan possessed higher %E in the
range of 25.0 – 45.0%, showing flexibility and toughness suitable for packaging applications.
Begin and Calsteren (1999) prepared formate films with a tensile strength value of 74.3 MPa and
%E of 6.4. The low %E value suggests film brittleness. However, our formate films made from
crawfish chitosan possessed comparable tensile strength values of 76.8 – 114.8 MPa, but much
higher %E in the range of 31.5 - 54.9. This suggests that the formic acid films formed with
crawfish chitosan were tougher than the former.
The citric acid films made with crawfish chitosans were soft and brittle. However,
relative to previously reported tensile strength values of citric acid films, they presented higher
tensile strength values. This indicates that citric acid films made with crawfish chitosan are not
suitable for packaging applications.
5.3.5 Puncture Strengths of Crawfish Chitosan Films
Table 5.4 lists the puncture strength of chitosan films made with different types of
crawfish chitosans and acids. All crawfish chitosan films exhibited variations in puncture
strengths, depending on the type of acid and chitosan used. The puncture strengths of the films
108
made with different acids were significantly (p<0.05) different. In general, formate films
possessed highest puncture strength, followed by acetate and citrate films. Formate films had
higher puncture strength than acetate films but their tensile strength was lower.
The films formed with all chitosan types were significantly (p<0.05) different in puncture
strength, except for DMCA and DMA films. The acetic and formic films which were tougher in
terms of tensile strength values produced a clean hole upon puncture. The citric acid films, which
were brittle in terms of tensile strength values, were splitted along radius upon puncture. This
again indicates that citric acid films are not suitable for packaging film formation.
Table 5.4: Puncture strength of crawfish chitosan films
Solvent Chitosan Type* Puncture (N/mm)
Acetic acid DPMCA 342.75g
DPMA 1409.46b
DMCA 1074.08cd
DMA 1105.00bc
Formic acid DPMCA 571.48fg
DPMA 1871.13a
DMCA 1191.19bc
DMA 1232.92bc
Citric acid DPMCA 510.76fg
DPMA 727.80ef
DMCA 787.97def
DMA 956.19cde *DPMCA = Deproteinized + demineralized + decolorized + deacetylated, DPMA = Deproteinized + demineralized + deacetylated, DMCA = Demineralized + decolorized + deacetylated, DMA = Demineralized + deacetylated. Means within the same column sharing same letters are not significantly different at p >0.05.
109
5.4 CONCLUSIONS
Crawfish chitosans can be formulated into thin and flexible films that are suitable for
packaging applications using acetic and formic acids without any plasticizers. Unplasticized
films made with crawfish chitosans were sufficiently hard and tough enough for packaging
applications. The type of chitosans and acids used to form the films significantly influenced the
tensile properties of the films. While acetic and formic acids form hard and tough films with
crawfish chitosans, citric acid forms weak and soft films that are unsuitable for packaging
application. Among the crawfish chitosans used, those produced by the traditional extraction
method (DPMCA) always resulted in poor mechanical strength.
Mechanical strength of crawfish chitosan films can be optimized by selecting the type of
film casting solvent and chitosan. Chitosan extracted from crawfish shell by the traditional
extraction method yielded highly degraded chitosan and thus mechanically weaker films. All
chitosan extracted by the nontraditional method formed films with acetic and formic acids and
had mechanical properties appropriate for packaging purposes. However, an unusually higher
swelling phenomenon observed in crawfish chitosan films limits their suitability for packaging
purposes. Since absorption of moisture and swelling would impair mechanical properties of
crawfish chitosan films, those films exhibiting minimum swelling should only be selected for
packaging applications. Hence, DMCA (chitosan extracted without deproteinization) chitosan
with acetic acid and DPMCA chitosan with formic acid, which showed minimal swelling and
adequate mechanical strengths, can be considered for packaging purposes.
The mechanical properties of crawfish chitosan films found to be superior to many
biopolymers and comparable to synthetic polymers. Selection of acid solvents and chitosan
extraction methods, and omission of plasticizers are critical to optimize mechanical properties of
110
crawfish chitosan film. If suitably scaled-up, the process may lead to the production of crawfish
chitosan based biopolymeric films for packaging purposes.
5.5 REFERENCES
AOAC. 1990. Official methods of analysis of AOAC International. 15th ed. Arlington, VA: AOAC International.
ASTMa. 1998. Standard test methods for high speed puncture properties of plastics using load
and displacement sensors (D3763-97a). In Annual Book of ASTM Standards. Philadelphia, Pa.: ASTM. p 430-7.
ASTMb. 1998. Standard test methods for tensile properties of thin plastic sheeting (D882-97). In
Annual Book of ASTM Standards. Philadelphia, Pa.: ASTM. p 159-67. Averbach BL. 1978. Film-forming capability of chitosan. In: Muzzarelli RAA, Pariser ER.
editors. Proceedings of the First International conference on Chitin/Chitosan. MIT: Cambridge, MA. p 199-209.
Aydt TP, Weller CL, Testin RF. 1991. Mechanical and barrier properties of edible corn and
wheat protein films. Transactions of the ASAE 34(1):207-17. Banker NH. 1966. Film coating theory and practice. J Pharm Sci 55(1):81-9. Begin A, Calsteren MRV. 1999. Antimicrobial films produced from chitosan. Int J Biol
Macromol 26:63-7. Benerjee RH, Chen G, Hendricks G, Levis JE. 1994. Functional properties of edible films from
whey protein concentrates. J Dairy Sci 77(Suppl. 1):7 [abstract]. Billmeyer FW. 1984. Textbook of polymer science. New York: Wiley. 276 p. Brandenburg AH, Weller CL, Testin RH. 1993. Edible films and coating from soy protein. J
Food Sci 58(5):1086-93. Brinston, J.H. 1988. Plastic films. 3rd ed. New York: Wiley. p 380. Butler BL, Vergano PJ, Testin RF, Bunn JM, Wiles JL. 1996. Mechanical and barrier properties
of edible chitosan films as affected by composition and storage. J Food Sci 61: 953-61. Caner C, Vergano PJ, Wiles, JL. 1998. Chitosan film mechanical and permeation properties as
affected by acid, plasticizer, and storage. J Food Sci 63(6): 1049-53. Chen H. 1993. Functional properties and applications of edible films made of milk proteins. J
Dairy Sci 78:2563-83.
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Chen M, Yeh GH, Chiang B. 1996. Antimicrobial and physicochemical properties of methylcellulose and chitosan films containing a preservative. J Food Process Preserv 20:379-90.
Dutkiewiez J, Tuora M, Judkiewicz L, Ciszewski R. 1992. In: Brine CJ, Standford PA, Zikakis
JP, editors. Advances in chitin and chitosan. Oxford: Elsevier. p 496-505. Gennadios A, Weller CL, Testin RF. 1993. Property modification of edible wheat, gluten-based
films. Am Soc Agri Eng 36(2):465-70. Hoagland PD, Parris N. 1996. Chitosan/Pectin Laminated Films. J Agric Food Chem 44(7): 1915-19. Karel M. 1975. Protective packaging of foods. In: Fennema O. editor. Physical principles of
food preservation. New York: Marcel Dekker. p 339. Khan TA, Peh KK, Ch’ng HS. 2000. Mechanical, bioadhesive strength and biological
evaluations of chitosan films for wound dressing. J Pharm Pharmaceut Sci 3(3):303-11. Kienzle-Sterzer CA, Rodriguez-Sanchez D, Rha C. 1982. Mechanical properties of chitosan
films: Effect of solvent acid. Makromol Chem 183:1353-9. Kittur FS, Kumar KR, Tharanathan RN. 1998. Functional packaging properties of chitosan films.
Z Lebensm Unters Forsch A 206:44-7. Lai HM, Padua GW. 1997. Properties and Microstructure of Plasticized Zein Films. Cereal
Chem 74(6):771-5. Maghami GG, Roberts GAF. 1988. Evaluation of the viscometric constants for chitosan.
Makromol Chem 189:195-200. McHugh TH, Krochta JM. 1994. Sorbitol- vs glycerol-plasticized whey protein edible films:
integrated oxygen permeability and tensile property evaluation. J Agri Food Chem 42(4):841-5.
Miranda SP, Garnica1 O, Lara-Sagahon V, Cárdenas G. 2004. Water vapor permeability and
mechanical properties of chitosan composite films. J Chil Chem Soc 49(2):173-8. Muzzarelli RAA, Isolati A, Ferrero A. 1974. Chitosan membranes: Ion exchange and
membranes. Vol. 1. London: Gordon and Breach Science Publishers. p 193-6. Muzzarelli, RAA. 1977. Chitin. Oxford. Pergamon Press. 326 p. Nadarajah K. and Prinyawiwatkul W. 2002. Filmogenic properties of crawfish chitosan
[abstract]. In: 54th Pacific Fisheries Technologists Annual Meeting Book of Abstracts; 2002 February 24-27; Reno, NV.
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No HK, ChoYI, Kim HR, Meyers SP. 2000. Effective deacetylation of chitin under conditions of 15 psi/121ºC. J Agric Food Chem 48(6):2625-7.
No HK, Meyers SP. 1995. Preparation and characterization of chitin and chitosan-A review.
Journal of Aquatic Food Product Technology. 4(2):27-52. Park HJ, Jung ST, Song JJ, Kang SG, Vergano PJ, Testin RF. 1999. Mechanical and barrier
properties of chitosan-based biopolymer film. Chitin and Chitosan Research 5(1):19-26. Park S, Daeschel MA, Zhao Y. 2004. Functional properties of antimicrobial lysozyme–chitosan
composite film. J Food Sci 69(8):215-21. Park SY, Lin XQ, Sano Y. 2000. Characterization of chitosan film and structure in solution. In:
Nishinari K, editor. Hydrocolloids Part 1. Amsterdam: Elsevier Science. p 199-204. Park SY, Marsh KS, Rhim JW. 2002. Characteristics of different molecular weight chitosan
films affected by the type of organic solvents. J Food Sci 67(1) 194-7. Rhim JW, Weller CL, Ham KS. 1998. Characteristics of chitosan films as affected by type of
solvent acid. Food Sci Biotech 7(4):263-8. Sabnis S, Block LH. 1997. Improved infrared spectroscopic method for the analysis of degree of
N-deacetylation of chitosan. Polym Bull 39:67-71. SAS, Release 9.0 (TS MO), 2002. SAS Institute, Inc. Cary, NC, USA. Wiles JL, Vergano PJ, Barron FH, Bunn JM, Testin RF. 2000. Water vapor transmission rates
and sorption behavior of chitosan films. J Food Sci 65(7):1175-9.
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CHAPTER 6
EFFICACY OF ANTIMICROBIAL EDIBLE FILMS
MADE OF ORGANIC SALTS OF CRAWFISH CHITOSAN
114
6.1 INTRODUCTION
Chitosan, the deacetylated derivative of chitin, possesses numerous versatile properties
suitable for antimicrobial edible films. The inherent antimicrobial activity against a variety of
bacteria, fungi, and viruses, good film-forming property, excellent biocompatibility, and non-
toxicity of chitosan are ideal for the development of antimicrobial edible films. In addition,
chitosan can also possibly be used to carry active substances such as antioxidants, nutrients,
colorants, and flavors to form multifunctional films. The ability of chitosan to release added
active substances in a controlled manner (Zambito and Colo 2003) is advantageous to formulate
active antimicrobial edible films.
Contamination of food products by pathogenic bacteria has emerged as a serious public
concern. Bacteria such as Listeria monocytogenes, Bacillus cereus, Escherichia coli (O157:H7),
Staphylococcus aureus, Salmonella typhimurium, Shigella sonnei and Vibrio vulnificus are
identified as serious and most frequently occurring food-borne pathogens in U.S.A (Mead and
others 1999). Antimicrobial packaging is one promising approach to prevent growth of
contaminated food-borne pathogens and spoilage microorganisms on the surface of food.
Chitosan may be combined with other antimicrobial substances for enhancing its
antimicrobial efficacy. Since chitosan dissolves in slightly acidic solutions, a simple and
economical way of enhancing antimicrobial properties of chitosan films would be to dissolve it
in organic acids that possess antimicrobial properties (Begin and Calsteren 1999). Various
organic acids that naturally occur in fruits and vegetables and possess general antimicrobial
activity such as acetic, lactic, malic, and citric, sorbic, benzoic and succinic acids can be used
for this purpose (Beuchat 1998).
115
Addition of different organic acids and their interaction with chitosan affect the film
properties such as permeability and mechanical strength (Chen and others, 1996). Further,
different acids cause different levels of antimicrobial activity in the presence of chitosan. The
types of chitosan used to form films also affect film properties (Rout and Prinyawiwatkul 2001;
Nadarajah and Prinyawiwatkul 2002). Therefore, it is necessary to select a chitosan and organic
acid combination that provides maximal antimicrobial activities without compromising their
mechanical properties.
Chitosan films containing acetic and propionic acids were effective against
Enterobacteriaceae and Serratia liquefaciens on bologna, regular cooked ham, and pastrami
(Ouattara and others 2000). Coma and others (2002) reported complete inhibition of Listeria
monocytogens inoculated onto a chitosan film containing acetic acid for 8 days. Similarly, 100%
inhibition of Staphylococcus aureus, and Listeria monocytogenes, and 70% inhibition of
Pseudomonas aeruginosa on a model agar medium by a chitosan film with acetic acid were
reported by Coma and others (2003). Rodriguez and others (2003) showed that chitosan film
with acetic acid delayed growth of Alternaria sp, Penicillium sp, and Cladosporium sp
(Deuteromycetes) in precooked pizza. Review of publications on chitosan films and their
antimicrobial properties reveal that (1) studies focused on the effect of organic acids on the
antimicrobial properties fail to address physicochemical changes of chitosan films due to
addition of organic acids, and (2) studies focused on the effect of organic acids on
physiochemical and other properties but not the antimicrobial properties that may be changed
due to addition of organic acids.
This study was intended to determine the antibacterial effect of various crawfish chitosan
film formulations on selected food pathogenic bacteria. Since many properties of chitosan films
116
such as antimicrobial, physicochemical, permeability and mechanical properties are affected by
the type of chitosan and acid used, all these properties should be individually studied. Results
obtained from such studies can help formulate crawfish chitosan films with greater antimicrobial
activity and desirable packaging properties.
6.1.1 Objective
The overall objective of the research was to assess antibacterial properties of crawfish
chitosan films, made of different organic acids and chitosans, on selected food pathogenic
bacteria: Listeria monocytogenes, Bacillus cereus, Shigella sonnei, Escherichia coli (O157:H7),
Staphylococcus aureus, Salmonella typhimurium and Vibrio vulnificus.
6.2 MATERIALS AND METHODS
6.2.1 Materials
All pathogenic bacteria Listeria monocytogenes, Bacillus cereus, Shigella sonnei,
Escherichia coli (O157:H7), Staphylococcus aureus, Salmonella typhimurium and Vibrio
vulnificus were obtained from the Food Microbiology Laboratory, Department of Food Science,
Louisiana State University, LA, U.S.A. Microbiological media, brain heart infusion (BHI),
University of Vermont Medium (UVM) Listeria enrichment broth and nutrient agar were
purchased from Difco Laboratories (Deitroit, MI, U.S.A.). Salmonella Shigella (SS) agar was
purchased from Troy Biologicals, Inc. (Troy, MI, U.S.A). Citric (anhydride), formic and acetic
acids were purchased from Sigma Chemical Co. (St. Louis, MO). Nisin (Nisaplin®) was
obtained from Alpin and Barrett Ltd. (Dorset, U.K.). Polyvenyl chloride wrapping film (Catolog
number 15-610) was purchased from Fisher Scientific (Tustin, CA). Crawfish chitosans were
derived from crawfish shell waste using the traditional method and/or modified methods. The
traditional method involves deproteinization (DP), demineralization (DM), decoloration (DC)
117
and deacetylation (DA) (No and Meyers 1995; No and others 2000). Four different chitosan
samples were prepared using the traditional method involving all above steps (DPMCA), the
traditional method excluding deprotienization (DMCA), the traditional method excluding
decoloration (DPMA), and the traditional method excluding both deproteinization and
decoloration (DMA).
6.2.2 Characterization of Crawfish Chitosan
The viscosity average molecular weight of crawfish chitosans was determined by the
intrinsic viscosity using the Mark-Houwink equation ([η] = KMa) where [η] is the intrinsic
viscosity, M is the molecular weight, K=1.81 x 10-3 and a=0.93 at 25oC (Maghami and Roberts
1988). Crawfish chitosan films deprotonated by methanolic ammonia was used to determine the
degree of deacetylation with IR spectroscopy (M2000 FTIR spectrophotometer, Midac Corp.,
Irvine, CA, USA) and the equation proposed by Sabnis and Block (1997) was used to calculate
degree of deacetylation values. Viscosity was determined for 1% chitosan in 1% acetic acid
(w/v) using the Brookfield viscometer at 50 rpm and 25oC with a spindle number RV5.
The moisture content (%) s of the chitosans was determined gravimetrically in triplicate
by drying samples at 100oC for 48 hours. Nitrogen (%) was determined by the combustion
method using the Leco CHN analyzer (Model # FP-428, Leco Corporation, St. Joseph, Michigan,
USA). The ash content (%) was determined according to the standard method # 923.03 (AOAC
1990).
6.2.3 Film Preparation
Four types of crawfish chitosans (DPMCA, DPMA, DMCA, and DMA) were separately
dissolved with 1% of film casting solvents (acetic, ascorbic, lactic, formic, citric or malic acids).
Each solution was vigorously agitated for 30 minutes, immersed in boiling water for 10 minutes,
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cooled down to room temperature, and filtered through glass-wool to remove undissolved
particles. The film casting solution (300 mL) was cast onto a 31 × 31 cm Teflon coated plate and
allowed to dry at room temperature (23oC) for 48 hours. Dried films were carefully peeled out
and stored in desiccators containing saturated NaBr until further tested.
A micrometer (Model 293-766; Mitutoyo, Tokyo, Japan) was used to measure the film
thickness to the nearest 0.001 mm. Thickness of each film (mm) was measured at room
temperature (23oC and 45% RH) and expressed as an average of 10 random measurements and
standard deviation.
6.2.4 Conditioning of Films
All film specimens used for the antimicrobial tests were preconditioned at 23°C and
57.6% relative humidity inside desiccators containing saturated solutions of sodium bromide.
Prior to their use, all films were exposed to UV light for only 5 minutes to sterilize them, as
prolonged exposure to UV light could affect physicochemical properties of chitosan (Andrady
and others 1996; Maria and Adam 2002). To avoid post aging impact on films, films of 3 days
old were used.
6.2.5 Organisms and Culture Maintenance
Pure cultures of Listeria monocytogenes strain V7 (serotype ½ a), Shigella sonnei (P
6129), Escherichia coli (O157:H7), Staphylococcus aureus, Salmonella typhimurium, Bacillus
cerous, and Vibrio vulnificus were maintained at -70°C. The bacteria were transferred to 10 mL
BHI broth by a sterile inoculation loop and inoculated at 37oC for 24 hours. On the second day,
10 µL of the bacterial suspension was transferred into 10 mL BHI broth and incubated at 37oC
for 18 hours.
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6.2.6 Zone Inhibition Assays
Zone inhibition assay was performed as a preliminary step to screen the antibacterial
activity of all crawfish chitosan film formulations, in an effort to select film formulations with
high antibacterial activity against majority of test pathogens.
Soft agar was prepared by mixing 0.8 g nutrient agar and 3.75 g BHI in 100 mL of
distilled water. After boiling, 10 mL of solutions were dispensed into test tubes and autoclaved.
Before using, tubes containing soft agar were melted in a boiling bath, then temperanted to 46oC.
A 10 µL of bacterial inoculum from the 2nd-day culture grown in BHI broth was mixed with 10
mL soft agar and poured onto a prepoured BHI plate (10 mL/plate). After hardening, circular
film disks (6 mm diameter) cut from each chitosan formulation were placed onto the soft agar.
Based on the results of zone inhibition assay and physicochemical and mechanical
properties, at least one film from each film casting organic acid was selected for quantitative
microbial analysis. The selected film formulations were also further subjected to zone inhibition
tests with controls before being tested quantitatively. Chitosan solution prepared with 1%
chitosan and 1% acetic, formic or citric acids (w/v), an acid solution prepared with 1% acetic,
formic, or citric acid (w/v), and the nisin solution (positive control) prepared with approximately
0.2 mg/mL concentration served as controls. A 88 µL volume of above controls, calculated based
on the material requirement to form 6 mm diameter film, was spotted on the surface of bacterial
lawns. A 6 mm diameter polyvenyle chloride film was also used as a control. All lawns were
incubated at 37oC for 24 hours and the bacterial lawn was visually examined for inhibition of the
inoculated pathogenic bacteria. The thickness of the inhibition zone around the film disks was
measured using a micrometer (Model 293-766; Mitutoyo, Tokyo, Japan).
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6.2.7 Direct Inoculation Assay
Crawfish chitosan films selected based on the preliminary zone inhibition assay were
taken for quantitative analysis. Direct inoculation assays were performed with selected Gram
positive food pathogenic bacteria; Listeria monocytogenes and Staphylococcus aureus and Gram
negative food pathogenic bacteria; Salmonella typhimurium and Shigella sonnei. The 2nd day
bacterial cultures were diluted 10-fold using a phosphate buffer solution (PBS) of pH 7 and 15
µL aliquot of the diluted culture was inoculated onto each film disk (9 mm diameter) that had
been previously placed aseptically into stomacher bag. The control was 15 µL of the diluted
culture placed into stomacher bag without any disk. The stomacher bags were incubated at 37oC
for 0, 2, 4, 8, 24 hours. These stomacher bags were stomached for 2 minutes after adding with a
985 µL PBS (pH 7). The suspension formed in the stomacher was drawn and serially diluted
with PBS (pH 7) for plating.
The selective media used during this study was UVM spread plates for Listeria
monocytogenes, SS agar spread plates for Salmonella typhimurium and Shigella sonnei and 3M
Petrifilm plates for Staphylococcus aureus. These plates were incubated at 37oC for 24 hours.
The 3M Petrifilms were kept at 62oC for 2 hours, inserted with reactive disks and incubated at
37oC for another 2 hours. Inhibition of bacteria by chitosan film was measured by the log
reduction numbers. Bacterial counts were obtained and expressed as log number of colony-
forming units per mL (CFU/mL).
6.2.8 Statistical Analysis
All zone inhibition assays were carried out in duplication. All other experiments were
repeated 3 times with 2 replications per experiment. Treatments were considered significantly
different at P > 0.05 using the ANOVA procedure (SAS, 2002). Where significant differences
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were detected, treatment means were separated using the Tukey’s studentized range test at α =
0.05.
6.3 RESULTS AND DISCUSSIONS
6.3.1 Zone Inhibition Test
Antibacterial activity of crawfish chitosan film formulations against 7 pathogenic bacteria
was expressed in terms of zone inhibition. The zone inhibition assay revealed primarily three
types of observations namely; defaced films without any clear or inhibition zones which could be
attributed to the absence of any inhibitory activity, clear zones without inhibitory zones which
could be attributed to bacteriostatic activity, and clear inhibition zone representing bacterialcidal
inhibition by films.
It was observed that the chitosan acetate and chitosan formate film disks placed on lawns
absorbed moisture, swelled and expanded radially, which could easily be mistaken for an
inhibition zone. Hence straightforward measurement of the inhibition zone thickness based on
an outer diameter of the inhibition zone and an initial film diameter would give erroneous
readings. Therefore, thickness of clear zone was measured using a micrometer with stringent
visual observation and the inhibition zone thickness was measured from the edge of the film.
The thickness of the inhibition zones measured is given in Table 6.1.
Among the films studied, the chitosan acetate films showed minimum inhibitory activity.
All chitosan acetate films were defaced with Listeria monocytogenes. This is in agreement with
the report of Coma and others (2002) who stated that the chitosan acetate films tested on Listeria
monocytogenes lawns failed to produce an inhibition zone. Also reported was the inability of
chitosan solutions (chitosan in acetic acid) to produce inhibitory zones in agar medium
containing Listeria monocytogenes, which was attributed to the limitation of chitosan solution to
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Table 6.1: Effect of chitosan films on selected food pathogenic bacteria.
Thickness of inhibition zone (mm) Film casting solvent
Chitosan typeb Listeria
monocytogenesStaphylococcus aureus
Bacillus cereus
Shigella sonnei
Salmonella typhimurium
Escherichia coli O157:H7
Vibrio vulnificus
DPMCA - + - + + + -
DPMA - + - + + + -
DMCA - + - + + + -
Acetic acid
DMA - + - + + + -
DPMCA + 0.55 - 1.56 + + -
DPMA + 1.64 - 0.89 0.83 0.99 -
DMCA + 1.56 - 0.49 + 1.12 -
Formic acid
DMA + 1.48 - 1.17 1.52 + -
DPMCA 2.26 1.33 1.40 1.03 1.03 0.78 6.00
DPMA 2.33 4.05 1.36 1.36 1.53 1.24 5.88
DMCA 1.85 4.13 1.52 0.52 1.19 1.11 5.77
Citric acid
DMA 1.75 5.75 1.01 1.54 1.31 1.04 5.74 a Values are means of duplicate analysis. b DPMCA = Deproteinized + demineralized + decolorized + deacetylated, DPMA = Deproteinized + demineralized + deacetylated, DMCA = Demineralized + decolorized + deacetylated, DMA = Demineralized + deacetylated. + clear zone without any inhibition. - no inhibition.
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diffuse in agar medium. However, it was reported that 8% film-forming solution (chitosan in
acetic acid) incorporated in agar medium (v/v) completely inhibited Listeria monocytogenes.
Chitosan acetate films also defaced with Bacillus cereus, and Vibrio vulnificus lawns indicating
they were ineffective in controlling these bacteria. However, all chitosan acetate films showed
bacteriostatic effects against Staphylococcus aureus, Shigella sonnei, Salmonella typhimurium,
and Escherichia coli O157:H7 as indicated by their clear zones in lawns. The chitosan formate
films were also ineffective in controlling Bacillus cereus, and Vibrio vulnificus as indicated by
defaced films by these bacterial lawns. Nevertheless, the chitosan formate films showed
inhibitory effects against Staphylococcus aureus, Shigella sonnei, Salmonella typhimurium, and
Escherichia coli O157:H7 and bacteriostatic activity against Listeria monocytogenes. However,
compared to the chitosan citrate films, the inhibitory effect of chitosan formate films were lower
and the thickness of the inhibitory zone was in the range of 0.49 to 1.64 mm compared to 0.78 to
6.0 mm of chitosan citrate films.
Among the crawfish chitosan films studied, all chitosan citrate films exhibited prominent
inhibitory effect on all 7 pathogenic bacteria (Table 6.1). All chitosan citrate films showed
distinctive inhibition zones against all pathogenic bacteria tested (Figure 6.1) and the inhibition
zones were considerably thicker than those produced by chitosan formate films. Also, the
inhibitory effects of chitosan citrate films were remarkably higher for Staphylococcus aureus and
Vibrio vulnificus as indicated by thicker inhibition zones accounting for more than 4 mm (Figure
6.2). The chitosan citrate films were the only films with antimicrobial effects against Bacillus
cereus, and Vibrio vulnificus. The higher inhibitory activity shown by all chitosan citrate films
can be attributable to complete solubility of chitosan which could make them more reactive
against bacterial cells.
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(1) Staphylococcus aureus lawn (2) Bacillus cereus lawn
(3) Salmonella typhimurium lawn (4) Escherichia coli O157:H7 lawn
Figure 6.1: Inhibition of food pathogenic bacteria by chitosan citrate films. In each lawn, DPMA (top left), DPMCA (top right), DMA (bottom left), and DMCA (bottom right) chitosan citrate films are shown.
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(1) Vibrio vulnificus lawn
(2) Staphylococcus aureus lawn
Figure 6.2: Inhibition zones produced by crawfish chitosan films. In each bacterial lawn, top row – acetate films, middle row – citrate films, and bottom row – formate films. In each row from left to right DMA, DMCA, DPMA, and DPMCA chitosan films.
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Based on higher antibacterial activity, lower swelling ratio and higher mechanical strengths,
acetate films with DMCA chitosan, formate films with DPMCA chitosan and citrate films with
DMA chitosan were selected for quantitative analysis of antimicrobial activity. The above
selected films were also tested more with inhibition zone assays with more controls. The nisin
spots used as the positive control produced more prominent clear zones with Listeria
monocytogenes, and Staphylococcus aureus lawns representing the Gram positive bacteria and
vague spots with Shigella sonnei and Salmonella typhimurium lawns representing the Gram
negative bacteria. Regardless of the type of bacteria, controls such as chitosan solutions, acid
solutions and the polyvinyl chloride plastic failed to produce any clear or inhibition zones
indicating there were ineffective in inhibiting the above food pathogenic bacteria. This
substantiates the claim that the direct application of antimicrobial agents, such as chitosan and
acids solutions used in our studies, onto food surfaces is less effective due to loss of
antimicrobial activity caused by leaching onto the food, enzymatic activity, and reaction with
other food components (Jung and others 1992; Ray 1992; Ouattara and others 2000). Hence, use
of packaging films or coating as a matrix to deliver antimicrobial agents becomes important.
Such packaging or coating can maintain a high concentration of antimicrobial agents on a food
surface and allows low migration into food (Torres and others 1985; Siragusa and Dickson 1992;
Ouattara and others 2000).
6.3.2 Direct Inoculation Assay
Results of the direct inoculation study were in agreement with the inhibition zone assays.
Table 6.2 shows the survivor log number CFU/mL of Listeria monocytogenes inoculated onto
the surface of selected chitosan films (DMCA acetate, DPMCA formate and DMA citrate).
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Listeria monocytogenes was more susceptible to chitosan citrate film than chitosan
formate or chitosan acetate films. Chitosan citrate film reduced the bacterial count by 5.34 log
CFU/mL within 4 hours of incubation (Table 6.3). Chitosan citrate films accounted for more than
4.47 log CFU/mL reduction of inoculum in 24 hours. Chitosan acetate films caused only
marginal reduction of the inoculum, accounting for less than 1 log CFU/mL reduction over the
entire 24-hour period incubation. The chitosan formate films caused about 1 log CFU/mL
reduction of inoculum at 2 hours of incubation and maintained a 1 log CFU/mL reduction over
24 hours of incubation. The rate of reduction of microbial count was poor with both chitosan
acetate and formate films as there was no significant difference in microbial count between 2
and 4-hours incubation and between 4 and 8-hour incubation as shown in Table 6.3.
Organic acids with smaller molecular weight have higher antimicrobial activity and
undissociated smaller molecules of formic (46.03 Dalton) and acetic (60.05 Dalton) acids may
Table 6.2: Recovery of Listeria monocytogenes survivors from crawfish chitosan film with different organic acids incubated at 37°C for 24 hours.
Log CFU/mL* Treatment
0 Hour 2 Hour 4 Hour 8 Hour 24 Hour
Control† 7.24 ± 0.10a 7.48 ± 0.12a 7.34 ± 0.05a 7.15 ± 0.21a 6.47 ± 0.08a
Acetate film 7.14 ± 0.09ab 7.16 ± 0.13b 7.09 ± 0.13b 6.87 ± 0.28a 6.44 ± 0.10a
Formate film 7.09 ± 0.07b 6.33 ± 0.08c 6.26 ± 0.08c 6.06 ± 0.09b 5.82 ± 0.09b
Citrate film 6.80 ± 0.09b 4.02 ± 0.20d ND ND ND
*All analyses were based on three separate experiments with each mean ± standard deviation being an average of three determinations. Means within each vertical column followed by the same letters are not significantly different (P> 0.05). †An inoculum of Listeria monocytogenes at 7.24 ± 0.10 (mean ± standard error) CFU/mL without any film was used as control. ND = Not detected.
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enter into the bacterial cells easily to change the internal pH of the organisms (Eswaranandam
and others 2004). Undissociated larger molecules of citric acid (192.13 Dalton) may not enter
into the cells effectively. However, this trend was not observed in the present study and the result
was in contrary. It indicates that chitosan films made of organic acids may behave as one entity
rather than separate entities, i.e., as a career matrix containing an antimicrobial agent.
Several studies have demonstrated that antimicrobial edible films can reduce bacterial
levels on meat products. Siragusa and Dickson (1992, 1993) showed that organic acids were
more effective against L. monocytogenes on beef carcass tissue when immobilized in calcium
alginate than when used as a spray or dip. In another study, Hoffman and others (2001) reported
that zein films, impregnated with nisin, lauric acid and EDTA and tested with broth cultures of L.
monocytogenes, reduced the bacterial counts over 5 logs after 48 hours. Janes and others (2002)
reported zein films containing nisin produced a 4.5 to 5 log reduction on L. monocytogenes
Table 6.3: Log reduction of Listeria monocytogenes survivors from crawfish chitosan film with different organic acids incubated at 37°C for 24 hours.
Log CFU/mL* Treatment
0 Hour 2 Hour 4 Hour 8 Hour 24 Hour
Control† 7.24 ± 0.10 7.48 ± 0.12 7.34 ± 0.05 7.15 ± 0.21 6.47 ± 0.08
Acetate film 0.10 ± 0.09a 0.32 ± 0.13c 0.24 ± 0.13c 0.28 ± 0.28c 0.03 ± 0.10c
Formate film 0.15 ± 0.07a 1.15 ± 0.08b 1.08 ± 0.08b 1.09 ± 0.09b 0.65 ± 0.09b
Citrate film 0.21 ± 0.09a 3.46 ± 0.20a > 5.34a > 5.15a > 4.47a
*All analyses were based on three separate experiments with each mean ± standard deviation being an average of three determinations. Means within each vertical column followed by the same letters are not significantly different (P> 0.05). † Log survivors of Listeria monocytogenes inoculated at 7.24 ± 0.10 (mean ± standard error) CFU/mL without any film was used as control.
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inoculated onto chicken breast tenders refrigerated for 16 days. Ming and others (1997)
impregnated the surface of meat casing with pediocin powder to produce a 1 to 3 log reduction
of L. monocytogenes on ham, turkey breast, and beef compared to inoculated controls. In most of
these studies, antibacterial activity against L. monocytogenes was attempted with added
antimicrobials. Considering this, the chitosan citrate film producing more than a 4.4 log
reduction in L. monocytogenes is a commendable achievement.
Effect of crawfish chitosan films on Staphylococcus aureus is shown in Table 6.4. As
with the case Listeria monocytogenes, the chitosan citrate films showed higher antibacterial
activity against Staphylococcus aureus. The chitosan citrate films produced more than a 5 log
reduction in Staphylococcus aureus within 4 hours of incubation and maintained its inhibitory
effect through out the incubation period (Table 6.5). The chitosan acetate films produced a poor
inhibition with less than 1 log reduction at 24 hours. The chitosan formate films maintained
about 1 log reduction for up to 4 hours. At 24-hour incubation chitosan formate films produced
more than a 5 log reduction similarly observed for chitosan citrate films.
Relatively very little research work has been dedicated to formulate edible films active
against Staphylococcus aureus. Ha and others (2001) reported that polyethelene film containing
grapefruit seed extract showed an inhibitory effect against Staphylococcus aureus as indicated by
a 2.5 to 7.0 mm inhibition zone by the agar diffusion method. Scanell and others (2000) reported
about 1.5 log and 2.8 log reduction of Staphylococcus aureus in cheese and ham by nisin-
absorbed bioactive inserts. Coma and others (2001) reported that an edible packaging made of
cellulosic esters, fatty acids and nisin produced up to 88 mm diameter inhibition zone on
Staphylococcus aureus. Further, they reported that addition of fatty acid reduced the inhibitory
activity. All these studies indicate the importance of having added antimicrobials in the films to
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Table 6.4: Recovery of Staphylococcus aureus survivors from crawfish chitosan film with different organic acids incubated at 37°C for 24 hours.
Log CFU/mL* Treatment
0 Hour 2 Hour 4 Hour 8 Hour 24 Hour
Control† 7.29 ± 0.10a 7.26 ± 0.14a 7.19 ± 0.06a 7.10 ± 0.11a 7.06 ± 0.11a
Acetate film 7.24 ± 0.09a 7.15 ± 0.16a 7.05 ± 0.14a 6.82 ± 0.25b 6.51 ± 0.16b
Formate film 7.10 ± 0.18a 6.20 ± 0.07b 6.08 ± 0.09b 4.91 ± 0.11c ND
Citrate film 6.81 ± 0.08b 5.81 ± 0.12c ND ND ND
*All analyses were based on three separate experiments with each mean ± standard deviation being an average of three determinations. Means within each vertical column followed by the same letters are not significantly different (P> 0.05). †An inoculum of Staphylococcus aureus at 7.29 ± 0.10 (mean ± standard error) CFU/mL without any film was used as control. ND = Not detected.
Table 6.5: Log reduction of Staphylococcus aureus survivors from crawfish chitosan film with different organic acids incubated at 37°C for 24 hours.
Log CFU/mL* Treatment
0 Hour 2 Hour 4 Hour 8 Hour 24 Hour
Control† 7.29 ± 0.10 7.26 ± 0.14 7.19 ± 0.06 7.10 ± 0.11 7.06 ± 0.11
Acetate film 0.06 ± 0.09b 0.11 ± 0.16c 0.13 ± 0.14c 0.29 ± 0.25c 0.55 ± 0.16
Formate film 0.19 ± 0.18b 1.06 ± 0.07b 1.10 ± 0.09b 2.19 ± 0.11b > 5.06
Citrate film 0.48 ± 0.08a 1.45 ± 0.12a > 5.19a > 5.10a > 5.06
*All analyses were based on three separate experiments with each mean ± standard deviation being an average of three determinations. Means within each vertical column followed by the same letters are not significantly different (P> 0.05). † Log survivors of Staphylococcus aureus inoculated at 7.29 ± 0.10 (mean ± standard error) CFU/mL without any film was used as control.
131
control Staphylococcus aureus. However, the crawfish chitosan citrate and formate films which
contained no added antimicrobials could produce more than a 5 log reduction. Further, the
inhibitory effect of chitosan citrate and formate films against Staphylococcus aureus was higher
than that of Listeria monocytogenes.
Along with Listeria monocytogenes, Salmonella typhimurium has been considered as a
microbiological hurdle for a long time. The effect of crawfish chitosan films against Salmonella
typhimurium is shown in Table 6.6. As with Listeria monocytogenes and Staphylococcus aureus,
a similar trend of inhibition was observed with Salmonella typhimurium. The chitosan citrate
films produced more than 3.4 log reduction in Salmonella typhimurium within 2 hours of
incubation, and reduction in counts reached 3.85 log at 4-hour and 4.83 log at 8-hour incubation
(Table 6.7). The chitosan acetate films were less effective with about 1 log reduction at 24 hours.
Table 6.6: Recovery of Salmonella typhimurium survivors from crawfish chitosan film with different organic acids incubated at 37°C for 24 hours.
Log CFU/mL* Treatment
2 Hour 4 Hour 8 Hour 24 Hour
Control† 6.97 ± 0.06a 6.91 ± 0.05a 6.83 ± 0.04a 6.76 ± 0.10a
Acetate film 6.11 ± 0.16b 6.00 ± 0.16b 5.86 ± 0.05b 5.71 ± 0.10b
Formate film 4.26 ± 0.09c 4.05 ± 0.11c 3.86 ± 0.07c 3.06 ± 0.32c
Citrate film 3.47 ± 0.25d 3.05 ± 0.40d ND ND
*All analyses were based on three separate experiments with each mean ± standard deviation being an average of three determinations. Means within each vertical column followed by the same letters are not significantly different (P> 0.05). †An inoculum of Salmonella typhimurium at 6.98 ± 0.06 (mean ± standard error) CFU/mL without any film was used as control. ND = Not detected.
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There was no significant (p>0.05) change in the Salmonella typhimurium count from 2-hour to
24-hour for chitosan acetate film. The chitosan formate films maintained about 2.7 log reduction
up to 8 hours and then produced a significant by (p<0.05) increased inhibition (3.7 log) between
8-hour and 24-hour incubation.
Effects of edible films on Salmonella typhimurium have been reported earlier. Natrajan
and Sheldon (2000) demonstrated a 4.3 log reduction of Salmonella typhimurium on inoculated
broiler skin exposed to nisin coated polyvinyl chloride film. Sheldon and others (1996) reported
a 4.23 log reduction of Salmonella typhimurium in pads treated with nisin formulations. Sheldon
(2001) applied nisin formulations to Salmonella typhimurium inoculated on tray pads and
demonstrated 3.1 log reduction. Compared to these published data, reduction of Salmonella
typhimurium in the present study by more than 4.7 log by chitosan citrate film and 3.7 log by
chitosan formate film is outstanding.
Table 6.7: Log reduction of Salmonella typhimurium survivors from crawfish chitosan film with different organic acids incubated at 37°C for 24 hours.
Log CFU/mL* Treatment
2 Hour 4 Hour 8 Hour 24 Hour
Control† 6.97 ± 0.06 6.91 ± 0.05 6.83 ± 0.04 6.76 ± 0.10
Acetate film 0.86 ± 0.14c 0.91 ± 0.15c 0.96 ± 0.05c 1.05 ± 0.11c
Formate film 2.71 ± 0.09b 2.86 ± 0.10b 2.87 ± 0.07b 3.7 ± 0.29b
Citrate film 3.49 ± 0.24a 3.85 ± 0.37a > 4.83a > 4.76a
*All analyses were based on three separate experiments with each mean ± standard deviation being an average of three determinations. Means within each row followed by the same letters are not significantly different (P> 0.05). †Log survivors of Salmonella typhimurium inoculated at 6.98 ± 0.06 (mean ± standard error) CFU/mL without any film was used as control.
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Effect of crawfish chitosan films against Shigella sonnei is shown in Table 6.8. As with
Listeria monocytogenes, and Staphylococcus aureus, chitosan acetate films produced minimal
inhibition against Shigella sonnei. The chitosan formate films accounted for about 1 log
reduction at 4 hours of incubation and 2.6 log reduction at 24 hours. The citrate films showed the
highest antibacterial activity against Shigella sonnei with more than 5 log reduction at 8 hours of
incubation (Table 6.9).
6.4 CONCLUSIONS
This study confirms that crawfish chitosan can be used to develop antimicrobial edible
films effective against both Gram positive and Gram negative food pathogenic bacteria. Chitosan
acetate films showed poor inhibitory effect against Listeria monocytogenes, Staphylococcus
aureus, Salmonella typhimurium, and Shigella sonnei. Although chitosan acetate films
Table 6.8: Recovery of Shigella sonnei survivors from crawfish chitosan film with different organic acids incubated at 37°C for 24 hours.
Log CFU/mL* Treatment
2 Hour 4 Hour 8 Hour 24 Hour
Control† 7.04 ± 0.08a 7.08 ± 0.06a 7.10 ± 0.04a 7.13 ± 0.04a
Acetate film 6.88 ± 0.09a 6.83 ± 0.09b 6.90 ± 0.11b 7.00 ± 0.07b
Formate film 6.71 ± 0.14 a 6.03 ± 0.10c 5.88 ± 0.06c 4.54 ± 0.22c
Citrate film 4.94 ± 0.36b 3.30 ± 0.43d ND ND
*All analyses were based on three separate experiments with each mean ± standard deviation being an average of three determinations. Means within each vertical column followed by the same letters are not significantly different (P> 0.05). †An inoculum of Shigella sonnei at 7.01 ± 0.06 (mean ± standard error) CFU/mL without any film was used as control. ND = Not detected.
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outweighed other films in terms of their mechanical properties, they demonstrated minimal
antibacterial effects similar to bacteriostatic effects with negligible bacterial reduction over a
period of 24 hours.
Chitosan formate films were effective against Staphylococcus aureus, Salmonella
typhimurium, and Shigella sonnei, causing more than 5, 3.7 and 2.5 log reduction at 24 hour
incubation, respectively. Chitosan formate films produced poor inhibitory effect against Listeria
monocytogenes with less than 1 log reduction at 24-hour incubation. Based on antibacterial and
packaging properties, chitosan formate films can be used as antibacterial packaging to control
Staphylococcus aureus, Salmonella typhimurium, and Shigella sonnei, except Listeria
monocytogenes.
Chitosan citrate films were highly effective against Listeria monocytogenes,
Staphylococcus aureus, Salmonella typhimurium, and Shigella sonnei. The effect of chitosan
Table 6.9: Log reduction of Shigella sonnei survivors from crawfish chitosan film with different organic acids incubated at 37°C for 24 hours.
Log CFU/mL* Treatment
2 Hour 4 Hour 8 Hour 24 Hour
Control† 7.04 ± 0.08 7.08 ± 0.06 7.10 ± 0.04 7.13 ± 0.04
Acetate film 0.16 ± 0.09b 0.24 ± 0.09c 0.20 ± 0.11c 0.13 ± 0.07c
Formate film 0.33 ± 0.14b 1.04 ± 0.10b 1.21 ± 0.06b 2.59 ± 0.22b
Citrate film 2.11 ± 0.36a 3.78 ± 0.43a > 5.10a > 5.13a
*All analyses were based on three separate experiments with each mean ± standard deviation being an average of three determinations. Means within each row followed by the same letters are not significantly different (P> 0.05). †Log survivors of Shigella sonnei inoculated at 6.98 ± 0.06 (mean ± standard error) CFU/mL without any film was used as control.
135
citrate films against Listeria monocytogenes and Staphylococcus aureus was prominent with
more than 5 log reduction within 4 hours of incubation. Furthermore, chitosan citrate films
indicated its potential antibacterial effect against Bacillus cereus and Vibrio vulnificus as
indicated by the zone inhibition tests. This study indicates the possibility of formulating an
antibacterial edible film, especially crawfish chitosan citrate film, active against a broad
spectrum of bacteria.
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CHAPTER 7
CONCLUSIONS
139
Development of antimicrobial films from biopolymers has been a challenge. Many
critical factors such as film-forming ability, mechanical strength, gas and water barrier
properties, storage stability, and antimicrobial properties have to be optimized in order to
successfully develop an antimicrobial film.
The film-forming ability of crawfish chitosans varied with different film casting organic
acids. Acetic, formic, and citric acids could be used as a film casting solvent to produce flexible
and transparent films, resembling plastic films. Malic, lactic, and ascorbic acids were not
recommended due to the undesirable hydrophilic nature or brittleness of the film produced.
Among many factors, the swelling and solubility of chitosan films in water were very
critical in selecting film formulations for packaging requirements. Chitosan citrate films, which
completely solubilized in water, may be utilized for edible film formation but not for packaging
applications. Films formed with DMCA acetate and DPMCA formate, which had the lowest
swelling can be selected for packaging applications.
Film casting solvents and chitosan types significantly influenced the sorption behavior of
crawfish chitosan films. On average, chitosan formate films adsorbed more moisture than
chitosan acetate or citrate films. While acetate and formate films maintained their flexibility, the
citrate films exhibited brittleness at the lower water activity levels. The sorption behavior of
crawfish chitosan films could be best predicted by the GAB model (R2 = 0.97-0.98). Analysis of
sorption isotherms indicated that clustering of water molecules took place when water activity
exceeded 0.57.
The water vapor permeability of crawfish chitosan films was significantly influenced by
the chitosan types and film casting solvents used to form the films. The chitosan citrate films
140
showed the lowest WVP, followed by chitosan acetate films. The highest WVP was obtained for
DPMCA chitosan formate films.
The type of chitosan and acid used to form the films significantly influenced the tensile
properties of the films. While acetic and formic acids formed hard and tough films with crawfish
chitosans, citric acid formed weak and soft films that are unsuitable for packaging application.
The mechanical properties of crawfish chitosan films found to be superior to many biopolymers
and comparable to synthetic polymers.
The crawfish chitosans can be used to develop antimicrobial edible films effective against
both Gram positive and Gram negative food pathogenic bacteria, especially when citric acid was
used as a film casting solvent. Chitosan acetate (DMCA) films showed poor inhibitory effect
against Listeria monocytogenes, Staphylococcus aureus, Salmonella typhimurium, and Shigella
sonnei. Chitosan formate (DPMCA) films were effective against Staphylococcus aureus,
Salmonella typhimurium, and Shigella sonnei, causing more than 5, 3.7 and 2.5 log reduction at
24 hour, respectively.
Chitosan citrate films were effective against Listeria monocytogenes, Staphylococcus
aureus, Salmonella typhimurium, and Shigella sonnei, and produced 4.4, 5.6, 4.7, and 5.1 log
reduction in 24 hours, respectively. The effect of chitosan citrate films against Listeria
monocytogenes and Staphylococcus aureus was prominent with more than 5 log reduction
observed within 4 hours of incubation. Furthermore, chitosan citrate films indicated its potential
antibacterial effect against Bacillus cereus and Vibrio vulnificus as indicated by the zone
inhibition tests.
This study indicated the possibility of formulating antibacterial edible films active against
a broad spectrum of bacteria using selected crawfish chitosans and formic and citric acid as film
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casting solvents. Crawfish formate films, with desirable mechanical properties, would be
recommended for the formation of antimicrobial packaging film while the citrate films with their
complete solubility were more suitable for antimicrobial edible film formation.
142
VITA
The author is native to Hatton, a small town in the hill county of Sri Lanka. He received
his primary and secondary education from the Highlands College, Hatton, Sri Lanka, and entered
University of Peradeniya in 1990. He received his Bachelor of Science degree in agricultural
science while concentrating in food engineering in 1994, and his Master of Science degree in
post harvest technology of fruits and vegetables in 1997 both from the University of Peradeniya,
Peradeniya, Sri Lanka.
The author served as a lecturer in food science and technology at the Sabaragamuwa
University of Sri Lanka from 1996 to 2000. He joined the Department of Food Science,
Louisiana State University, in August 2000, to pursue his doctoral studies. He received a Doctor
of Philosophy degree in food science with areas of concentration in food engineering and food
microbiology.
The author is a son of Mr. and Mrs. Annamalay Kandasamy and the brother of
Puwaneswary, Manohar, Shivakumar, and Bharathy. He is also the husband of Radha
Subburathinam.
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