Biotechnological Manipulations of Chitosan Polymer for …prr.hec.gov.pk/jspui/bitstream/123456789/6757/1/Ijaz... · 2018-07-23 · Biotechnological Manipulations of Chitosan Polymer

Ijaz Bano

2015

Department of Biotechnology

Pakistan Institute of Engineering and Applied Sciences

Nilore, Islamabad, Pakistan

Biotechnological Manipulations of Chitosan

Polymer for Biomedical Applications

Thesis Submission Approval

This is to certify that the work contained in this thesis entitled Biotechnological

Manipulations of Chitosan Polymer for Biomedical Applications, was carried out by

Ijaz Bano, and in my opinion, it is fully adequate, in scope and quality, for the degree of

Ph.D. Furthermore, it is hereby approved for submission for review and thesis defense.

Supervisor: _____________________

Dr. M. A. Ghauri (DCS)

Date: 28 May, 2015

Place: NIBGE, Faisalabad.

Co-Supervisor: __________________

Dr. Tariq Yasin (DCS) Date: 28 May, 2015

Place: PIEAS, Islamabad.

Head, Department of Biotechnology: ___________________

Dr. Shahid Mansoor, S.I Date: 28 May, 2015

Place: NIBGE, Faisalabad.

Biotechnological Manipulations of Chitosan

Polymer for Biomedical Applications

Ijaz Bano

Submitted in partial fulfillment of the requirements

for the degree of Ph.D.

2015

Department of Biotechnology

Pakistan Institute of Engineering and Applied Sciences

Nilore, Islamabad, Pakistan

ii

Dedications

Dedicated to the Women of my Family My Sweet Ammi Jaan,

Her Daughters

&

Grand Daughters

Sunshine of my Life…………………………………………

iii

Acknowledgments

In the name of Allah, Most Gracious, Most Merciful. I am grateful for all the bounties that

Allah has showered on me which enabled me to complete this doctoral thesis. I would like to

express my appreciation to all the individuals without whom the completion of this thesis

would not be possible. My heartfelt thanks go to my principal supervisor, Dr. Muhammad

Afzal Ghauri (DCS), for his continual and unwavering encouragements, guidance and support

over the course of my studies. My deepest thanks go to my co-supervisor Dr. Tariq Yasin

(DCS), encourage for his invaluable assistance. I have learnt a huge amount from you both

and it has been an honor to have each of you as a mentor. It is honor to acknowledge the

affectionate role of Dr. Shahid Mansoor, S.I (Director, NIBGE) who appreciate all

students during the period of research work.

My sincere gratitude to Dr. Nasrin (SS), Dr. Rizwan, Dr. Kalsoom (PS), Dr

Shazia (SS) and Dr. Munir (PS) for their immense help during my thesis writing. Thanks to

all the people in the industrial Biotechnology Division at NIBGE, who supported me in any

respect during the completion of my PhD.

I gratefully acknowledged the Higher Education Commission of Pakistan for the

award of scholarship, a necessary financial support for the execution of my PhD.

I am eternally grateful to my dearest Ami, Abu and sisters Mumtaz, Shahnaz,

Dr. Rabia and Chand, my caring brother in law Abdul Rashid, who have been a source of

encouragement and inspiration to me throughout my life. I am very thankful to Allah for

having a very understanding, loving and caring husband, Akmal Shahzad.

I feel immense pleasure to express my deepest gratitude and sincere thanks to all

my friends especially Ammara, Tehmina, Sadia, and Neelam for their time and company.

Ijaz Bano

iv

Declaration of Originality

I hereby declare that the work contained in this thesis and the intellectual content of this

thesis are the product of my own work. This thesis has not been previously published in

any form nor does it contain any verbatim of the published resources which could be

treated as infringement of the international copyright law. I also declare that I do

understand the terms ‘copyright’ and ‘plagiarism,’ and that in case of any copyright

violation or plagiarism found in this work, I will be held fully responsible of the

consequences of any such violation.

__________________

(Ijaz Bano)

14 May, 2014

PIEAS, Islamabad.

v

Copyrights Statement

The entire contents of this thesis entitled “Biotechnological Manipulation of Chitosan

Polymer for Biomedical Applications” by Ijaz Bano are an intellectual property of

Pakistan Institute of Engineering & Applied Sciences (PIEAS). No portion of the thesis

should be reproduced without obtaining explicit permission from PIEAS..

vi

Table of contents

Dedications ………………………………………………………………………... .ii

Acknowledgments ………………………………………………………………... iii

Declaration of Originality ……………………………………………………..….. iv

Copyrights Statement …………………………………………………………….... v

Table of Contents ………………………………………………………………..... vi

List of Figures ……………………………………………………………………... .x

List of Tables ………………………………………………………………….......xiii

Abstract ……………………………………………………………………………xiv

List of Publications and Patents …………………………………………………...xvi

List of Abbreviations and Symbols …………………………………………….....xvii

1 Introduction and Review of Literature …………………………………….. 1

1.1 Chitin ………………………………………………………………….. 1

1.2 Chitosan ……………………………………………………………… 2

1.2.1 Characterization of chitosan ………………………………… 4

1.2.2 Biological properties of chitosan ……………………………. 6

1.2.3 Potential applications of chitosan polymer ………………… 14

1.2.4 Biosafety/non-toxicity of chitosan ………………………… 18

1.3 Poly (vinyl alcohol), PVA; a synthetic polymer …………………….... 18

1.3.1 Physicochemical properties and applications of PVA …….... 19

1.4 Chitosan/PVA blend membranes ……………………………………… 20

1.5 Crosslinkers for chitosan blends with other polymers ……………….... 22

1.6 Microorganisms for antimicrobial studies ………………………...... 23

1.6.1 Pathogenic bacteria ………………………………………….. 23

1.6.2 Aflatoxins producing fungi ………………………………… 25

Objectives of present work ………………………………………………………... 29

2 Materials and Methods……………………………………………………... 30

2.1 Polymers and chemicals used in the study …………………………….. 30

vii

2.2 Microbial strains used in the study ……………………………….........30

2.3 Growth media and maintenance of microbial cultures ……………….. 31

2.3.1 Liquid and solid media for the growth of bacteria …………. 31

2.3.2 Liquid and solid medium for the growth of fungus ………… 31

2.4 Extraction of chitin and chitosan from crab shells ……………………. 32

2.4.1 Gamma irradiation of chitosan ……………………………….33

2.4.2 Extraction of chitosan from gamma irradiated chitosan

solutions …………………………………………………… 33

2.5 General procedure for preparations of chitosan/PVA membranes ……. 35

2.6 Characterization of chitosan and chitosan membranes ……………….. 36

2.6.1 Infrared spectroscopy (IR) …………………………………... 36

2.6.2 Determination of viscosity average molecular weight (Mv)

of chitosan …………………………………………………. 36

2.6.3 Antibacterial study of variable molecular weight chitosan

using agar well diffusion method…………………………… 37

2.6.4 Determination of minimum inhibitory concentration (MIC)

and 50% inhibitory concentration (IC50) of chitosan against

drug resistant bacteria ……………………………………. 38

2.6.5 Metabolite analysis of Pseudomonas aeruginosa SS29 ……. 39

2.6.6 Culture and inoculum preparation for antifungal studies

of chitosan (CHI50) ……………………………………….. 39

2.6.7 Liquid chromatography and mass spectrometric (LC-MS)

analysis of aflatoxins (Aspergillus flavus) ………………… 41

2.6.8 Morphological analysis of membranes ……………………… 45

2.6.9 Water absorption studies of membranes ……………………. 46

2.6.10 Mechanical properties of membranes ……………………… 46

2.6.11 Water barrier properties of membranes ……………………. 46

2.6.12 Contact angle measurements of membranes ………………. 47

2.6.13 Antibacterial study of chitosan membranes by membrane’s

disc diffusion and optical density (OD) method …..……….. 48

2.7 Controlled drug release study by membranes …………………………. 49

viii

2.7.1 Preparation of Buffer Solutions ……………………………... 49

2.7.2 Preparation of standard solutions for insulin ……………….. 49

2.7.3 Loading of insulin on membrane and its release analysis ….. 49

3 Results …………………………………………………………………….. 51

3.1 Gamma irradiation of chitosan ………………………………………… 51

3.2 Fourier Transform Infrared (FTIR) characterization of chitosan

and membranes ………………………………………………………. 52

3.3 Antibacterial activity of chitosan using agar wells diffusion method … 53


and 50% inhibition concentration (IC50) of un-irradiated

and irradiation chitosan against drug resistant bacteria …… 58

3.3.2 Analysis of metabolite of Pseudomonas aeruginosa ……….. 58

3.4 Antifungal activity of chitosan …………………………………………67


of chitosan and analysis of aflatoxins………………………. 67

3.4.2 Inhibition of aflatoxins (Aspergillus flavus) on Basmati rice... 69

3.5 Characterization of chitosan membranes ……………………………… 71

3.5.1 Morphological analysis of chitosan/PVA membranes ……… 71

3.5.2 Effect of time, temperature, pH, and electrolyte on water

absorption capacity of the membranes ………………………. 75

3.5.3 Mechanical properties ……………………………………….. 77

3.5.4 Water vapor transmission rate (WVTR) and water vapor

permeability (WVP) of membranes ……………………… 78

3.5.5 Contact angle measurement (surface properties) …………… 79

3.6 Antibacterial activity of chitosan membranes ………………………….79

3.7 Controlled release of insulin by membranes ………………………….. 82

4 Discussion …………………………………………………………………. 84

4.1 Antimicrobial studies of variable molecular weight chitosan and

chitosan membranes …………………………………………………. 84

4.2 Surface characteristics of membranes …………………………………. 89

4.3 Water absorption response of membranes in various media ………….. 90

ix

4.3.1 Water absorption capacity of membranes in distilled water … 90

4.3.2 Temperature dependent water absorption capacity of

membranes ………………………………………………… 91

4.3.3 Effect of pH on water absorption capacity of membranes …... 91

4.3.4 Effect of ionic concentration on water absorption

capacity of membranes ……………………………………. 92

4.4 Mechanical analysis of membranes …………………………………… 93

4.5 Water transmission analysis of membranes …………………………… 94

Conclusions ………………………………………………………………………... 95

Recommendations/Future work …………………………………………………… 96

5 References …………………………………………………………………. 97

x

List of Figures

Figure 1-1 Structures of chitin and chitosan ……………...………..........3

Figure 1-2 Structure of poly(vinyl alcohol) ………………………….. 19

Figure 1-3 A typical SEM image of chitosan/PVA membrane showing

distribution of pores ( )..……………………………….. 22

Figure 1-4 A representative structure of aflatoxin …………………… 26

Figure 1-5 The growth of Aspergillus flavus on rice………………… 27

Figure 2-1 Extraction of chitin, chitosan and gamma irradiation……. 33

Figure 2-2 Isolation of chitosan powder from gamma irradiated

chitosan solution …………………………………………... 34

Figure 2-3 Flow sheet for the preparation of chitosan/PVA membranes.36

Figure 2-4 Liquid drop on surface for measuring contact angle …….. 48

Figure 3-1 Infrared spectra of un-irradiated chitosan (CHIC) and

irradiated chitosan (CHI25, CHI50, CHI75) (4000-

500cm-1

) ……………………………………………………52

Figure 3-2 Infrared spectra of PVA, Mem25 and Mem75 (4000-

500cm-1

)…………………………………………………….53

Figure 3-3 Bioactivity of chitosan against Pseudomonas aeruginosa

(SS29) at concentrations 2% (a), 1% (b), 0.5% (c) and

0.25% (d)……………………………………………………54

Figure 3-4 Bioactivity of chitosan against E. coli (SS2) at concentrations

2% (a), 1% (b), 0.5% (c) and 0.25% (d) …………………. 55

Figure 3-5 Bioactivity of chitosan against E. coli (SS9) at concentrations

2% (a), 1% (b), 0.5% (c) and 0.25% (d) ………………….. 55

Figure 3-6 Bioactivity of chitosan against Proteus mirabilis (SS77) at

concentrations 2% (a), 1% (b), 0.5% (c) and 0.25% (d….….56

Figure 3-7 Bioactivity of chitosan against Staphylococcus aureus

(LM15) at concentrations 2% (a), 1% (b), 0.5% (c) and

xi

0.25% (d)….............................................................................56

Figure 3-8 Zone of bacterial growth inhibition by chitosan (CHIC,

CHI25, CHI50, CHI75) concentrations at 2% (a), 1%

(b), 0.5% (c) and 0.25% (d) against SS29 (Pseudomonas

aeruginosa), SS1,SS2, SS9 (Escherichia coli), SS77

(Proteus mirabilis), LM15 (Staphylococcus aureus)

and Bacillus subtilis ………………………………………..57

Figure 3-9(a) Mass spectra of Pseudomonas aeruginosa metabolites in the

absence of chitosan ……………………………………... 60

Figure 3-9(b) Mass spectra of Pseudomonas aeruginosa metabolites in the

presence of 0.5% chitosan concentration …………………. 61

Figure 3-9(c) Mass spectra of Pseudomonas aeruginosa metabolites in the

presence of 1% chitosan concentration ……………………. 62

Figure 3-9(d) Mass spectra of Pseudomonas aeruginosa metabolites in the

presence of 4% chitosan concentration …………………… 63

Figure 3-10(a) MS/MS analysis of hydroquinolones (mass 244.17)

compounds ……………………………………………… 64

Figure 3-10(b) MS/MS analysis of hydroquinolones (mass 258.2)

compounds …………………………………………….... 65

Figure 3-10(c) MS/MS analysis of hydroquinolones (mass 272.2)

compounds ………………………………………………… 66

Figure 3-11 Plate showing MIC of CHI50 against Aspergillus flavus… 67

Figure 3-12 Antifungal activity (agar well diffusion) of CHI50

(2%, 1%, 0%) at day 3 (a) and day 7 (b) …………………. 68

Figure 3-13 Aflatoxin inhibition by chitosan from agar wells diffusion

method….……………………………………………….. 69

Figure 3-14 Aflatoxin inhibition by CHI50 solution at concentration

0% (C), 2%, and 1.75%, 1.5% and 1% at day 1, day 3

and day 5………………………………………………… 70

Figure 3-15 Aflatoxin inhibition by chitosan (CHI50) on rice ………… 71

xii

Figure 3-16 Camera images of chitosan membrane showing it

transparent (a) and (b) …………………………….. ………72

Figure 3-17 SEM analysis of chitosan membranes at different

magnifications. Shown are SEM micrographs of

MemC (panels A1 and A2), Mem25 (Panels B1and B2),

Mem50 (panels C1 and C2) and Mem75 (panels D1 and

D2). In each case the first panel shows micrographs taken

at 1990x magnification and the second at 3300x …………73

Figure 3-18 AFM images of uncrosslinked (a) and crosslinked (b)

chitosan/PVA membranes ……………………………….....74

Figure 3-19 Time dependent water absorption capacity of MemC,

Mem25, Mem50 and Mem75 ……………………………. 75

Figure 3-20 Temperature dependent water absorption capacity of

MemC, Mem25, Mem50 and Mem75……………………... 76

Figure 3-21 pH dependent water absorption capacity of MemC,

Mem25, Mem50 and Mem75 ……………………………. 76

Figure 3-22 Water absorption capacity of MemC, Mem25, Mem50 and

Mem75 in different conc. of NaCl (solid symbols) and

BaCl2 (hollow symbols) ………………………………….. 77

Figure 3-23 Rate of decrease of contact angle for chitosan membranes... 79

Figure 3-24 Antibacterial activities of chitosan membranes (Mem75)

against pathogens …………………………...…………….. 80

Figure 3-25 Inhibitory zones by CHI25, CHI50, CHI75, solvent and

un-irradiated chitosan (CHIC) against E. coli (a) and

B. subtilis (b) ……………………………………………..... 81

Figure 3-26 Antibacterial activity of MemC, Mem25, Mem50, Mem75

against E. coli culture from st. phase (a) and log phase (b)…82

Figure 3-27 Antibacterial activity of MemC, Mem25, Mem50, Mem75

against B. subtilis culture from stationary phase (a) and log

phase (b) …………………………………………………. 82

Figure 3-28 Insulin release in SGF and SIF solutions ………………….. 83

xiii

List of Tables

Table 1-1 Relationship between characteristics of chitosan and its biological

properties …………………………………………………......... 7

Table 1-2 Potential applications of chitin and chitosan ………………….. 15

Table 3-1 Viscosity average molecular weight of chitosan and membranes

composition…………………………………………………... 51

Table 3-2 The values (%) for MICs and IC50 of chitosan against human

bacterial isolate………………………………………………. 59

Table 3-3 Aflatoxins analysis from agar wells diffusion plate (µg/mL) ….. 68

Table 3-4 Concentration of aflatoxin B1, B2, G1 and G2 (µg/mL) ………… 70

Table 3-5 Mechanical properties of chitosan membranes ………………… 78

Table 3-6 Water vapor transmission rate (WVTR) and water vapor

permeability (WVP) of chitosan/PVA membranes ………….. 78

xiv

Abstract

Chitosan is a deacetylated derivative of chitin found in a wide range of natural sources.

Chitosan as a natural polymer has been modified into a number of formulations based on

its important characteristics such as biodegradability, biocompatibility, antibiotic activity

and antitumor activity etc. Naturally available chitosan of high molecular weight has

limited the efficiency of these polymers for antimicrobial activities. One of the

techniques for improving chitosan antimicrobial efficiency is reducing its molecular

weight. In this regard, irradiation is a widely used method for achieving reduction in

molecular weight of polymers, which may improve some of its characteristics. In this

study, chitosan was extracted from crab shells and irradiated by gamma radiations at

different doses. Effect of radiation dose on chitosan structure was analyzed by Fourier

Transform Infrared (FTIR) spectroscopy. Furthermore, un-irradiated and irradiated

chitosans were blended with poly(vinyl alcohol) and crosslinked with

tetraethylorthosilicate to form membranes. The membranes were found to be transparent

and crosslinked macroporous in structure, exhibiting high tensile strength (TS: 27-

47MPa) and elongation at break (EB: 292.6-407.3%). The response of membranes

towards water absorption capacities at different temperatures, pHs and salt solutions were

studied. Chitosan membranes were found to be temperature and pH responsive. So,

chitosan membrane was used for controlled release of insulin as a model drug at

intestine’s pH value (6.8). Un-irradiated and irradiated chitosan and their membranes

were studied for their antibacterial properties against bacterial pathogens i.e.,

Pseudomonas aeruginosa (SS29), Escherichia coli (SS1, SS2, and SS9), Proteus

mirabilis (SS77), Staphylococcus aureus (LM15) and Bacillus subtilis. Irradiated low

molecular weight chitosan and its membranes showed higher antibacterial activities.

Analysis of bacterial metabolites by Liquid Chromatography Mass Spectrometry (LC-

MS) exhibited the suppression of virulence factors by chitosan in Pseudomonas

aeruginosa. The production of aflatoxins B1, B2, G1 and G2 by Aspergillus flavus was

xv

considerably reduced by irradiated chitosan (CHI50) as validated by LC-MS analysis. It

was found that low molecular weight chitosan inhibited the production of aflatoxin and

Aspergillus flavus which increased with increasing concentrations of chitosan.

xvi

List of Publications

Bano, I., Ghauri, M.A., Yasin, T., Huang, Q., Palaparthi, A.D.S., 2014. Characterization

and potential applications of gamma irradiated chitosan and its blends with poly(vinyl

alcohol). International Journal of Biological Macromolecules. 65, 81-88.

Islam, A., Yasin, T., Bano, I., Riaz, M., 2011. Controlled release of aspirin from pH

sensitive chitosan / Poly(vinyl alcohol) hydrogel. Journal of Applied Polymer Science

124, 4184-4192.

xvii

List of Abbreviations and Symbols

Mv viscosity average molecular weight

Mw molecular weight

DDA degree of deacetylation

d density

WAC water absorption capacity

NaOH sodium hydroxide

NaCl sodium chloride

BaCl2 barium chloride

LB Luria-Bertani broth

g gram

mg milligram

mL milliliter

kGy kilo gray

WVTR water vapor transmission rate

WVP water vapor permeability

TS tensile strength

EB elongation at break

SGF simulated gastric fluid

SIF simulated intestine fluid

ppm parts per million

MPa mega Pascal

LC-MS liquid chromatograph and mass spectrometer

MS/MS tandem mass spectrometry

FTIR fourier transform infrared spectroscopy

PET polyethylene tetraphthalate

PVA poly(vinyl alcohol)

xviii

TEOS tetraethylorthosilicate

HPLC high performance liquid chromatography

PDA photodiode array detector

FLD fluorescence detection

FAO food and agriculture organization

FDA food and drug administration

AFB1 aflatoxin B1

AFB2 aflatoxin B2

AFG1 aflatoxin G1

AFG2 aflatoxin G2

YES yeast extract sucrose

ATR attenuated total reflection

MIC minimum inhibitory concentration

IC50 50% inhibitory concentration

ESI-MS/MS electron spray ionization tandem mass spectrometry

[η] viscosity

1. Introduction and Review of Literature

Chitosan is the only pseudo-natural cationic polymer and important derivative of chitin. It

finds many important applications in agriculture, industry and biomedical fields that

result from its unique cationic character. Every year approximately 100 billion tons of

chitin is produced from different sources. According to a report, the global market for

chitosan will reach more than $21 billion by the year 2015 (Chitin and Chitosan: A

Global Strategic Business Report, 2012). Extensive research is continuing to improve

chitosan characteristics for specific applications. Chitosan mostly find its applications in

scaffolds, wound dressings, antimicrobial coatings, food packaging, drug delivery and

waste water treatment [1]. Owing to its properties and applications, chitosan is used alone

or blends with other polymers [2-4].

1.1. Chitin

Chitin, a biopolymer [β-(1-4)-N-acetyl-D-glucosamine] is a natural polysaccharide of key

importance in different fields. Annual production of chitin in the world makes it as the

most abundant biopolymer and polysaccharide after cellulose [1, 5]. Chitin biopolymer is

synthesized by many living organisms and it occurs in nature as ordered crystalline

microfibrils forms. Chitin is the major component of the cell walls of fungi, exoskeletons

of orthropods including crabs, lobsters, shrimps and insects and the radulas of mollusks.

Besides fishery wastes, chitin is also produced by a number of other living organisms in

the lower plant and animal kingdoms. Regardless of its extensive occurrence, the highest

commercial sources of chitin have been exoskeleton of crabs and shrimps. Extraction of

chitin from exoskeleton of crustaceans needs to follow the processing steps carefully for


2

commercial purpose. The industrial processing involves the chitin extraction by acid

treatment to dissolve calcium content (CaCO3) and alkaline extraction to solubilize the

proteins. In addition, leftover pigments are removed by decolorizing step to obtain a

colorless product. Each chitin source must be passed through above mentioned steps. The

resulting chitin needs to be sorted according to its purity and color because residual

protein and pigment can cause problems for its utilization in different applications,

especially for biomedical purposes. Chitin under alkaline conditions produce partial

deacetylated derivative and another biopolymer of great importance, named “Chitosan”

which is the most important derivative of chitin in terms of commercial applications [6-

9].

1.2. Chitosan

Poly [β (1,4)-2-amino-2-deoxy-D-glucose], known as chitosan, is obtained from chitin by

deacetylation process [1]. Crustacean waste is the largest source of chitin or its

deacetylated derivative, chitosan, whose isolation process involves deproteinization,

demineralization and bleaching [1]. The structural difference between chitin and chitosan

is the presence of –NH2 group in chitosan replaced by –NHCOCH3 group in chitin (Fig.

1-1). Chitosan has been extensively studied for its multiple industrial applications and,

more recently, also for its, antimicrobial, antioxidant and film forming properties [10]. In

addition to the chitinous material, the remains of heads and exoskeletons are also rich in

other compounds of high nutritional value and functionality, primarily proteins, which

can be up to 40 % of the total waste weight, as well as lipid soluble carotenoid pigments,

responsible for the typical crustacean orange pink color. Chitosan polymer is considered

as one of the most investigated biopolymer in recent years [3, 11-13]. Chitosan has

potential applications in biomedical field due to its non-toxicity, high biocompatibility

and antigenicity. These properties of chitosan have driven the interest of the scientific

community [14]. Chitosan has been used to develop ecofriendly food packaging

materials. Generally, biopolymers based films or membranes are sensitive to

environmental conditions and usually present a low mechanical resistance. Therefore, a

number of studies have been carried out to develop the membranes/films using blends of


3

biopolymers with synthetic polymers [2, 15]. Due to biocompatibility of chitosan

polymer and its blend membranes, it is widely used in tissue engineering and drug

delivery systems as a biomaterial [1, 16]. Crosslinked chitosan membranes are three

dimensional networks that can absorb and retain a large amount of water while retaining

their structures. Chitosan membranes have various applications in the field of burn

wound dressings, food coatings and packaging materials [17-19]. Depending on the

source of chitin and the method of its deacetylation, chitosan varies greatly in its

molecular weight (Mw) and degree of deacetylation (DDA). The typical DDA of chitosan

is over 70 %, making it soluble in dilute acetic acid and hydrochloric acid. Furthermore,

due to its biodegradability and non-toxicity, much attention is being paid to the

modification of chitosan for its utilization, especially in biomedical field [20-22].

Fig. 1-1. Structures of chitin and chitosan.

CH2OH

OHNHCOCH3 NHCOCH3

OH

OH

NH2

OH

CH2OH

CH2OH

OHNH2

OH

OHOH

CH2OH

Chitin

Chitosan


4

1.2.1 Characterization of chitosan

Chitosan is most versatile biopolymer for numerous applications in different fields due to

variation in its degree of deacetylation (DDA) and molecular weight (Mw). Most of the

chitosan’s applications based on above mentioned two characteristics.

1.2.1.1. Degree of deacetylation (DDA) of chitosan

Chitosan’s degree of deacetylation is one of the most important chemical characteristics

of chitosan, which influences the functioning of chitosan in several applications. Also,

the degree of deacetylation, which reveals the content of free -NH2 in the polysaccharides

chain, can be used to distinguish chitin and chitosan. Chitosan exists in a variety of

degree of deacetylation (≥70 %). In general, chitin with a DDA of above 70 % is

considered as chitosan. The process of deacetylation involves the conversion of acetyl

groups from the chitin into amino groups [23]. However, some other parameters are also

needed to determine such as purity (ash content) of chitosan, moisture content, the

content of heavy metals, endotoxin and proteins. Above mention parameters are

important to determine for chitosan applications, relevant to human food and medical [23,

24]. Earlier, it has been reported, that the origin of chitin/chitosan affects its crystallinity

and purity as well as arrangement of polymer chain [5]. Crystallinity of chitosan has been

related to DDA of chitosan. Chitosan is crystalline in nature and its DDA and

crystallinity may vary significantly depending on the origin of the chitosan and its

treatment during extraction from raw resources of chitin. There are a number of methods

which have been adopted to determine the DDA, such as infrared spectroscopy, linear

potentiometric titration, nuclear magnetic resonance (NMR) spectroscopy, titrimetry and

first derivative UV-spectrophotometry [24]. Infrared spectroscopy is a frequently used

technique to determine the DDA of chitosan because of its simplicity in operating.

However, other methods can be tedious and costly for routine analysis including nuclear

magnetic resonance spectroscopy and even can be damaging to polymer samples. It is

understandable from literature, that the DDA values of chitosan are highly associated

with the analytical methods employed.


5

1.2.1.2. Molecular weight (Mw) of chitosan

Molecular weight of chitosan is another most important characteristic which varies

depending on the sources and methods of preparations [23]. The chitosan obtained from

different raw material sources are usually of high molecular weight. There are methods

available to lower the molecular weight of chitosan. Irradiating by gamma radiations is

very convenient method for reducing the molecular weight of chitosan through

degradation. Carbohydrates such as chitosan can be easily degraded into smaller chains

by gamma radiation in liquid and solid state [25]. The effect of average molecular weight

on the viscosity of aqueous solutions of chitosan plays important role in the biochemical

and bio-pharmacological applications [26]. Various studies about bacteriostatic and

bactericidal activities of chitosan have generated ambiguous results regarding a

correlation between bioactivity and Mw of chitosan [27]. Some studies reported that high

molecular weight chitosan possesses lower antibacterial activity of chitosan against E.

coli, while in some other studies, high molecular weight chitosan showed greater

antibacterial activity [28]. In addition, activities still were found to be equal against E.

coli and Bacillus subtilis regardless of molecular weight [27]. Although, very limited

available results on antibacterial activities of low Mw chitosan were comparable

depending on bacterial strains, biological conditions for testing and Mw of relevant

chitosan, the results were not analogous with each other. For example, high molecular

weight chitosan inhibits growth of E. coli while low molecular weight stimulates its

growth [29]. In some other studies, low Mw chitosan and its derivatives showed better

antibacterial and antifungal activities [27]. For reducing the molecular weight of chitosan

without altering its chemical structure, different degradation processes have been

developed [30]. These degradation methods include acid hydrolysis [31], oxidative

degradation, enzymatic degradation [32] and radiation degradation [33]. Among these,

radiation degradation is relatively simple and an eco-friendly process and can be

employed for the large scale production of oligochitosan [34]. However, yet the

applications of radiation degraded chitosan have not been fully realized. The Mw of

chitosan can be determined by various methods like chromatography, viscosity, and light

scattering [28, 35]. Among these methods, viscosity method is the simple and rapid for


6

the determination of molecular weight [35]. The intrinsic viscosity of a chitosan solution

is related to its molecular weight according to the Mark-Houwink equation [35]. The

viscosity average molecular weight of chitosan can be determined using following

equation:

[η]=KMvα (1-1)

Where “Mv” is the viscosity average molecular weight, “[η]” is the intrinsic

viscosity of the solution, and “K” and “α” are constants for a given solute-solvent system

and temperature.

1.2.2. Biological properties of chitosan

Chitosan has fascinating properties which make them suitable for use in biomedical

applications. Due to their biodegradability, biocompatibility and non-toxic nature, chitin

and chitosan currently have received a great interest in medical and pharmaceutical

applications. Moreover, some reports also display these polysaccharides as antimicrobial,

analgesic, antitumor, haemostatic, hypocholesterolemic and antioxidant [36, 37]. The

majority of the biological properties of chitosan are related to its cationic behavior and

size of the polymer chain. But, in some cases, the molecular weight has a prime role.

Other than the degree of deacetylation and molecular weight, some other properties such

as chitosan chain conformation, its solubility and degree of substitution have also been

considered to affect the biological properties of chitosan [1]. Chitosan produced by

heterogeneous deacetylation, where acetylated and deacetylated units are arranged

alternatively have a tendency to accumulate in aqueous solutions. However excess

aggregation and intermolecular interactions may reduce availability of functional groups

on the chitosan molecule. This may be the reason for some of the variances between

reported properties of chitosan. So, one should pay close attention to the preparation of

chitosan dispersions or/and preparation procedure [37-40]. The relationship between

some characteristics of chitosan and its biological properties are shown in Table 1-1.


7

Table. 1-1. Relationship between characteristics of chitosan and its biological

properties. [1]

Characteristics

Properties

Molecular Weight

Antimicrobial

Degree of Deacetylation

Biocompatibility, Analgesic

Degree of Deacetylation and Molecular Weight

Haemostatic, Biodegradability,

Antitumor,

Anticholesterolemic

1.2.2.1. Biodegradability of chitosan

Chitosan from many studies has been proved to be non-toxic and biodegradable.

Although, chitin and chitosan both are not present in mammals but still there are certain

enzymes like lysozyme, papain, pepsin etc., which have significant ability to degrade

chitosan into non-toxic oligosaccharides of variable lengths. Their biodegraded products

subsequently are incorporated to glycoproteins and glycosaminoglycans, and ultimately

to metabolic pathways or excreted out of the body [41]. Lysozyme is a non-specific

protease found in all mammalian tissues which seems to play major role in the

degradation of chitosan. The degradation also depends upon the crystallinity of chitosan

which is mainly controlled by the DDA. Furthermore, the biodegradation can be affected

by distribution of amino groups since the absence of these groups and their homogeneous

distribution reduces the rate of enzymatic degradation [7, 42].


8

Moreover, several studies reported that the chain length of chitosan also affects

the rate of degradation [43-45]. The process of degradation is very essential in many

controlled release system of biomolecule and in functional tissue regeneration

applications. Ideally, the rate of degradation of chitosan scaffold should reflect the rate of

new tissue formation or be sufficient for the bioactive molecules to be released. Thus, it

is of prime importance to understand the rate by which each material is degraded as well

as the mechanism involved in the process of degradation. Biocompatibility is also

affected by degradation rate as amino sugars are accumulated by fast degradation which

ultimately originates an inflammatory reaction. Chitosan with more acetyl groups

involves in acute inflammatory response while chitosan with low acetyl groups produces

a minimal response because of slow degradation rate. The rate of degradation has been

shown to increase as DDA of chitosan decreases [46-48]. Some scientists have explored

the process of enzyme degradation of various chitosans by examining the viscosity

change of chitosan solution [49]. These scientists observed that low DDA chitosan was

degraded more rapidly as compared to high DDA chitosan samples. However, other

workers reported that variations in the degradation may be due to the distribution of

amino groups on the chitosan polymer [42, 50]. The distribution of acetamide groups

occurs due to variant conditions during process of deacetylation [48]. Conclusively, it can

be assumed that estimating rate of biodegradation from the DDA alone is not possible.

1.2.2.2. Biocompatibility of chitosan

Both chitin and chitosan are believed to have effective biocompatibility. Chitosan has

excessive variety on the basis of degree of deacetylation, molecular weight, source of

origin and method of preparation which affects the biocompatibility. Chitosan does not

get absorbed when administered orally though the gastrointestinal enzymes which can

degrade chitosan partially. For this reason, chitosan is considered as non-bioavailable.

Toxicity of chitosan reported mainly depends on its DDA. Some scientists reported that

chitosan with degree of deacetylation above 35 % showed low toxicity, while a chitosan

with DDA less than 35 % produced dose dependent toxicity. On the other hand,

molecular weight of chitosan, either low or high does not impart any toxicity [51].


9

Chitosan presents higher cytocompatibility in vitro than chitin because of positive

charge produced by amino groups. The biocompatibility of chitosan has also been proved

in vitro with hepatocytes, endothelial, epithelial, fibroblast, myocardial, condrocytes and

keratinocytes cells [52]. This property appears to be related to the DDA of the chitosan

polymer. The chitosan interactions with cells increase with increase in positive charge on

chitosan. The adhesion of keratinocytes and fibroblasts cells to chitosan and their

proliferation depend on DDA and cell type. For example, extra negative charge surface of

fibroblasts than keratinocytes make them favorable for adhesion with chitosan.

Sometimes the chitin and chitosan could be allergic to cells due to residual proteins. The

presence of protein content in any sample mainly depends on the method adapted for its

preparation and the source of the chitin and chitosan [1].

1.2.2.3. Antimicrobial activity of chitosan

Chitosan as antimicrobial agent has got considerable attention in recent years. Chitosan

and its derivatives have showed reasonable antimicrobial activity against different

microorganisms, such as bacteria, fungi and yeast. The antimicrobial activity of chitosan

is affected by molecular weight and degree of deacetylation. Low molecular weight

chitosan has strong antibacterial properties and is also harmless to human body [28, 53-

57]. For the food packaging industry, food quality and food safety to human health are

the two most important concerns as consumers choose fresh and nominally processed

products. Particularly, microbial contamination of ready to eat products is of major

concern [53]. Chitosan has proven a useful antimicrobial agent in food processing,

particularly for improving the shelf life of food materials [58, 59]. Different studies show

that chitosan's antibacterial efficacy depends on many factors, like microorganism

species, cell age, concentration of chitosan, chelating capacity, hydrophilic/hydrophobic

characteristic, solubility and solid state of chitosan etc. [28, 60]. Being a cationic

polymer, most chitosan blends have the ability to respond to external stimuli such as

temperature, pH, and electric fields [28]. On the basis of above mentioned factors, two

core antimicrobial mechanisms of chitosan have been proposed by various scientists [61,

62]. The first mechanism demonstrated that there may be some interaction of anionic


10

groups on the cell surface with cationic groups on the chitosan polymer chains. This

interaction may develop impermeable layer around the bacterial cells and inhibit the

transport of vital solutes. The other mechanism involves the invasion of chitosan into the

cell nucleus which inhibit the RNA and synthesis of protein especially low molecular

weight chitosan and chitosan oligomers [61]. Moreover, Liu et al. [62] reported that

labeled chitosan oligomers of molecular weight from 8 to 5 kDa inside the E. coli cell

showed significant antibacterial activities. Here, the molecular weight of polymer has

played the key role in inhibiting the growth of microbial growth. These findings suggest

that Mw of chitosan is one of the important factors which play a key role as antimicrobial

activity of polymer. However, some other mechanisms have also been suggested [28].

Chitosan may also act like a chelating agent for microbes. Consequently microbial

growth can be inhibited by extracting vital elements thus making them unavailable for

their critical growth. These elements can be metals, some trace elements and essential

nutrients. Some reports express that chitosan interact with flocculating proteins, however,

this interaction was dependent on pH of the medium [63]. Numerous studies have

suggested that the antifungal action of chitosan against filamentous fungi might be due to

the disturbance of membrane functions [64]. However, this is not clear whether the

antimicrobial activity of chitosan is caused by cell death, growth inhibition or both.

Antibacterial activities of chitosan also vary with substituent groups on chitosan. It was

found that antibacterial activities increase in the order, N,-O-CM-chitosan, chitosan and

O-CM-chitosan [1]. In pure chitosan, when -NH2 and -OH groups were replaced by

carboxymethyl groups, it remained with fewer amino (-NH2) residues which affect its

antimicrobial property. In the case of -O-CM-chitosan, its number of amino groups

remained same. Furthermore, its carboxyl group may react with the -NH2 groups intra- or

inter-molecularly and charged these groups. Subsequently, Liu et al. [62] also concluded

that, in actual effective number of -NH3+ groups play major role for antimicrobial actions

of chitosan and CM-chitosan derivatives. Several other studies also demonstrate that

increase in the number of positive charge in the form of -NH3+ on chitosan makes it bind

more strongly to the cell walls of bacteria and hence high antibacterial activity [65]. The

correlation between molecular weight, number of positive charge and antimicrobial


11

activity has been demonstrated by Kim et al. [66]. They observed that -O-CM chitosan

derivative of short chain chitosan polymer was more effective than normal chitosan. The

increased antibacterial activity of O-CM-chitosan was attributed due to increase in

number of -NH3+ groups by interaction of the carboxyl group with the amino group. In

the case of native chitosan, antibacterial efficiency reduced because extra -NH2 groups on

-O-CM chitosan stimulates a crosslinked network through strong intra-molecular

hydrogen bonding. Thus, the number of amino groups available to bacterial surfaces

decreased due to hydrogen bonding. In contrast, a strong correlation between the degree

of deacetylation of chitosan and its antimicrobial activity has not built yet. Some authors

propose that the antimicrobial activities of chitosan depend on the particular chitosan

samples and the microorganism to be examined [67].

1.2.2.4. Haemostatic activity of chitosan

Haemostasis is a process which causes bleeding to stop and keep blood within a damaged

blood vessel. Haemostatic agents are important in pharmaceutics because a haemostasis

is the first stage of wound healing. Chitosan, as well as sulphated chitosan oligomers

have been reported as anticoagulant [68]. Again, the anticoagulant activity of chitosan

seems to be positive charge dependent due to amino groups on chitosan which actually

interact with negatively charged membranes of red blood cells [69, 70]. Molecular weight

of chitosan may also affect the binding or clumping of red blood cells [71]. A

comparative study was conducted among chitosan in solid state and its solution in acetic

acid by Yang et al. [72]. For this purpose, several chitosan samples with different

molecular weights (from 2000 to 400 kDa) and degree of deacetylation (from 90 to 70 %)

were tested for coagulation mechanism. It was found that chitosan in solid state and

solution state followed quite different mechanisms for haemostasis. During this study,

when blood sample was mixed with chitosan solution, the erythrocytes were distorted

after aggregation. Also, the degree of deacetylation, particularly chitosan solution with a

high DDA had a considerable effect on the aggregation and deformation of erythrocytes.

However, chitosan in solid state with a high degree of deacetylation bounded more

platelets and had greater haemostatic affect [72].


12

1.2.2.5. Analgesic effect of chitosan

Some scientists have earlier documented that both chitin and chitosan have analgesic

effect on inflammatory pain [73, 74], but in the later years, others have studied that

analgesic effect by chitin and chitosan is due to intra-peritoneal administration of acetic

acid [75]. Moreover they proposed that chitosan showed a greater analgesic ability than

chitin due to its polycationic nature. The difference in analgesic activity of chitosan

involves the absorption of proton ions by chitosan which are released in the inflammatory

area. The free primary –NH2 groups on chitosan protonate in the presence of acidic

conditions which reduce the pH of effected area and cause the effective analgesic

influence [75].

1.2.2.6. Antitumor activity of chitosan

Chitosan, although, as antitumor agent has some controversial reports. However, chitosan

has been identified as antitumor polymer by inhibition of tumor cells growth largely due

to an immune stimulation effect. Studies were conducted on mice as model animal that

were ingested with low molecular weight chitosan which showed a significant

antimetastatic effect of chitosan against lungs carcinoma [31]. Chitosan with a

carboxymethyl group has also been proved to be effective in reducing tumor

development. The proposed mechanism involves immune-stimulating effects of chitin,

chitosan and carboxymethyl chitosan by stimulation of cytolytic T-lymphocyte cells [76].

This antitumor activity increases with lower molecular weight of polymer and it was

proposed that they had immune-stimulating effects. This effect activates the peritoneal

macrophages and stimulates non-specific host resistance. However, higher molecular

weight chitosan oligomers have also showed antitumor activity [1]. Similar type of

mechanism was described for their activity through increased production of lymphokines

by activated lymphocyte cells. The activation of macrophage cells by chitosan was

proposed to facilitate in vivo antitumor properties. However, the angiogenic prompting

properties of chitosan may produce harmful effects, such as promotion of tumor growth

and its invasion all over the body [71, 76, 77].


13

1.2.2.7. Anticholesterolemic activity of chitosan

Chitosan as cholesterol reducing agent involves several proposed mechanisms.

Muzzarelli et al. [78] proposed the formation of insoluble chitosan salts from bile acids.

The hydrophobic nature of bile acids permits the accumulation of cholesterol and lipids

through hydrophobic interaction. Three types of reactive groups in chitosan are

responsible for interaction between cationic groups of chitosan and anionic surface active

materials of phospholipids and bile acids. These three groups include one amino group at

the C-2 position on chitosan and a primary and a secondary hydroxyl groups at the

carbon positions-3 and -6, respectively [79, 80].

Another study demonstrated that chitosan get adsorbed to the emulsified lipid

surface, hence a protective coating is formed that might inhibit the lipase and co-lipase

adsorption to the droplet surfaces and access of lipids inside the droplets may be denied

[81]. There are some studies in which the fat binding capacity was positively correlated

with the molecular weight of chitosan [82]. In an experiment, the fat binding capacity of

five chitosans of increasing molecular weight (range 500-800 kDa) with same DDA was

studied and found that the sample with the second lowest molecular weight showed

significantly higher activity [9, 83]. Similarly, a chitosan sample was degraded with

gamma radiations and five chitosans of decreasing molecular weight were produced in

the range 25-400 kDa and these showed an increasing trend of fat binding activity with

decreasing molecular weight [84]. It was also observed that the chitosan binding capacity

for bile acids was selective and varies with the type of bile acid. Although, no correlation

was identified between individual bile acid binding capacity to chitosan and any other

tested physicochemical properties of chitosan. These studies suggested that molecular

weight, degree of deacetylation or swelling capacity might not be useful to calculate the

bile acid binding capacity of the chitosan [85, 86].


14

1.2.3. Potential applications of chitosan polymer

The development of commercial applications of chitosan with range of molecular weight

and degree of deacetylation has expanded rapidly in recent years. There is great potential

for exploitation of its properties in numerous marketplaces. The best potentials for its

applications are in pharmaceutics, food industry, agriculture, cosmetics, waste water

treatments etc. (Table. 1-2).

1.2.3.1. Biomedical applications of chitosan

Antibacterial polymers like chitosan and its derivatives can be applied in health care and

hygienic applications. Due to bactericidal and bacteriostatic nature of chitosan it may be

incorporated in the form of membrane, hydrogel, fibers, beads etc. and used for contact

disinfectants in many biomedical applications which include wound dressing,

orthopedics, tissue engineering and drug delivery carrier. Polysaccharides like chitosan,

possessing hydrogel forming properties have been considered to be beneficial in wound

dressing materials. Evaluating the eligibility and capability of the dressing material,

antimicrobial assessment of chitosan is a key parameter regarding wound dressing. It is

reported that an ideal wound dressing material should have following properties: (1)

Allows water evaporation at a certain level, (2) capable of absorbing the exuded liquids

from wound and (3) finally does not allow the microbial transport to wound areas [87-

89]. Therefore chitosan based materials have received much attention in this regard due

to their above mentioned properties. Typically, chitosan in the form of fiber, membrane,

sponge and hydrogel provides better antimicrobial effect to wound dressings [58, 90].


15

Table. 1-2. Potential applications of chitin and chitosan

Industry

Applications

Health care

Wound curing and healing,

Surgical suture,

Ophthalmology,

Orthopedics,

Pharmaceutics

Food and beverages Antimicrobial preservatives,

Food stabilizers

Flavors and taste,

Anticholestrol and fat binding

Animal feed preservative

Cosmetics and toiletries Hair, Skin and Oral care

Agriculture Feed ingredients,

Seed treatment

Nematocides, Insecticides

Waste water treatment Chelating agent,

Purification of drinking water,

Treating of food waste,

Pools and sanatoria

Product separation and recovery Membrane separation,

Chromatographic columns,

Coagulation

New applications of chitin and chitosan Imprinted chitosan based matrixes

Chitosan-metal nanocomposits


16

1.2.3.2. Applications of chitosan in food industry

There is a wide range of chitosan applications in the food industry. Food applications

include preservation of foods from microbial deterioration. Chitosan also acted as a

dietary fiber and as functional food ingredient and carrier for nutraceuticals [11]. Due to

its fat binding ability, chitosan and its derivatives have been used in several nutritional

supplements. Recently, it has been reported that fed diets for rats that contained highest

DDA chitosan has significantly decreased the plasma cholesterol in blood [91]. Chitosan

with high molecular weight significantly controlled the gain in body weight of adult rats

[91]. Considering the DDA and polymer chain length, chitosan with higher Mw showed

better cholesterol binding capacity in vitro [91]. However, it was concluded that when the

particle is finer, and degree of deacetylation and molecular weight of chitosan were

relatively high, the effect was better. Zeng and coworkers [92], examined the absorption

phenomena of different molecular weight chitosan inside the mice and found that low

molecular weight chitosan had greater absorption capacity and the effect was greater with

greater water solubility [92]. Chitosan with very high Mw obviously was very difficult to

absorb and enter into the blood. However, chito-oligomers were easily degraded and

quickly absorbed and distributed.

1.2.3.3. Chitosan as food preservative

Chitosans have been known as versatile biopolymers for food preservation due to their

bactericidal and fungicidal efficacies against food borne pathogens. Chitosan solubility is

pH-dependent and allows them to be modified in the form of beads, films and membranes

for applications in food industry [93]. Some experiments report that generally, low to

medium molecular weight (5-78 kDa) chitosans with high DDA (85-98 %) efficiently

inhibit the growth of both Gram’s positive and Gram’s negative bacteria [65, 94].

Contrary to these results, a study on chitosan obtained from cuticles of housefly larvae

describes that the antibacterial effect of chitosan decreases with higher molecular weight.

Chitosans with 21 to 44 kDa molecular weight were more effective than chitosans with 8

and 476 kDa molecular weight [95]. Higher degree of deacetylation is related with better


17

antimicrobial results. Tsai et al. [40] compared the antimicrobial activities of chitin (from

shrimp shells) and chitosan attained by chemical and biological treatments. The minimum

inhibitory concentrations were in the range of 50-200 ppm concentration which became

reduced with higher degree of deacetylation [40].

1.2.3.4. Chitosan as edible coatings and films

Chitosan coatings are used to enhance the shelf life of fruits and vegetables. Coatings

generally retard the ripening of fruits and reduce the microbial decay. Semi permeable

coatings of chitosan may generate a modified atmosphere at a lower cost very similar as

used in storage for controlled atmosphere [96]. Although many studies on chitosan as

coating material are available, very few of them considered the effect of the

physicochemical properties of chitosan on their activity [97]. In a study, membranes were

prepared from shortfin squid chitosan and shrimp chitosan and tested for their water

vapor permeability, water absorption capacity and tensile properties. The influence of the

molecular weight and degree of deacetylation of chitosan were evaluated on these

properties [1, 96]. The results showed that lowering the acetyl groups on polymer led to

more intermolecular interactions by hydrogen-bonding. Due to this interaction a compact

polymer network structure is established. Therefore water vapor permeability and water

absorption capacity are found to be low while mechanical properties are improved [96].

Chitosan with reduced molecular weight, without change in degree of deacetylation, led

the membranes with lower water vapor transmission rate, water vapor permeability and

water absorption capacity [98]. This significant effect is related to the effective packing

of polymer chains within the membrane matrix formed from lower molecular weight

chitosan chains. However, formation of a looser network by high molecular weight

chitosan weakens the tensile and elongation properties as a result of decreasing

entanglement and degree of crosslinking [99]. It has been accepted world widely that

chitosan coatings are used as a barrier to protect and enhance the storability of fruits and

vegetables [100-102].


18

1.2.3.5. Chitosan use in waste water treatment

The chemical contamination of water from a wide range of toxic products is a serious

environmental problem. The contaminating components can be metals, aromatic

molecules, dyes and so on, creating potential human toxicity. The usage of inexpensive

polymers to remove water pollutants is of great attention [103]. In this regard, chitin and

chitosan have been widely used to remove a great variety of water pollutants [9]. Guibal

and co-workers have extensively worked on adsorption properties of chitosan for metals,

dyes and organic compounds [104-106]. Chitosan has been used to remove anionic dyes

has been studied by Crini and Badot [6]. The chitosan proved to be more efficient for

metal ions and anionic dyes removal than chitin. Also, the coagulation and flocculation

process as well as the adsorption process depends on the degree of deacetylation [9, 104,

107].

1.2.4. Biosafety/non-toxicity of chitosan

Chitosan is non-toxic and has been approved as functional food in some countries

including Japan and Korea [1]. The addition of chitin and chitosan as food gradient was

deliberated in 2003 by the Codex Alimentarius Commission, Italy. However, chitosan is

not currently included in general standards for food additives and is not authorized as a

food ingredient in Europe. Although, studies have shown that this compound is non-

toxic, still no studies on long term human safety has been described [108].

1.3. Poly(vinyl alcohol), PVA; a synthetic polymer

Poly(vinyl alcohol) is a colorless, non-toxic and water soluble synthetic polymer and has

good physical and chemical properties. As a blending agent, poly(vinyl alcohol) has

excellent miscibility and film forming properties. It is synthetic and inexpensive polymer

used for membranes preparations. It contributes better tensile strength and flexibility to

the blends. The PVA blended chitosan has been employed in many biomedical

applications due to its biodegradable, non-carcinogenic and biocompatible nature [16, 59,


19

109-112]. This polymer has been used in many important applications such as controlled

drug delivery systems, membrane preparation and packaging materials. The PVA is

commonly used in making adhesives, paper coatings, textiles, films, sponges and

building products such as ceramics and cements. Some important properties including

anti-thrombogenicity and biocompatibility of PVA have been studied extensively [113,

114]. The degree of hydrolysis affects the solubility of PVA in water. It has been reported

that PVA grades with high degree of hydrolysis have low solubility in water. The

molecular weight distribution is an important characteristic of poly(vinyl alcohol)

because it affects many of its properties such as crystallizability, adhesion and

mechanical strength [115]. In biomedical field, use of poly(vinyl alcohol) with chitosan

blends have been reported for its antimicrobial and sustained drug release applications

[111, 112]. Chemical structure of PVA unit is given below (Fig. 1-2).

Fig. 1-2. Structure of poly(vinyl alcohol).

1.3.1. Physicochemical properties and applications of PVA

Poly(vinyl alcohol) is an attractive material that exhibits crystallinity. It is composed of

1,3-diol linkages but a few percent of 1,2-diols also occurs. The vinyl ester is used as

precursor for the preparation of PVA. Poly(vinyl alcohol) has excellent film forming

CH2

PVA


20

properties and is inexpensive polymer. It is also resistant to grease, oil and some solvents.

Poly(vinyl alcohol), a synthetic polymer is water soluble at higher temperature (80ºC). It

possesses higher mechanical and elastic properties and contributes strength to blending

polymers. Chitosan also possess high oxygen and aroma barrier properties. However

these properties are humidity dependent, with higher humidity more of the water is

absorbed [15, 116].

Poly(vinyl alcohol) is generally non-toxic and safe for biomedical applications up

to 5 % PVA solutions. It is biodegradable but rate of degradation of PVA is slow [15].

Some of the important uses of PVA are as following:

Good blending agent with chitosan for different biomedical applications

Used as thickener, modifier and in poly(vinyl acetate) glues

Used as textile sizing agent in textile industry

Applied as paper coatings

It forms water-soluble film useful for packaging materials

The PVA is used as CO2 barrier in polyethylene terephthalate (PET) bottles

It is used as a lubricant in eye drops and hard contact lens solution

The PVA fiber, as reinforcement in concrete

Used in protective chemical resistant gloves

Used as artificial tears for treatment of dry eyes

1.4. Chitosan/PVA blend membranes

Chitosan can be modified in the form of membranes, coating films, nanofibers or beads

by physical or chemical crosslinking for some particular applications. As a blending

agent, poly(vinyl alcohol) has excellent miscibility and film forming property. It

contributes better tensile strength and flexibility to the blends. It is synthetic and

inexpensive polymer used for membrane preparations. The PVA blended chitosan has

been employed in many biomedical applications due to its biodegradable, non-


21

carcinogenic and biocompatible nature [16, 59, 109, 111, 112]. The antibacterial

properties of numerous chitosan blends, including PVA/chitosan [111, 112],

cellulose/chitosan [56], starch/chitosan [2, 117], banana flour/chitosan [118],

gelatin/chitosan [119], pullulans/chitosan [120], guar gum/chitosan [121] and many more

have been investigated. Polymer modification by blending two or more different

polymers to obtain desired properties is now in common practice. Chitosan and

poly(vinyl alcohol) must be crosslinked in order to be useful for a wide variety of

applications, specifically in the area of medicine and pharmaceuticals. For preparations of

chitosan blends with synthetic polymers, different methods can be applied including

physical, chemical or by radiations.

Some of the polymer blends exhibit superior and rare properties, unexpected from

homopolymers. Chitosan/PVA crosslinked membranes possess uniform pores with

modifiable water absorption capacity (Fig. 1-3). These properties are crucial for wound

healing materials. Therefore blends of chitosan and PVA have been used as

biocompatible and biodegradable antimicrobial wound dressings [122, 123]. While

referring chitosan and its blends to pharmaceutical and surgical materials for humans

including tissue engineering and drug release system. There arise some complications

from infections caused by microorganisms and it is a universal problem. The infected

materials can cause high morbidity and mortality after introduction into the body.

Therefore, combating these challenges, chitosan is being used in combination with

antibiotics and developing some materials resistant to bacteria [124]. For example,

chitosan /PVA hydrogel coated grafts, crosslinked by ultraviolet irradiation, exhibited a

resistance against E. coli in vitro and in vivo [28].

The textile materials made from natural polymers are usually considered to be

more prone to microbial attack as compared to synthetic fibers. This might be due to their

porous structure, hydrophilic nature and moisture transport characteristics. Chitosan

alone or blends with PVA use as antimicrobial agent is gradually becoming a standard

finish for medical and hygienic textile products [28]. Recently, it has become popular in

women's wear and sportswear to impart antimicrobial and anti-odor properties [28].


22

Moreover, chitosan/PVA membranes have been used for controlled drug release systems

[16, 109].

Fig. 1-3. A typical SEM image of chitosan/PVA membrane showing distribution of

pores ( ). [98]

1.5. Crosslinkers for chitosan blends with other polymers

Chitosan polymer is needed to be crosslinked in order to modify their general properties.

Crosslinking occurs when the backbone or the functional groups of polymer chains form

bonds between same or different polymer chains. The polymer chains interlinked by ionic

bonds or covalent bonds and sometimes by secondary interactions. In physically

crosslinking polymers chains are interlinked by week forces which exist between

different polymer chains. In chemically crosslinking, a covalent bond is formed between

the different polymer chains. In natural and synthetic polymer based membranes,

interlinks between polymers chain are formed by crosslinkers. Different methods are used

to crosslink polymers for hydrogel applications.


23

Several reagents have been used for crosslinking the chitosan alone or with other

polymers using boric acid, glutaraldehyde, ethylene glycol, tripolyphosphate,

diisocyanate and diglycidyl ether as crosslinkers [125]. But, synthetic crosslinkers are

reported as more or less cytotoxic [126]. Hence it is necessary to use a non-toxic

crosslinker for biomedical applications.

Tetraethylorthosilicate (TEOS) is considered as biocompatible, biodegradable and

harmless molecule [127]. This compound also has health benefits and can be used as a

non-toxic crosslinking agent. The chemical formula of TEOS is Si(OC2H5)4 that consists

of four ethyl groups attached to orthosilicate (SiO44-

) ion. As an ion in solution,

orthosilictae does not exist. Although, TEOS is mainly used as crosslinking agents, other

applications of tetraethylorthosilicate are as coatings for carpets. The TEOS is also used

in the production of aerogels. These applications exploit the reactivity of the Si-OR bonds

[16, 128].

1.6. Microorganisms for antimicrobial studies

1.6.1. Pathogenic bacteria

P. aeruginosa, E. coli, S. aureus and B. subtilis etc. are commonly associated with

hospital-acquired infections and food born pathogenicity [129]. The infections caused by

some strains of P. aeruginosa and E. coli are increasing in hospitals as well as in general

community and is known pathogen for nosocomial infections, particularly among

immune compromised individuals [130]. P. aeruginosa were investigated for drug-

chitosan interactions by Tin et al. [124]. In this study chitosans with different DDA and

chitosan oligosaccharide were combined with antibiotics to show the synergistically

effect of antibacterial property. All the chitosans showed synergistic activity with

sulfamethoxazole, a sulfonamide antibiotic [124].

The antimicrobial activities of chitosan with cellulose blend membranes were

examined against E. coli and S. aureus. The chitosan/cellulose blend membranes


24

characteristics were presented suitable as a wound dressing with antibacterial properties

[56]. Similarly a hydrogel sheet composed of chitosan with honey and gelatin with

different ratios were developed as dressings for burn wounds. The hydrogel sheets

showed powerful antibacterial efficacy up to 100 % to Staphylococcus aureus and E. coli

with increasing concentration of chitosan. Therefore, chitosan hydrogel sheets

demonstrated their potential as a wound treatment [90]. Also, antimicrobial activity of

chitosan biopolymer films against E. coli-0157:H7 were investigated by agar diffusion

method. The chitosan biopolymer films were more effective at inhibition of E. coli-

O157:H7 with low molecular weight chitosan [53]. Some studies against E. coli and

Staphylococcus aureus were conducted to analyze the antimicrobial activity of different

molecular weights chitosan.

The effect of the concentration and molecular weight of chitosan were

investigated [53, 57]. For chitosan with molecular weight below 300 kDa, the

antimicrobial effect on E. coli was reduced as the molecular weight was increased. In

contrast, the same effect on S. aureus was increased [57]. In a study, an antimicrobial

film was prepared by chitosan and poly(vinyl alcohol) with cross-linker glutaraldehyde.

The effect of above membrane was studied against few pathogenic bacteria including E.

coli, Staphylococcus aureus and Bacillus subtilis. The obtained results indicated that film

might be a promising material for packaging applications in food industry [59]. In

another study, chitosan blend films with poly(vinyl alcohol) and pectin were prepared for

antibacterial studies by Tripathi and coworkers. The microbiological screening of these

films had demonstrated the antimicrobial activity of the film against pathogenic bacteria

E. coli, S. aureus, B. subtilis and Pseudomonas by measuring the diameter of zone of

bacterial inhibition (included diameter of film strips), the values of which were always

higher than original diameter of tested films. Overall, the chitosan/PVA/pectin films

happened to be a suitable material as antimicrobials for wound dressings and food-

packaging applications [131]. Similarly, banana flour with chitosan blend films were

produced using different ratios of banana flour and chitosan. The presence of chitosan

gave them the antimicrobial property which enhanced with increased concentration of

chitosan. The banana-chitosan composite bags were found to protect asparagus, baby


25

corn and Chinese cabbage against Staphylococcus aureus activity by serving as a

antimicrobial agent [118].

Different molecular weight chitosan with the same degree of deacetylation (80 %)

obtained by acetic acid hydrolysis method were investigated against Escherichia coli by

Liu et al. [132]. All of the chitosan samples with molecular weight from 55-155 kDa had

antimicrobial activities at the concentrations above 200ppm. But the chitosan sample with

molecular weight 90 kDa promoted the growth of bacteria [132]. Also, chitosan added at

earlier stage of cultivation showed greater effect. The mechanism of antibacterial activity

in this case was the flocculation of E. coli culture [132].

1.6.2. Aflatoxins producing fungi

Certain fungi produce toxic metabolites, called aflatoxins in/on foods and feeds during

storage. During harvest or storage, crops get contaminated with mycotoxins, the

secondary metabolites of many saprophytic fungi. The ubiquitous nature of fungi makes

food vulnerable to fungal contamination. Aflatoxins are unavoidable contaminants of

food components which even exist where very good manufacturing practices are

followed [133]. Perhaps, they are the best identified researched mycotoxins in the world.

Specific guidelines are established for acceptable levels of aflatoxins in food components

by food and drug administration (FDA). The acceptable levels were established on the

basis of action levels that allow for the removal of violating lots from import/export

products. Even at very low concentrations, the intake of aflatoxin B1 for a long period of

time may be highly dangerous [134, 135].

Aflatoxins have been related to various kinds of diseases throughout the world

including aflatoxicosis in domestic animals, livestock and humans. The Food and

Agriculture Organization (FAO) has reported the presence of mycotoxins in 25 % of the

world’s crop [136]. Aflatoxin B1, produced by Aspergillus flavus and Aspergillus

parasiticus, is one of the most toxic and common contaminants in food and feed.


26

Among the myotoxins, aflatoxins have received greater attention because of their

verified and influential carcinogenic effect in susceptible laboratory animals and their

acute toxicological effects in humans. Ingestion of aflatoxin contaminated food leads to

acute and chronic toxic effects, which may be hepatocarcinogenic, mutagenic, teratogenic

or genotoxic [137-140]. The growth of aflatoxins producing fungi is influenced by certain

environmental factors therefore extent of contamination varies accordingly [141]. The

four naturally occurring aflatoxin are aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin

G1 (AFG1) and aflatoxin G2 (AFG2). A representative chemical structure of aflatoxin is

given in Fig. 1-4. Among them, AFB1 is the most common, as well as the most dangerous

for its ability to cause liver cancer in human [142].

Fig. 1-4. A representative structure of aflatoxin.


27

Fig. 1-5. The growth of Aspergillus flavus on rice.

One of the studies by Ghadeer and Khalaf [143] proves that among several foods, rice

appeared more prone to aflatoxins production (Fig.1-5). Production of AFB1 by

Aspergillus flavus in natural crushed food samples (rice, peanut, wheat and corn) was

compared in a set of glass flasks and a set of clay pots incubated at 28ºC for 10 days.

Flasks were more favorable for the production of AFB1 with rice than wheat and peanuts.

The lowest AFB1 was with using corn and clay pots with the same medium while

produced less AFB1 than those in the glass flasks [143].

1.6.2.1. Antifungal and antiaflatoxin properties of chitosan

One of the most important and attractive feature of chitosan is its antifungal property.

Chitosan and its derivatives have the ability to inhibit the growth of certain fungi

producing toxic metabolites [144]. Though aflatoxins are very stable and do not degrade

up to 270ºC (their melting temperature) in dry conditions, several physical and chemical

methods have been suggested for the degradation of aflatoxins [145]. Moreover, several

biological methods have been adopted for the degradation or removal of aflatoxins in

food and feed components [133]. On the other hand biologically aflatoxin degrading

microbes may also be harmful to human as well.


28

Natural polymers can be the efficient and alternative source to inhibit the

aflatoxin. Although, natural polymers are inexpensive and easily available, but there is

need to unveil their potential antifungal activities. The antifungal effect of chitosan, a

deacetylated form of chitin, on the growth of Aspergillus spp. and the aflatoxin

production was evaluated by many researchers [64, 146, 147]. A great inhibitory effect of

chitosan against Aspergillus parasiticus was found to be 3.0-5.0 mg.mL-1

. Moreover, 4.0-

5.0 mg.mL-1

of chitosan could completely prevent aflatoxin production by A. parasiticus

[64].


29

Objectives of Present Work

Main objectives of these studies are manipulation of chitosan for biomedical applications

as narrated below:

Irradiation of crab shells chitosan at different doses of gamma radiations.

Preparation and characterization (structure, morphology, water absorption

capacity, mechanical, water barrier, and surface properties) of novel

biocompatible membranes from chitosan.

Antimicrobial studies of chitosan and chitosan membranes for biomedical

applications.

Applications of the prepared membranes for controlled release of drug(s).

2. Materials and Methods

2.1. Polymers and chemicals used in the study

Variable molecular weight chitosan (CHIC; Mv 220000, CHI25; Mv 801, CHI50; Mv

473 and CHI75 Mv 259 g.mol-1

) were indigenously produced from crab shells chitin

which was obtained from fishery waste, Karachi, Pakistan. The extraction of chitosan

from chitin was performed following standard procedures. A chitosan; CHI15 (DDA

98.0% and Mw 15 kDa) was procured from Kunpoong Bio. Co. Ltd. (South Korea) and

PVA (degree of hydrolysis 98.0-98.8 mol%, degree of polymerization ~4300, Mw 195

kDa) was purchased from Aldrich, Germany. The Basmati rice (Oryza sativa) were

acquired from local market of Pakistan. Tetraethylorthosilicate (TEOS, d 0.933 g.ml-1

)

was obtained from Fluka, Germany. Glycerol (98% reagent grade) from Fisher Scientific

(Pittsburg, PA) and biological grade insulin (Bovine pancreas) was obtained from Sigma

Aldrich. All other chemicals including yeast extract, sucrose, chloroform, acetonitrile,

ethanol, Tween80, KH2PO4, CH3COOH (99.7%), NaCl, BaCl2, NaOH, HCl,

ZnSO4.7H2O, CuSO4.5H2O, MgSO4.7H2O were of analytical grade. The distilled water

was used throughout the experiments.

2.2. Microbial strains used in the study

Following bacterial and fungal strains were obtained from NIBGE culture collection and

used in experiments.

Pseudomonas aeruginosa (SS29)

Escherichia coli (SS1, SS2, SS9)


31

Proteus mirabilis (SS77)

Staphylococcus aureus (LM15)

Bacillus subtili

Aspergillus flavus

2.3. Growth media and maintenance of microbial cultures

2.3.1. Liquid and solid media for the growth of bacteria

The commonly used medium for the growth of bacteria is Luria-Bertani (LB) broth [148].

The medium was prepared according to the following composition in 100 mL.

1. Tryptone (1 g)

2. Yeast Extract (0.5 g)

3. Sodium Chloride (1 g)

For solid medium, agar (2 g) was added to LB medium. The media were prepared

in distilled water and the pH was adjusted approximately at 7±0.5 and sterilized by

autoclaving at 121°C temperature and 15 psi pressure for 15 min. The solution was

cooled down to room temperature before inoculation. For solid medium, the solution was

cooled down to 40°C before pouring into sterilized Petri plates. The Petri plates were

kept safe for further use after solidification of the medium.

2.3.2. Liquid and solid media for the growth of fungus

The general medium used for the growth of Aspergillus flavus is Yeast Extract Sucrose

(YES). The medium was prepared according to the following composition in 100 mL.

1. Yeast Extract (2 g)

2. Sucrose (1.5 g)


32

3. ZnSO4.7H2O (0.001 g)

4. CuSO4.5H2O (0.0005 g)

5. MgSO4.7H2O (0.05 g)

For solid medium, agar (2 g) was added to YES medium. Yease extract sucrose

media were prepared by adding the above mentioned chemicals into small amount of

distilled water, after dissolution, volume was made up to 100 mL, its pH was

approximately 6.3±0.2 and sterilized by autoclaving at 121°C temperature and 15 psi

pressure for 15 min. The solution was cooled down to room temperature before

inoculation. For solid medium, the solution was cooled down to 40°C before pouring into

sterilized Petri plates. The Petri plates were kept safe for further use after solidification of

the medium.

All the bacterial and fungal cultures were refreshed regularly in liquid and solid

media as described above at required physical conditions. The growth was also monitored

under phase contrast microscope (Zeiss MC 80, Germany). The fresh culture was used

always as inocula throughout the studies.

2.4. Extraction of chitin and chitosan from crab shells

The procedure for the extraction of chitosan was followed as described by Rege and

Block (1999). The crab waste was washed with water to remove dust particles and soaked

in HCl (2N) with 1:10 ratio for 24 hours to remove the mineral salts. The crab shells were

then removed by filtration and subsequent filtrate was mixed with NaOH (1N) as 1:10

ratio for further 24 hours to remove proteins. The obtained chitin was subjected to

deacetylation process by soaking chitin in NaOH (50%) and refluxed for 10 hours. The

obtained chitosan was dried and stored for use in experiments.


33

2.4.1. Gamma irradiation of chitosan

Chitosan solution (2% in acetic acid) was mixed with hydrogen peroxide (1%) and

irradiation was performed at ambient temperature in air at Pakistan Radiation Services

using 60

Co gamma irradiater (Model JS-7900, IR-148 ATCOP) at dose rate of 1.02

kGy.hour-1

at 25 kGy, 50 kGy and 75 kGy (Fig. 2-1). The chitosan irradiated at different

doses were extracted from solution and used for further studies.

Fig. 2-1. Extraction of chitin, chitosan and gamma irradiation.

2.4.2. Extraction of chitosan from gamma irradiated chitosan solutions

The pure chitosan was recoverd from individual gamma irradiatated chitosan solution

following steps (Fig. 2-2) described as below:


34

i) The irradiated chitosan solution (900 mL) was mixed with concentrated NH3 solution

(30 mL) for overnight. The chitosan residues were isolated by filtration and washed with

distilled water untill a neutral pH was attained. Chitosan precipitaes were vaccum dried at

50-60ºC and preserved for analysis while filtrate was saved for further precipitation of

chitosan.

ii) The above filtrate was mixed with 50 mL of NaOH (3M) solution for further 24 hours.

The precipitates were seprated by filtration and washed with distilled water untill

neutralized. The washed precipitaes of chitosan were vaccum dried at 60ºC and saved for

analysis while filtrate was saved to remove water soluble chitosan from it. Un-irradiated

chitosan and chitosan irradiated at different doses will be referred to as CHIC, CHI25,

CHI50 and CHI75, respectively.

Fig. 2-2. Isolation of chitosan powder from gamma irradiated chitosan solution.


35

2.5. General procedure for preparations of chitosan/PVA

membranes

Chitosan was dissolved in acetic acid (0.5M) to prepare 2% (w/v) chitosan solution.

Poly(vinyl alcohol, PVA) was separately dissolved in deionized water at 80oC to make

4% (w/v) solution. Both the solutions were mixed with few drops of glycerol. The

mixture was stirred thoroughly for 30 min. at 55-60oC. The crosslinker;

tetraethylorthosilicate (TEOS) was then added drop wise to the above mixture (Fig. 2-3).

After vigorous stirring of about an hour, the resulting mixture was poured into plastic

dishes and kept at room temperature for drying in dust free environment. The dried films

were removed, rinsed with deionized water to eradicate any foreign particles, vacuum

dried at 60°C and stored in desiccator. All formulations contained fixed mass ratio of 5

CHI : 95 PVA. The tetraethylorthosilicate solution for crosslinking (6%) the membranes

was prepared separetly by mixing 3-5 mL of ethanol (99.9%) in 10 mL measuring flask.

To this added required volume of TEOS and a drop of HCl (3M). Afterwards, volume

was made up to mark with ethanol and used dropwise during membrane preprations. The

membranes prepared from un-irradiated chitosan will be refferd as MemC while with

chitosan irradiated at 25, 50 and 75 kGy as Mem25, Mem50 and Mem75 respectively.

OEt

OEt

OEt

OEt

OH

OH OH

OH Hydrolysis/Acid

Si Si + 4C2H4OH


36

Fig. 2-3. Flow sheet for the preparation of chitosan/PVA membranes.

2.6. Characterization of chitosan and chitosan membranes

2.6.1. Infrared spectroscopy (IR)

The IR spectra of all chitosan and membranes were recorded in the range of 4000 to

400cm-1

using Fourier Transform Infrared (FTIR) spectrophotometer (Thermo Electron

Corp., Nicolet 6700, Waltham, Massachusetts, USA) at room temperature. The reported

spectra were recorded with 200 scans at 6.0cm-1

resolution using an attenuated total

reflectance (ATR) technique with a diamond crystal tip.

2.6.2. Determination of viscosity average molecular weight (Mv) of

chitosan

Molecular weights of un-irradiated and irradiated chitosan were determined using

Ubbelohde viscometer [35]. The sample solutions were prepared by adding 0.25 g of


37

chitosan powder in 15-20 mL of acetic acid (0.5M), dissolved untill clear solution was

obtained and added 0.3662 g of NaCl (solid) to above solution. After complete

dissolution of solutes, volume was made upto 25 mL by acetic acid (0.5M). For reference

solution, only 0.3662 g of solid NaCl was added to falsk and volume was made upto 25

mL by acetic acid (0.5M).

To determine the rate of flow of solutions, the viscometer was washed well and

dried before the start of experiment. Average of 5 values was taken at 25ºC for samples

and reference solutions. The Mv was determined according to the following equation.

Mvα = [η]/k (2-1)

Where,

„Mv‟ is viscosity average molecular weight of sample

[η] = [2(η sp – ln η r)]1/2

/Conc.(g.mol-1)

η r = t/to

η sp = η r – 1

„t‟ is time flow of sample solution (Sec)

„to‟ is time flow of reference solution (Sec)

„k‟ is constant (2.14x10-3

)

„α‟ is constant (0.657)

2.6.3. Antibacterial study of variable molecular weight chitosan using

agar well diffusion method

A modified agar well diffusion method [119] was used to evaluate qualitative

antibacterial activity of un-irradiated and irradiated chitosan. Antibacterial activity was

performed against few pathogen bacteria i.e., Pseudomonas aeruginosa (SS29)

Escherichia coli (SS1, SS2, SS9), Proteus mirabilis (SS77), Staphylococcus aureus

(LM15) and Bacillus subtilis. Chitosan solutions (2, 1, 0.5, 0.25%, w/v) were prepared in

acetic acid (0.5M) and pH was adjusted at 5.0-5.5 before pouring into wells. Then 100 µL

of un-irradiated and irradiated chitosan solutions at different concentrations were poured


38

into wells (7 mm diameter) on agar plates, which were previously seeded with 20 µL of

inocula containing approximately 105-10

6 CFU.mL

-1. The plates were then incubated at

37oC for overnight and, afterwards, examined for width of inhibition zones. The clear

zone of inhibition was calculated by Vernier calipers which included well‟s diameter.

The inhibitory zones surrounding the wells were compared with reference to the effect of

molecular weight and irradiation dose. Experiments were performed and repeated at least

for three times.

2.6.4. Determination of minimum inhibitory concentration (MIC) and

50% inhibitory concentration (IC50) of chitosan against drug resistant

bacteria

For quantitative assay, MIC of chitosan was assessed using a dilution method [13]. The

MIC is the minimum sample concentration which completely inhibits the growth of

bacteria while the concentration which inhibits 50% bacterial growth is called IC50. To

determine the MIC and IC50 of chitosan samples, the bacterial cultures were freshly

grown in Luria Broth (LB). The corresponding bacterial culture was diluted with sterile

LB media with ratio 1:4 in sterile glass tubes and thoroughly blended for homogeneous

bacterial suspension. Inoculated media (200 µL) were distributed in sterile 96 wells plate

and 100 µL dilution of each chitosan concentration was added in the wells. The final

chitosan concentrations in mixture obtained were 1.20, 0.66, 0.32, 0.25, 0.16, 0.08, 0.06,

0.04, 0.02 and 0%. The plates were incubated at 37oC for 18-20 hours. The same

procedure was adopted for all bacterial strains. The micro titer plate was visually

observed for MIC of the chitosan. For exact calculation of MIC and IC50, the culture was

further plated (after appropriate dilution) on agar plates for counting CFU.mL-1

of

bacteria with each concentration of chitosan. The initial bacterial count for inoculation

was maintained at ~106-10

7CFU.mL

-1. The experiments were performed in triplicate and

repeated for at least three times.


39

2.6.5. Metabolite analysis of Pseudomonas aeruginosa SS29

The effect of chitosan on metabolic profiling of Pseudomonas aeruginosa was

determined. As Pseudomonas aeruginosa is highly pathogenic among other bacteria so

was used for metabolite analysis. For this purpose, chitosan from Kunpoong Bio.

(CHI15) was mixed with acetic acid to give different concentrations (6, 4, 2, 1, 0.5, 0.25,

0.125 and 0%). The bacterial growth in the absence and presence of chitosan was

conducted as described above (section 2.6.4). The bacterial mass was isolated from

metabolites by centrifugation and 20 mL of chloroform was then added to the above

filtrate for 12-14 hours with shaking (120rpm) at room temperature. Afterwards, extract

was separated and concentrated by rotary evaporator. The concentrates were then added

to acetonitrile (1 mL) and afterwards metabolic analyses were conducted on liquid

chromatography mass spectrometry.

2.6.6. Culture and inoculum preparation for antifungal studies of

chitosan (CHI50)

The Aspergilllus flavus was grown in YES medium in a flask with stirring at 28ºC and

afterwards spread on YES agar medium. The plates were kept for 5-7 days at 28ºC

temperature for the growth of fungus untill sporolation occured. For the preparation of

inoculum, 1 mL of each of chitosan concentration was taken separately into eppendorfs

tubes. After adding small pallet of spores and two drops of Tween80 to above solution,

shaked vigorously for 1-2 min and counted the number of spores.mL-1

(Zeiss MC 80,

Germany). Final number of spores in each flask were in the range of 1.1-1.9x107

spores.mL-1

.

2.6.6.1. Minimum inhibitory concentration of CHI50 against Aspergillus flavus

The minimum inhibitory concentration of CHI50 against Aspergillus flavus was

determined according to method applied to filamentous fungi (Clinical and Laboratory

Standard Institute., 2008). For this purpose, chitosan (CHI50) solutions of different


40

concentrations (2, 1.75, 1.625, 1.50, 1.37, 1.25, 1 and 0%) were prepared in acetic acid

(pH-5). For determination of MIC, the inoculum was prepared as described above

(section 2.6.6) and 1 mL of inoculum was added to wells tube. Then added 100 uL of

each chitosan solution to the above inoculum and whole assembly was gently mixed and

incubated at 28ºC temperature for 7 days for the growth of fungus. The experiment was

carried out in triplicate and MIC was determined by visual inspection of the wells.

2.6.6.2. Production of aflatoxins on rice by Aspergillus flavus

A modified method [149] was used for the production of aflatoxins by Aspergillus flavus

on rice. For our study, 5 g of Basmati rice were taken in Erlenmeyer flask and soaked in 2

mL distilled water for two hours and were autoclaved. The inoculum with chitosan

(CHI50) solution (1.75, 1.50, 1 and 0%) was prepared as described earlier (section 2.6.6)

and added separately into flasks and incubated at 28oC with shaking (300rpm) for 120

hours. During incubation, the moisture content inside the flasks was maintained by

adding 100 µL sterile water, not enough to cause individual rice kernels to adhere to each

other. After first 50 hours of incubation, flasks were covered with aluminium foil and

incubated at 26oC without shaking for further 70 hours. During incubation, the growth of

Aspergillus flavus was observed and documented at regular time intervals till it was

constant. All experiments were done in triplicate and under extremely aseptic conditions.

2.6.6.3. Extraction of aflatoxins

Most of the analytical methods require the extraction of analytes from solid samples into

liquid phase which termed as liquid-solid extraction. Organic solvents like chloroform,

methanol, ethanol, acetone and acetonitrile were used for extraction. Solvent and sample

were mixed by blending or shaking. Special care was taken to ensure that the entire

sample was in intimate contact with extraction solvent. For the extraction of aflatoxin and

metabolites, 25 mL of chloroform were used for each sample. Each sample was made to

stand for 12-14 hours with shaking (120rpm) at room temperature. Afterwards, extract

was filtered through Whatmann No.1 (Maidstone, UK) filter paper and chloroform was


41

evaporated by rotary evaporator. The residues were then dissolved in acetonitrile (1 mL)

and were kept at -20oC in an amber glass vial for analysis on LC-MS/MS.

2.6.7. Liquid chromatography mass spectrometric (LC-MS) analysis of

aflatoxins (Aspergillus flavus)

The LC-MS/MS (liquid chromatography with mass spectrometer from Thermo Fisher

Scientific, Waltham (MA), USA) was used with a) Finnigan Surveyor Autosampler Plus;

b) Finnigan Surveyor LC Pump Plus; c) Finnigan Surveyor MS Pump Plus; d) Finnigan

Surveyor PDA Plus Detector. e) Finnigan LTQXL a Linear Quadrapole Ion Trap MS/MS

with Electrospray Ionization (ESI) Probe and Atmospheric Pressure Chemical Ionization

(APCI) Probe; f) Analytical column: C18 Luna (250x4.6 mm, 3 µm) Phenonomenex

Thermo Scientific; USA.

2.6.7.1. Operational standard solution for calibration

Stock solutions for individual reference standards; B1, G1, B2 and G2 were prepared (1

mg/1 mL = 1000 ppm) and preserved at 4oC. Working solutions were prepared from

stock solution as 2.5, 5, 10, 20, 40, 80 (ppb) for B1 and G1 and 0.75, 1.5, 3, 6, 12, 24

(ppb) for B2 and G2 in acetonitrile and stored in refrigerator. These standards were used

for calibration in order to determine the response versus the injected amount of analyte.

The linearity of the instrument was checked by means of line regression.

2.6.7.2. Determination and quantification of aflatoxins by LC-MS

The high performance liquid chromatography (HPLC) separates the mixture of

compounds by relative retention of compounds on a stationary and mobile phase (mixture

of polar solvents). Afterwards, the compounds pass through a detector normally photo

diode array (PDA), fluorescence, or MS detectors. The analyses of the cleaned extracts

were carried out using Surveyor HPLC system equipped with mass spectrometer and

PDA plus detectors (Thermo, USA).


42

The system was validated for analysis with known standards individually and in

the form of mixture. The analyses were performed in triplicate using luna phenomenex

C18 column (150x4.6 mm, 3 µm), in isocratic mode. Before LC-MS/MS analysis both of

the components LC and MS were optimized in prior to performing the real sample

analysis. Each of these components can be independently used and optimized. In the

current study, mass spectrometer with LC has been used. An analytical method has been

generated after getting the optimization of each component of the LC-MS/MS. The

optimization parameters for each component are given below.

2.6.7.3. Mass spectrometry parameters

Two types of analyses for aflatoxins were performed on LC-MS/MS. In the first type of

experiments, samples and standards were injected directly to the mass spectrometer using

syringe pump. While in second type of experimentation, samples and standard have to

pass through HPLC system before going to MS. For each type of these experimentations

different parameters on MS have been set, which are given below.

Following parameters for direct insertion of aflatoxins samples/standards to MS

were used. By employing these parameters, MS was tuned involving automatic tuning

process by selecting molecular masses of each mycotoxin. Aflatoxin ions at positive

modes [M+Na]+ have m/z values as: B1 m/z=335; G1 m/z=351; B2 m/z=337; G2

m/z=353. Following are the ESI-MS/MS conditions for aflatoxins through direct

insertion pump.

Syringe pump speed: 3-10 µls-1

Source voltage: 5.0 kV

Sheath gas flow: 15 (arb)

Aux gas flow: 2 (arb)


43

Capillary temperature: 275ºC

Capillary voltage: 49 V

Tube lens voltage: 110 V

2.6.7.4. Tuning

The liquid chromatograph and mass spectrometer (LC-MS) system was tuned-up for

aflatoxins before starting their study to optimize the analysis procedure which includes

ESI source parameters, voltage for ion optics and MS parameters. Probe position was set

manually to the point where maximum intensity value of ion signal with least variation

was achieved. Sheath and auxiliary gas flow rates were subjected to get stable spray.

Voltages of ion optics were optimized automatically and semi-automatically to get

focusing of particular analyte ions (AFB1, AFG1, AFB2, AFG2) towards analyzer. In

order to get maximum signal strength of aflatoxins in MS and MSn mass spectra, its

parameters such as microscans, maximum injection time, ion isolation width and

collision energy were optimized. When tuning has been achieved the tune file was saved

and used for the analysis of aflatoxins.

2.6.7.5. LC-MS conditions

There is lot of difference in conditions for HPLC when it is hyphenated with MS detector

as compared to the system where HPLC is linked with other detectors such as FLD or

PDA detectors. Because many of the parameters like sample loading amount, type of

mobile phase, flow of mobile phase, position of the ionization probe, flow of

sheath/auxillary gases, temperature of ion transfer capillary have to be changed

drastically in case if MS detector along HPLC are being applied. Following are the LC-

MS conditions for aflatoxins.

Injection volume: 10 µl

Needle height from bottom: 2 mm

Syringe speed: 8 µls-1


44

Flush volume: 1000 µls-1

Wash volume: 2000 µl

Flush speed: 500 µls-1

Loop loading speed: 10 µls-1

Column temperature: 30ºC

Column: C18 Luna (250x4.6mm, 3 µm)

Phenomenex Thermo Scientific

Surveyor LC pump Flow rate: 0.5 mLmin-1

Run time: 25 min

Mobile phase. Combination of mobile phases at different ratios was used during

optimization of good resolution of all types of aflatoxins in LC. However, the best

possible combination of these parameters have yielded the retention times of AFB1,

AFG1, AFB2, AFG2 at 18.14, 15.29, 13.89, 11.87 mins respectively and their ion

detection on MS. Mobile phase used was methanol:acetonitrile:water (22.5:22.5:55.0

v/v). Following are the MS conditions.

Source type: ESI

Capillary temperature: 335ºC

Sheath gas flow: 70 (arb)

Aux gas flow: 20 (arb)

Source voltage: 5 kV

Capillary voltage: 49 V

Tube lens voltage: 120 V

Concentration of aflatoxins (B1, G1, B2, G2) was calculated by the following formula:

Conc. in samples = [conc. in standard × peak area of samples]

peak area of standard samples

For standard curve linear equation was adopted: Y = MX+C (2-2)


45

2.6.7.6. LC-MS conditions for metabolites

The mobile phase used for the analysis of metabolites was same as used for aflatoxin

analysis. Following are the MS conditions for the metabolites analysis.

Source type: ESI

Spray Voltage: 4.02 kV

Spray Current: 0.13 µA

Sheath Gas Flow Rate: 19.01 (arb)

Aux Gas Flow Rate: 1.99 (arb)

Capillary Voltage: 19.01 V

Capillary Temp: 287.27°C

Tube Lens: 110.10 V

Flow Rate : 10.00 µl/min

Syringe Diameter: 2.30 mm

2.6.8. Morphological analysis of membranes

The morphology of chitosan/PVA membranes surfaces were examined using a model

JSM-7500F Field Emission Scanning Electron Microscope (Jeol, Japan) and ordinary

phase contrast microscope (Zeiss MC 80, Germany). The membranes were prepared by

freeze-drying samples after swelling in distilled water. The images were examined at

different magnifications. The surface of dried chitosan membranes were also imaged

using atomic force microscope using a Commercial Nanoscope IIIa Multi-Mode AFM

(Veeco Instruments, USA) by tapping mode in air. The topographies were attained at the

scanning size of 3µm and scanning frequency of 1.2Hz (data collection at 512×512

pixels) by silicon nitride cantilever. The membranes for analysis were selected as

chitosan/PVA without crosslinker and with crosslinker.


46

2.6.9. Water absorption studies of membranes

Small piece of membranes were cut, weighed and immersed in deionized water (30 mL),

until the equilibrium was reached. The swollen membranes were removed and extra

water was carefully wiped out. The content of water absorbed in membranes was

calculated as:

WAC (%) = [(Ws-Wd) / Wd)] x 100 (2-3)

Where “Wd” is the weight of dry membrane and “Ws” is the weight of swollen

membrane at equilibrium (Kim et al., 2003b). The results were reported as average of

three readings.

The water absorption capacity of membranes prepared with variable molecular

weight chitosan and PVA were also investigated at different temperature conditions

(28ºC, 37ºC, 50ºC), pH conditions (2-8) and different concentrations (0.2, 0.4, 0.6, 0.8,

1mol.L-1

) of salt solutions (NaCl, BaCl2).

2.6.10. Mechanical properties of membranes

The tensile strength (TS) and elongation at break (EB) of the membranes were measured

on a TA.XT2 Texture Analyzer (Texture Technologies, New York, USA) at a speed of

0.1 mm.sec-1

. The appropriate sized membranes (1.0x10.0 cm) were cut using a razor

blade and the gauge length was set at 10.0 mm. For each sample, the measurements were

replicated by four times.

2.6.11. Water barrier properties of membranes

The water vapor transmission rate (WVTR) and water vapor permeability (WVP) of the

membranes were analyzed according to ASTM Method E96/E96M. Briefly, circular test


47

cups were filled with calcium chloride (10.0±0.5 g) as desiccant at 0% relative humidity

(RH), and sealed with test membranes. The membranes were tightly attached and the

initial weights of the cups were recorded. The cups were placed in an environmental

desiccators set at 25oC and 53% RH. After reaching at equilibrium state in the

desiccators, cups were weighed daily up to 14 days. The WVTR was calculated as the

slope of the regression line drawn between elapsed time and the weight change of test

cups. The actual WVTR and WVP of the membranes were calculated according to the

following equations:

WVTR = (G/t) / A

WVP = WVTR /ΔP = WVTR/S(R1-R2) (2-4)

Where “G” is weight change (g), “t” is time (hrs), “A” is the test area (m2), and

“ΔP” is vapor pressure difference (Pa), “S” is saturation vapor pressure at test

temperature, mmHg (1.333x102Pa).

2.6.12. Contact angle measurements of membranes

It quantifies the wettability of a solid surface by a liquid. The static water contact angle

measurements of the membranes were carried out at room temperature (25oC) by drop

method using a VCA Optima XE Dynamic Contact Angle Analyzer (AST Products Inc.

Billerica, MA). The values for contact angle (θ) were calculated using Young‟s equation

(Fig. 2-4). The rate of change of surface wettability was taken at different points on the

membranes. A CCD camera recorded the images, immediately after the water drop was

deposited onto the membrane surface. Each reported value of contact angle was the mean

value of 10 measurements.

Ɣsv

= Ɣsl + Ɣ

lvCOSθ (2-5)


48

Fig. 2-4. Liquid drop on surface for measuring contact angle.

2.6.13. Antibacterial study of chitosan membranes by membrane’s disc

diffusion and optical density (OD) method

Antibacterial activities of chitosan membranes were performed by placing small pieces of

membranes on preoculated agar plates and also embeded in semi solid LB agar media.

Then these plates were incubated at 37oC for 18-20 hours. The clear zones of inhibition

were calculated for qualitative analysis. The antibacterial activities of the chitosan

membranes were also performed according to the method described by Zhai et al. [117]

with slight modifications. Due to curling nature of membrane on absorption of moisture,

these were embedded in semi-solid media before complete solidification and then

inoculated with corresponding bacterial culture. Chitosan membranes were washed with

sterile distilled water before the experiment to remove any foreign agents. Each

membrane was weighed (50mg±0.2 mg) and added into test tubes having nutrient broth

along with bacterial culture (0.5%) containing approximately 105-10

6 CFU.mL

-1. The

culture tubes were incubated overnight at 37oC with shaking (150rpm). During

incubation, the turbidity of the medium was measured at 600nm after regular time

intervals till it was constant. All experiments were done in triplicate under aseptic

conditions. Moreover, in order to determine the effect of bacterial growth phase on the


49

antibacterial activity of the chitosan membranes, the bacterial cultures were evaluated at

two growth phases i. e. stationary phase and log phase.

2.7. Controlled drug release by membranes

2.7.1. Preparation of buffer solutions

Simulated gastric fluid (SGF) at pH=1.2 was prepared by mixing 1 g of NaCl in 3.5 mL

of HCl (37%) and diluted up to 500 mL with distilled water. Simulated intestinal fluid

(SIF) at pH=6.8 was prepared by mixing 250 mL of KH2PO4 (0.2M) with 118 mL of

NaOH (0.2M) (Islam and Yasin, 2012).

2.7.2. Preparation of standard solutions for insulin

A 10 ppm stock solution of insulin was prepared by dissolving 1 mg of insulin in 100 mL

of solvent. The solvent system used for insulin solubility consists of methanol, conc. HCl

(2-3 drops) and NH3SO4 solution (for neutralization). For calibration line, the solutions at

2, 4, 6, 8 and 10ppm were prepared from stock using methanol as dilution solvent. The

amount of released insulin was determined by a Perkin Elmer Lambda 40 UV-Vis

spectrophotometer at 276nm and calibration line was drawn by absorption values versus

insulin concentration.

2.7.3. Loading of insulin on membrane and its release analysis

The dried sample (~30 mg) was loaded by 20 mL of commercially available insulin

solution of 10ppm (0.01 mg.mL-1

). The insulin solution was added drop wise on pre

swollen membrane. The membranes showed swelling in the aqueous solvent system and

drug was loaded by the process of sorption. These membrane samples were dried in dark

conditions. The drug loaded membrane sample was placed in a beaker at 37°C containing

100 mL of SGF solution. After 2 hours, it was transferred to SIF solution at same


50

temperature for 4 hours. Aliquots of 3 mL were taken from beaker for analysis after every

30 min. and 3 mL of fresh solutions (SIF, SGF) was inserted back into the beaker to

make up the liquid volume. The concentration of the released insulin was determined by

spectrophotometer using a Perkin Elmer Lambda 40 UV–Vis spectrophotometer at

276nm. The simulated gastric fluid and simulated intestine fluid were used as reference

standards. Calibration line was drawn using insulin solutions ranging from 2 ppm to 10

ppm. The amount of insulin released by the membrane (Mem25) was determined using

calibration line. The Mem25 was chosen for drug release study because this membrane

has significant water absorption and larger pore size. So that insulin can adsorb in

membrane pores in dry state and release as it swell in simulate gastric fluid.

3. Results

3.1. Gamma irradiation of chitosan

Chitosan obtained from crab shells was subjected to gamma radiations at different doses

and its effect was evaluated by determining viscosity average molecular weight (Mv) of

chitosan. The results show that the viscosity average molecular weight of chitosan

decreased with increasing dose of gamma radiations (Table 3-1). The Mv and codes of

chitosan and membranes prepared from un-irradiated and irradiated chitosan blending

with poly(vinyl alcohol, PVA) are given in Table 3-1.

Table 3-1. Viscosity average molecular weight of chitosan and membranes

composition.

Irradiationa

Chitosanb

Mvc (g.mol

-1)

Membranesd

No irradiation

CHIC

220000

MemC

25kGy

CHI25

801

Mem25

50kGy

CHI50

473

Mem50

75kGy

CHI75

259

Mem75 a irradiation dose b chitosan powders c viscosity average molecular weight of chitosan d chitosan:PVA- 5:95

3. Results

52

3.2. Fourier Transform Infrared (FTIR) characterization of

chitosan and membranes

The infrared (IR) spectra of the un-irradiated and irradiated chitosan recorded in solid

state, showed the overlap stretching of hydrogen bonded -OH and -NH2 band ranging

from 3070-3450cm-1

(Fig. 3-1). The stretching of C-H bond appeared at 2877cm-1

. In

addition, a band appeared at 870cm-1

representing pyranose ring of chitosan, while small

bands also appeared at 1050 and 1384cm-1

due to C-O-C stretching and methyl groups

respectively.

Fig. 3-1. Infrared spectra of un-irradiated chitosan (CHIC) and irradiated chitosan

(CHI25, CHI50, CHI75) (4000-500cm-1

).

However the IR spectra of the poly(vinyl alcohol) and chitosan/PVA membranes

revealed peaks at 1044-1100cm-1

representing siloxane linkage Si-O- in membrane form.

Also, there was a band with higher intensity at 3290cm-1

for chitosan membrane (Fig. 3-

2).

3. Results

53

Fig. 3-2. Infrared spectra of PVA, Mem25 and Mem75 (4000-500cm-1

).

3.3. Antibacterial activity of chitosan using agar wells diffusion

method

The antibacterial property of variable molecular weight chitosan was tested against

pathogenic bacteria which were initially isolated from human wound infections in the

Faisalabad region of Pakistan. For this purpose, agar wells diffusion method was adopted

to check the antibacterial activities of variable molecular weight chitosan at different

concentrations (2, 1, 0.5 and 0.25%). The antibacterial activity was conducted against

some of the Gram’s negative bacteria; Pseudomonas aeruginosa (SS29), E. coli (SS1,

SS2 and SS9), Proteus mirabilis (SS77) and Gram’s positive bacteria; Staphylococcus

aureus (LM15) and Bacillus subtilis. Antibacterial activity was assessed by pouring 100

µL of different molecular weight chitosan solutions with different concentrations into

wells on agar plates which were previously inoculated with corresponding culture. Un-

irradiated chitosan (CHIC) of high molecular weight showed no antibacterial activity

against Pseudomonas aeruginosa (SS29) at any concentration of chitosan (Fig. 3-3).

While, chitosan irradiated at different doses of gamma radiations (CHI25, CHI50,

3. Results

54

CHI75) showed zone of bacterial inhibition. Among irradiated chitosan, CHI75 had

larger zone of bacterial inhibition as compared to CHI50 and CHI25 at all concentrations

(Fig. 3-3).

Fig. 3-3. Bioactivity of chitosan against Pseudomonas aeruginosa (SS29) at

concentrations 2% (a), 1% (b), 0.5% (c) and 0.25% (d).

Similarly, antibacterial activities of chitosan against E. coli (SS2) were slightly

higher for CHI75 as compared to CHI50 and CHI25 (Fig. 3-4). Also, CHI75 and CHI50

had antibacterial activities against E. coli (SS9) only at higher concentrations (Fig. 3-5).

Both of the E. coli (SS2 and SS9) showed no effect on growth in the presence of CHIC.

The antibacterial test of chitosan against Proteus mirabilis (SS77) and

Staphylococcus aureus (LM15) showed evident growth inhibition near to the edges of

agar wells, while bacterial growth seemed to be reduced at a distance from the edges

(Figs. 3-6 and 3-7). However, CHI75 and CHI50 had almost equivalent antibacterial

efficiency against Proteus mirabilis (SS77) and Staphylococcus aureus (LM15). Overall,

the results indicated that antibacterial activity of chitosan was developed and improved

by increasing irradiation dose on chitosan. The calculated zone of inhibition also include

3. Results

55

wells diameter (~7 mm). The clear zones of bacterial growth inhibition by all types of

chitosans at different concentrations were calculated for comparison (Fig. 3-8).

Fig. 3-4. Bioactivity of chitosan against E. coli (SS2) at concentrations 2% (a), 1%

(b), 0.5% (c) and 0.25% (d).

Fig. 3-5. Bioactivity of chitosan against E. coli (SS9) at concentrations 2% (a), 1%

(b), 0.5% (c) and 0.25% (d).

3. Results

56

Fig. 3-6. Bioactivity of chitosan against Proteus mirabilis (SS77) at concentrations

2% (a), 1% (b), 0.5% (c) and 0.25% (d).

Fig. 3-7. Bioactivity of chitosan against Staphylococcus aureus (LM15) at

concentrations 2% (a), 1% (b), 0.5% (c) and 0.25% (d).

3. Results

57

SS29 SS1 SS2 SS9 SS77 LM15 B.subtilis

0

5

10

15

20

25

30

35

40

Zo

ne o

f in

hib

itio

n (

mm

)

CHIC

CHI25

CHI50

CHI75


0

5

10

15

20

25

30

35

40

Zo

ne o

f in

hib

itio

n (

mm

)

CHIC

CHI25

CHI50

CHI75


0

5

10

15

20

25

30

35

40

Zo

ne

of

inh

ibit

ion

(m

m)

CHIC

CHI25

CHI50

CHI75


0

5

10

15

20

25

30

35

40

Zo

ne

of

inh

ibit

ion

(m

m)

CHIC

CHI25

CHI50

CHI75

Fig. 3-8. Zone of bacterial growth inhibition by chitosan (CHIC, CHI25, CHI50, CHI75) concentrations at 2% (a), 1% (b),

0.5% (c) and 0.25% (d) against SS29 (Pseudomonas aeruginosa), SS1,SS2,SS9 (Escherichia coli), SS77(Proteus mirabilis), LM15

(Staphylococcus aureus) and Bacillus subtilis

a b c d

3. Results

58

3.3.1. Determination of minimum inhibitory concentration (MIC) and

50% inhibition concentration (IC50) of un-irradiated and irradiated

chitosan against drug resistant bacteria

The quantitative antibacterial activities of un-irradiated chitosan (CHIC) and irradiated

chitosan (CHI25, CHI50 and CHI75) were determined by calculating their MICs and IC50

against pathogenic bacteria (section 2.2). The MIC and IC50 of chitosan (CHI15) were

determined against only highly opportunistic Pseudomonas aeruginosa (Table. 3-2). The

results showed that MIC and IC50 of un-irradiated chitosan (CHIC) was indicated only

against B. subtilis as all other tested bacteria had no effect of CHIC on their bioactivities

(Table. 3-2). On the whole, CHI75 and CHI50 had lower MIC and IC50 against

Pseudomonas aeruginosa, E. coli and B. subtilis as compared to CHI25. The MIC and

IC50 values of CHI15 were much higher against Pseudomonas aeruginosa (Table. 3-2).

3.3.2. Analysis of metabolites of Pseudomonas aeruginosa

The analyses of metabolites produced by Pseudomonas aeruginosa in the absence of

chitosan and in the presence of different concentrations of chitosan (CHI15) were

conducted using liquid chromatography mass spectrometry (LC-MS). The analysis

showed the presence of some important peaks which were related to hydroquinolones.

The peak intensities were reduced in metabolite analysis when chitosan was added in

growth medium of Pseudomonas aeruginosa. The overall peak intensities gradually

decreased with the addition of increased concentration of chitosan (Fig. 3-9a-d). Also,

hydroquinolones and hydroquinolones related peaks were confirmed by their mass

spectral (MS/MS) analysis. The MS/MS analysis of hydroquinolones and related

compounds along with their structures (Fig. 3-10a-c) confirmed the production of

hydroquinolones. The results showed that production of hydroquinolones was decreased

in the presence of chitosan.

3. Results

59

Table. 3-2. The values (%) for MICs and IC50 of chitosan against human bacterial isolates.

Pathogenic bacteria

Different molecular weight chitosan

CHIC CHI25 CHI50 CHI75 CHI15

MIC IC50 MIC IC50 MIC IC50 MIC IC50 MIC IC50

P. aeruginosa (SS29)

NA

NA

0.66

0.0412

0.33

0.0206

0.33

0.0206

2.64

0.083

E. coli (SS1)

NA

NA

0.66

0.0412

0.33

0.0206

0.33

0.0206

ND

ND

E. coli (SS2)

NA

NA

1.32

0.0825

0.66

0.0412

0.66

0.0412

ND

ND

E. coli (SS9)

NA

NA

1.98

0.165

1.32

0.0825

0.66

0.0412

ND

ND

P. mirabilis (SS77)

NA

NA

1.32

0.0825

0.66

0.0412

0.66

0.0412

ND

ND

S. aureus (LM15)

NA

NA

1.98

0.165

0.66

0.0412

0.66

0.0412

ND

ND

B. subtilis

1.32

0.083

0.66

0.0206

0.33

0.0206

0.33

0.0206

ND

ND

NA= no activity

ND= not determined

3. Results

60

Fig. 3-9 (a). Mass spectra of Pseudomonas aeruginosa metabolites in the absence of chitosan.

Control-Arogenosa-Positive-Mode-24-9-13_24-9-13 #7 RT: 0.06 AV: 1 NL: 8.23E5T: ITMS + p ESI Full ms [50.00-2000.00]

200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

270.25

244.25272.25267.08

260.17

298.25288.25

286.25

258.25 283.08 328.17314.25300.25242.17 247.08 399.25381.17350.25330.33 373.25202.08 357.33211.17 232.17

NL: 8.23E5

3. Results

61

Fig. 3-9 (b). Mass spectra of Pseudomonas aeruginosa metabolites in the presence of 0.5% chitosan concentration.

0-25-Arogenosa-Positive130924144347 #1 RT: 0.00 AV: 1 NL: 7.33E5T: ITMS + p ESI Full ms [50.00-2000.00]

200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

260.17

270.17244.17

272.17

288.25298.25

314.25245.25330.25 355.42273.25

258.08 300.33 328.25282.08242.17 247.08 356.42 377.33312.25211.17 332.25 399.33342.17318.33 389.17230.17

NL: 7.33E5

3. Results

62

Fig. 3-9 (c). Mass spectra of Pseudomonas aeruginosa metabolites in the presence of 1% chitosan concentration.

1%-Arogenosa-Positive_25-9-13 #1 RT: 0.00 AV: 1 NL: 1.10E5T: ITMS + p ESI Full ms [65.00-2000.00]

200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

267.08

244.25

272.17260.17

288.33 301.25

211.08316.25283.08 291.08

349.25 391.33247.08 319.42 365.25333.08219.17203.00 306.25274.08 371.25242.17 399.08227.08

NL: 1.10E5

3. Results

63

Fig. 3-9 (d). Mass spectra of Pseudomonas aeruginosa metabolites in the presence of 4% chitosan concentration.

4%-Arogenosa-Positive_25-9-13 #1 RT: 0.00 AV: 1 NL: 1.88E5T: ITMS + p ESI Full ms [50.00-2000.00]

200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

319.25

301.25267.08 288.33244.17

326.33316.33

284.33245.17 333.25

272.25 349.17306.17381.33261.17233.08 393.33280.08 290.17 362.17 375.08 401.25338.25211.08 217.08

NL: 1.0E5

3. Results

64

Control-Arogenosa-MS-MS-Positive-Mode-24-9-13_130924131354 #131 RT: 0.88 AV: 1 NL: 1.06E4T: ITMS + p ESI Full ms2 [email protected] [65.00-500.00]

80 100 120 140 160 180 200 220 240 260 280 300

m/z

0

20

40

60

80

100

Re

lative

Ab

un

da

nce

159.00

172.08244.17

216.00146.00 188.08132.00 252.08106.00 280.0085.00

Fig. 3-10 (a). MS/MS analysis of hydroquinolone (mass 244.17) compound.

3. Results

65


80 100 120 140 160 180 200 220 240 260 280 300

m/z

0

20

40

60

80

100

Re

lative

Ab

un

da

nce

159.00

172.08

174.08240.08 258.08146.08

198.00 212.08132.08 275.92 294.00120.0081.08 96.92

Fig. 3-10 (b). MS/MS analysis of hydroquinolone (mass 258.2) compound.

3. Results

66


80 100 120 140 160 180 200 220 240 260 280 300

m/z

0

20

40

60

80

100

Re

lative

Ab

un

da

nce

159.08

172.00

272.17

186.08146.08 200.00 254.00132.00 228.1792.17 299.0099.92

Fig. 3-10 (c). MS/MS analysis of hydroquinolone (mass 272.2) compound.

3. Results

67

3.4. Antifungal activity of chitosan

3.4.1. Determination of minimum inhibitory concentration (MIC) of

chitosan and analysis of aflatoxins

The MIC of chitosan against Aspergillus flavus was determined in the range of 1-2%

solutions of CHI50. This showed clear growth of Aspergillus flavus at low concentration

of chitosan while at the concentration of 1.25% (MIC value) and above, there was

complete inhibition of fungal growth (Fig. 3-11).

Fig. 3-11. Plate showing MIC of CHI50 against Aspergillus flavus.

Antifungal activity of chitosan was tested by agar wells diffusion method. For this

purpose, 100 µL of 0%, 1% and 2% chitosan (CHI50) was poured into wells on Yeast

Extract Sucrose (YES) agar plates in duplicate, which were previously inoculated with

Aspergillus flavus.

The plates were incubated at 28ºC for 7 days. The results indicated that there was

a clear zone of inhibition around 2% chitosan wells and growth was inhibited around 1%

chitosan wells while there was heavy fungal growth around 0% chitosan wells (Fig. 3-

12). These observations preliminary confirmed the antifungal property of CHI50.

3. Results

68

Fig. 3-12. Antifungal activity (agar well diffusion) of CHI50 (2%, 1%, 0%) at day 3

(a) and day 7 (b).

Further, chitosan’s antifungal activity was analyzed quantitatively for aflatoxins

inhibition efficacy and values were determined by LC-MS analysis (Table 3-3). The

results indicated complete inhibition of aflatoxin B2, G1 and G2 at 2% chitosan

concentration while, 92% decrease in aflatoxin B1 was observed at the same

concentration. Likewise, with 1% chitosan, the aflatoxins production was decreased to

70%, 80%, 80% and 100% for aflatoxin B1, B2, G1 and G2 respectively (Fig. 3-13) in

comparison with control (absence of chitosan)

Table 3-3. Aflatoxins analysis from agar wells diffusion plate (µg.mL-1

).

Concentration of CHI50

Aflatoxin B1

Aflatoxin B2

Aflatoxin G1

Aflatoxin G2

0%

0.268

0.02005

0.098

0.00333

1%

0.079

0.00401

0.01901

0

2%

0.02052

0

0

0

3. Results

69

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Afl

ato

xin

(u

g/m

l)

Chitosan concentration (%)

Aflatoxin B1

Aflatoxin B2

Aflatoxin G1

Aflatoxin G2

Fig. 3-13. Aflatoxin inhibition by chitosan from agar wells diffusion method.

3.4.2. Inhibition of aflatoxins (Aspergillus flavus) on Basmati rice.

The quantitative production of Aspergillus flavus and inhibition of aflatoxin by chitosan

on rice was determined by LC-MS using different concentrations of chitosan (CHI50)

solutions (0-2%). The results indicated that there was dominant growth in control flask

(at 0% chitosan) and flask having 1% chitosan solution. Other flasks having 1.5% and

1.75% chitosan solution apparently showed inhibited growth of Aspergillus flavus (Fig.

3-14). This observation was validated when aflatoxin analysis were conducted on LC-MS

which showed considerable reduction in the production of aflatoxin (Table. 3-4).

Moreover, at 2% chitosan concentration complete inhibition of growth of Aspergillus

flavus was observed along with the production of aflatoxin B1, B2, G1 and G2 (Fig. 3-15).

3. Results

70

Fig. 3-14. Aflatoxin inhibition by CHI50 solution at concentration 0% (C), 2%, and

1.75%, 1.5% and 1% at day 1, day 3 and day 5.

Table 3-4. Concentration of aflatoxin B1, B2, G1 and G2 (µg.mL-1

).

Concentration of CHI50 (%)

Aflatoxin B1

Aflatoxin B2

Aflatoxin G1

Aflatoxin G2

0.00

50.30

9.10

3.22

1.22

1.00

23.75

3.18

1.13

0.34

1.50

3.70

0

0

0

1.75

0.81

0

0

0

2.00

0.12

0

0

0

3. Results

71

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25

0

10

20

30

40

50

Aflatoxin B1

Aflatoxin B2

Aflatoxin G1

Aflatoxin G2

Afl

ato

xin

(u

g/m

l)

Chitosan concentration (%) 1ml/5g rice

Fig. 3-15. Aflatoxin inhibition by chitosan (CHI50) on rice.

3.5. Characterization of chitosan membranes

3.5.1. Morphological analysis of chitosan/PVA membranes

Scanning electron and atomic force microscopies were used to study the morphological

features of the membranes in detail. The dried membranes appeared to be transparent,

smooth, elastic and strong (Fig. 3-16a-b).

The micrograph images of chitosan membranes by scanning electron microscope

(SEM) revealed a porous and crosslinked network structure with a variety of pore sizes.

The micrograph images showed that the pore size of the membranes decreased as chain

length of the chitosan polymers decreased in the membranes (Fig. 3-17).

3. Results

72

The surface topography of chitosan/PVA membranes without crosslinking and

with crosslinking were imaged by atomic force microscope (AFM). Both un-crosslinked

and crosslinked membranes showed the macromolecule aggregation on membrane

surfaces but the aggregation pattern was greater for crosslinked membranes. The

chitosan/PVA membranes without crosslinking possessed a dense surface structure with

uniform distribution of spherical particles (Fig. 3-18a), while chitosan/PVA membranes

with crosslinking exhibited aggregation structure with some protuberance on membrane

surface (Fig. 3-18b).

Fig. 3-16. Camera images of chitosan membrane showing it transparent Mem25 (a)

and Mem50 (b).

3. Results

73

Fig. 3-17. SEM analysis of chitosan membranes at different magnifications. Shown are SEM micrographs of MemC

(panels A1 and A2), Mem25 (Panels B1 and B2), Mem50 (panels C1 and C2) and Mem75 (panels D1 and D2). In each

case the first panel shows micrographs taken at 1990x magnification and the second at 3300x magnification.

3. Results

74

Fig. 3-18. AFM images of uncrosslinked (a) and crosslinked (b) chitosan/PVA

membranes.

a

b

3. Results

75

0 50 100 150 200 250 300 350

0

200

400

600

800

1000

1200

1400

1209%

622%

557%

Wate

r ab

so

rpti

on

cap

acit

y (

%)

Time (min)

MemC

Mem25

Mem50

Mem75

444%

3.5.2. Effect of time, temperature, pH, and electrolytes on water

absorption capacity of the membranes

The time dependent water absorption capacity (WAC) of the membranes was

investigated in distilled water. These membranes reached at equilibrium state within 195

min. at room temperature (28oC). Of the four membranes, MemC, prepared from un-

irradiated chitosan had highest water absorption capacity (1209%; Fig. 3-19), whereas the

membranes prepared from irradiated chitosan had lower WAC of Mem25 (almost 50%)

and it further decreased in Mem50 and Mem75.

Fig. 3-19. Time dependent water absorption capacity of MemC, Mem25, Mem50

and Mem75.

The effect of temperature on water absorption capacity of chitosan membranes

was studied at room (28oC), human body (37

oC) and higher (50

oC) temperatures. There

was an increase in the water absorption capacity of the membranes with increase in the

temperature of the swelling medium (Fig. 3-20).

3. Results

76

MemC Mem25 Mem50 Mem75

0

400

800

1200

1600

2000

2400

Wate

r a

bso

rpti

on

cap

acit

y (

%)

Membrane types

28oC

37oC

50oC

0 2 4 6 8 10

0

200

400

600

800

1000

Wate

r ab

so

rpti

on

cap

acit

y (

%)

pH

MemC

Mem25

Mem50

Mem75

The swelling behavior of the chitosan membranes was greatly influenced by the

pH of surrounding medium. The pH dependent swelling equilibrium of the membranes

was determined in various pH solutions (2.0 to 8.0) and results have been shown in Fig.

3-21. The absorption capacity of all the samples increased steadily with increase in pH

and reached to maximum (MemC, 963%; Mem25, 797%; Mem50, 718%; and Mem75,

524%) at neutral pH (7) while it decreased at higher pH values.

Fig. 3-20. Temperature dependent water absorption capacity of MemC, Mem25,

Mem50 and Mem75.

Fig. 3-21. pH dependent water absorption capacity of MemC, Mem25, Mem50 and

Mem75.

3. Results

77

0.0 0.2 0.4 0.6 0.8 1.0

0

100

200

300

400

500

600

700

800

900

W

ate

r a

bso

rpti

on

cap

acit

y (

%)

Concentration (mol/L)

MemC

Mem25

Mem50

Mem75

MemC

Mem25

Mem50

Mem75

The influence of electrolytes (NaCl and BaCl2) and their concentrations on

absorption capacity of chitosan membranes were also studied. The results showed a

decrease in water absorption with increase in salt concentration and charge density of the

electrolytes (Fig. 3-22).

Fig. 3-22. Water absorption capacity of MemC, Mem25, Mem50 and Mem75 in

different conc. of NaCl (solid symbols) and BaCl2 (hollow symbols).

3.5.3. Mechanical properties

Effects of molecular weight of chitosan on mechanical properties (tensile strength, TS

and Elongation at break, EB) of chitosan/PVA membranes were investigated. Tensile

strength and %age elongation of membranes increased with low molecular weight of

chitosan in the membranes. At further low molecular weight chitosan membrane

(Mem75), the TS and EB of membrane decreased. Over all membranes showed fairly

good mechanical properties, but membrane containing CHI50 had slightly higher tensile

3. Results

78

strength (47.19 MPa) and elongation at break (359.42%) values (Table 3-5) as compared

to other membranes.

Table 3-5. Mechanical properties of chitosan membranes.

Membranes

Tensile strength (MPa)

Elongation at break (%)

MemC

28.54

407.30

Mem25

35.04

359.30

Mem50

47.19

359.42

Mem75

27.07

292.60

3.5.4. Water vapor transmission rate (WVTR) and water vapor

permeability (WVP) of membranes

The water vapor transmission rate (WVTR) and water vapor permeability (WVP) of

irradiated chitosan membranes were investigated and recorded (Table 3-6). The WVTR

Table 3-6. Water vapor transmission and permeability of chitosan/PVA membranes.

Membranes

WVTR (g.h

-1.m

-2) x10

-3

WVP (g.Pa-1

.h-1

.m-2

) x10-6

Graphical values

Numerical values

Graphical values

Numerical values

MemC

ND

ND

ND

ND

Mem25

170

164

1.95

1.87

Mem50

150

144

1.72

1.67

Mem75

95

97

1.11

1.087 ND = not determined

3. Results

79

0 20 40 60 80 100 120

0

10

20

30

40

50

60

70

80

Co

nta

ct

an

gle

(O

)

Time (minutes)

Mem25

Mem50

Mem75

and WVP of the membranes decreased with low molecular weight chitosan

membranes.The WVTR and WVP of membranes with high molecular weight chitosan

were higher as 164.04x10-3

g.h-1

.m-2

and 1.87x10-6

g.Pa-1

.h-1

.m-2

respectively.

3.5.5. Contact angle measurement (surface properties)

For our study, rate of decrease in contact angle was measured through water to determine

the extent of hydrophilicity of the membranes. A very sharp decrease in contact angle

was observed with time for all the membranes and contact angle value decreased with the

passage of time (Fig. 3-23).

Fig. 3-23. Rate of decrease of contact angle for chitosan membranes.

3.6. Antibacterial activity of chitosan membranes

In present work, the antimicrobial properties of the membranes prepared from un-

irradiated and irradiated chitosan were studied against Gram’s negative bacteria

Pseudomonas aeruginosa (SS29), Escherichia coli (SS1, SS2 and SS9), Proteus

mirabilis (SS77) and Gram’s positive bacteria; Staphylococcus aureus (LM15) and

Bacillus subtilis using agar membrane disc diffusion method. The embedded membranes

in semisolid media showed antibacterial effect against different bacteria used in our

3. Results

80

study. Chitosan membranes having highest antibacterial activity (Mem75) in this way

showed antibacterial effect near the edges in a more pronounced way (Fig. 3-24).

Fig. 3-24. Antibacterial activities of chitosan membranes (Mem75) against

pathogens.

As, these membranes were not suitable for agar membrane disc diffusion method

due to their curling nature when come in contact with agar medium, optical density (OD)

method was adopted for antibacterial studies of chitosan membranes. For using OD

method, a representative Gram’s negative; Escherichia coli (SS1) and Gram’s positive;

B. subtilis bacteria were selected. Previously, these bacteria were also tested for

antibacterial activities of un-irradiated and irradiated chitosan. Low molecular weight

chitosan (CHI75) has fairly good antibacterial property while CHI50 and CHI25 have

almost equivalent antibacterial activities against these pathogens (Fig. 3-25). However,

3. Results

81

with un-irradiated chitosan (CHIC), only B. subtilis has showed prominent inhibition in

its growth.

Fig. 3-25. Inhibitory zones by CHI25, CHI50, CHI75, solvent and un-irradiated

chitosan (CHIC) against E. coli; SS1 (a) and B. subtilis (b).

For antibacterial activities of chitosan membranes with OD method, it was

observed that OD of the medium increased with time with un-irradiated chitosan

membranes (MemC) while it remained almost un altered for membranes with irradiated

chitosan (Fig. 3-26a and 3.27a). In another experiment, bacterial culture was harvested

from log phase to study the antibacterial characteristics of chitosan membranes against E.

coli and B. subtilis. These results indicated that, in general, all membranes prepared from

irradiated chitosan (Mem25, Mem50 and Mem75) had stronger antibacterial activity

against E. coli and B. subtilis bacteria (Fig. 3-26b and 3-27b), while, un-irradiated

chitosan membrane had antibacterial activity only against B. subtilis.

3. Results

82

Fig. 3-26. Antibacterial activity of MemC, Mem25, Mem50 and Mem75 against E.

coli culture from stationary phase (a) and log phase (b).

Fig. 3-27. Antibacterial activity of MemC, Mem25, Mem50 and Mem75 against B.

subtilis culture from stationary phase (a) and log phase (b).

3.7. Controlled release of insulin by membranes

Controlled release of insulin was studied at stomach pH (1.2) by simulated gastric fluid

(SGF) and at intestine pH (6.8) by simulated intestine fluid (SIF). For this purpose 10

0 2 4 6 8 10

0.0

0.1

0.2

0.3

0.4

0.5

Bacte

rial g

row

th (

OD

600n

m)

Time (h)

MemC

Mem25

Mem50

Mem75

0 2 4 6 8 10 12

0.0

0.1

0.2

0.3

0.4

0.5

Bacte

rial g

row

th (

OD

600n

m)

Time (h)

MemC

Mem25

Mem50

Mem75

a b

0 2 4 6 8 10 12

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Bacte

rial g

row

th (

OD

600n

m)

Time (h)

MemC

Mem25

Mem50

Mem75

0 2 4 6 8 10 12

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Bacte

rial g

row

th (

OD

600n

m)

Time (h)

MemC

Mem25

Mem50

Mem75

a b

3. Results

83

0 60 120 180 240 300 360

0

1

2

3

4

5

6

7

pH 6.8

pH 1.2

Rele

ase o

f in

su

lin

(p

pm

)

Time (minutes)

Mem25

ppm insulin was loaded on chitosan membranes (Mem25). The results indicated that

almost all of the insulin was released within first hour in simulated gastric fluid (Fig. 3-

28).

Fig. 3-28. Insulin release in SGF and SIF solutions.

4. Discussion

Chitosan is very important polysaccharide due to some of its unique characteristics.

Chitosan only required mild processing conditions for applications in different fields. In

recent years, chitosan is being used as antimicrobial agent in food processing, food

packaging, and burn wound dressings. The primer focus of scientists is improving the

antimicrobial property of chitosan by various methods as most of the raw chitosan has

very poor antimicrobial properties. In our study, chitosan was extracted from crab shells

and subjected to gamma irradiations to improve its various characteristics including

antimicrobial efficacy. Ionizing radiations induced the chain breaking which was

radiation dependent. Chain degradation was determined by measuring the viscosity

average molecular weight of chitosan. Increasing irradiation dose decreased the

molecular weight of chitosan which developed and improved its antimicrobial properties.

Chitosan molecular weight also affected membrane characteristics which will be

discussed subsequently.

4.1. Antimicrobial studies of variable molecular weight

chitosan and chitosan membranes

It is very important for biomedical materials to be sterile and free from microorganisms.

Chitosan exists in a variety of molecular weights as well as modified into various forms

for particular applications. So, antimicrobial characteristics of a particular chitosan and

its modified form are necessary for using in biomedical applications. In our daily life,

Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis, Staphylococcus aureus,

4. Discussion

85

Bacillus subtilis and many more are central bacterial pathogens most frequently

responsible for nosocomial infections. These pathogens gain resistance against numerous

antibiotics used in causative therapy. Therefore, resistance has been developed due to

ineffectiveness of many antibiotics [129]. Thus, improving the effectiveness of antibiotics

and decreasing their toxicity are the two basic purposes in the development of novel

antimicrobial agents. Utilization of natural materials as antibiotics is one of the current

approaches. The antibacterial activity of such biomaterials is the most valuable property

and has been employed in many biomedical applications. Investigating the antimicrobial

characteristics of natural polymers especially chitosan has gained significant importance

in recent years [13, 90, 98, 124, 150].

In case of irradiated chitosan powder, significant growth inhibition of the tested

bacteria was observed; however, the inhibitory effects were different for different

molecular weight chitosan and type of bacterium. Antibacterial activity of variable

molecular weight chitosan was investigated against Pseudomonas aeruginosa (SS29),

Escherichia coli (SS1, SS2, and SS9), Proteus mirabilis (SS77), Staphylococcus aureus

(LM15) and Bacillus subtilis. Un-irradiated chitosan of high molecular weight showed no

antibacterial activity while low molecular weight chitosan displayed antibacterial activity

against all bacterial strains under investigation. Chitosan showed little stronger

antibacterial effect against Gram’s negative bacteria than against Gram’s positive

bacteria, as similar type of results were also observed by Chung et al. [151]. However,

some results contrary to these studies have also been reported [54]. We observed that

Pseudomonas aeruginosa (SS29), Escherichia coli, and Bacillus subtilis were more

sensitive towards all types of gamma irradiated chitosan. In addition, chitosan having

lower molecular weight displayed larger zone of inhibition as compared to higher

molecular weight chitosan. In our studies, the antimicrobial activity of naturally available

chitosan from crab shells was developed and improved with gamma irradiations. Gamma

irradiation has infect shortened the chain length of chitosan polymer. Further, results

showed more antibacterial activity at high concentrations of low molecular weight

chitosan and vice versa. All of the gamma irradiated chitosan showed strong antibacterial

activity against pathogens at 2 µg.mL-1

of chitosan. The clear zone of inhibition against

4. Discussion

86

pathogens by all un-irradiated and irradiated chitosans at different concentrations was

calculated for comparison. Amongst irradiated chitosan, CHI75 had even more

antibacterial effect as compared to others. No antibacterial activity was seen by un-

irradiated chitosan against these bacteria at any concentration of chitosan except against

Bacillus subtilis. The minimum inhibitory concentration (MIC) assay revealed that

irradiated low molecular weight chitosan inhibited P. aeruginosa, E. coli, and Bacillus

subtilis growth at a much lower concentration comparing to un-irradiated chitosan. For S.

aureus, and Proteus mirabilis the MIC value was much lower (0.66%) while in P.

aeruginosa, E. coli, and Bacillus subtilis, the MIC value was reduced to (0.33% or 3300

µg.mL-1

) much lower value than some of the reported values (4096 µg.mL-1

) against P.

aeruginosa [124]. These observations have significantly developed the antibacterial

activity of chitosan against highly versatile opportunistic pathogens especially P.

aeruginosa, E. coli, S. aureus and Bacillus subtilis. The obtained results revealed the

importance of low molecular weight chitosan as they have greater antibacterial activity

against drug resistant bacteria and potential to be efficiently used for biomedical

applications.

Antibacterial activity of chitosan in the form of membranes was studied using

various methods including membrane disc diffusion and optical density method. In these

experiments we faced some problems due to curling nature of membranes when in

contact with agar media acted as a limiting factor for experimental observations. Also,

putting wet membranes on agar media, initially, membranes of same size vary in size

after swelling. Antibacterial experiments for membranes were also tried by setting

membranes in semi-solid medium before solidification but it was not stable as sometimes

membranes rolled up and extended in size due to moisture absorption (Fig. 3-24).

However, adopting these methods antibacterial activities were observed but results were

not stable and reproducible. For antibacterial activities of chitosan membranes, it is also

necessary for bacteria to come in contact with chitosan membranes. Further, it is reported

that antibacterial activity of chitosan influenced by number of factors including chitosan

itself and the tested bacterium, that act in orderly/independent way [28]. Generally,

bacteria have different sensitivities at different culture stages towards variable molecular

4. Discussion

87

weights of chitosan [89]. Therefore, the antibacterial activities of the chitosan membranes

were also tested by using the bacterial culture harvested from log and stationary phases.

So, keeping in mind these limitations factors for determining antibacterial activities,

optical density (OD) method was adopted for growth evaluation in the presence of

chitosan membranes. It was observed that chitosan membranes were very effective to

inhibit the growth of Gram’s negative E. coli and Gram’s positive B. subtilis culture

taken from the stationary phase. In other experiment, when bacterial culture were used

from log phase of E. coli and B. subtilis, the results showed that, in general, all

membranes exhibited antibacterial activities in the form of membranes too. However,

chitosan in the form of membranes revealed depressive antibacterial activity against B.

subtilis as compared to E. coli. One of the reasons might be the lesser availability of

chitosan in membrane forms and thick wall of peptidoglycan layers in Gram’s positive

bacteria.

Above experiments indicated that cell sensitivity was influenced by the growth

phase of the microbes and mode of availability of chitosan. According to numerous

authors [12, 152], one of the reasons for the antimicrobial character of chitosan is the

presence of -NH2 which interacts with negatively charges on cell membranes, which

leads to the leakage of intracellular constituents of the microorganisms. The possible

mechanisms for antibacterial activity are given by Zheng and Zhu [57]. Antibacterial

activity of chitosan primarily is due to its polycationic nature because formation of

impermeable membrane as a result of interaction between cationic groups on chitosan

and anionic groups on cell surface inhibited the transport of essential nutrients. Chitosan

also has chelating property due to its cationic nature. This chelating effect can also

embrace antibacterial mechanism. Low molecular weight chitosan can enter the bacterial

cells through pervasion and disrupt their metabolism [28, 57]. These probable

mechanisms may work simultaneously. In our experiments, all the irradiated chitosans in

pure and membrane form had significant antibacterial effect. However, chitosan’s

antibacterial effect was slightly reduced when studied in the form of membranes. The

reason might be the reduced quantity and less availability of irradiated chitosan in

membrane form. These results also showed that decrease in molecular weight enhanced

4. Discussion

88

the antibacterial effect. This may be due to the fact that the low molecular weight

chitosan can work in two ways i.e. destroying the microbial cells and disrupting the

metabolism of the microorganisms.

In addition to above mechanisms, we also have proposed another important factor

which might be helpful in understanding antibacterial mechanism. As we know, the broad

host opportunistic pathogen Pseudomonas aeruginosa is a major cause of debilitating

bacterial infections. These bacteria mostly produce infections in patients with severe

burns. The bacterium P. aeruginosa produces a wide array of extracellular compounds

which may functioning as virulence factors or have cell to cell communication to sense

their population density [153]. The structural identification of these extracellular

virulence factors is important in understanding and combating P. aeruginosa infections.

We studied the metabolic profile of P. aeruginosa in the absence and presence of

different concentration of antimicrobial chitosan. The LC-MS analysis showed the

suppression of virulence (hydriquinolones and related) compounds which may ultimate

reduced its growth in the presence of chitosan. This study might be helpful in

understanding the actual antibacterial mechanism of chitosan.

Mycotoxins and human diseases are largely ignored global health issues.

Aflatoxins are the mycotoxins which contaminate a large fraction of the world’s food like

cereals, groundnuts, maize and rice etc. Mycotoxins contaminate the food of a large

proportion of the world’s population. The low molecular weight chitosan showed a

significant antifungal activity and was also evaluated for reduction in the production of

aflatoxins. Very little is reported on anti-aflatoxin property of chitosan. We have shown

that 2% chitosan concentration is the amount which completely inhibited the growth of

Aspergillus flavus and production of aflatoxins B1, B2, G1 and G2 on 5 g of rice sample.

While chitosan concentration at 1.75% and 1.5% apparently inhibited the complete

growth of Aspergillus flavus and inhibition in the production of aflatoxin B1 up to 98%

and 92% respectively was observed while other aflatoxin were completely inhibited. The

1% chitosan concentration apparently did not inhibited the production of Aspergillus

flavus but afflatoxin B1, B2, G1 and G2 were significantly inhibited by 53%, 65%, 64%

and 72% respectively as compared to control. So, chitosan use as anti-aflatoxin appears

4. Discussion

89

to be a very important property and very little has been reported for aflatoxins inhibition.

Other natural and synthetic compounds such as flavonoids, phenolic compounds and

essential oils are able to reduce aflatoxin production in vitro [154, 155]. The antifungal

activity of chitosan is considered to be due to interaction between the cationic chain of

chitosan polymer and anionic groups of the residue of fungal cell surface

macromolecules, which lead to the leakage of intracellular constituents and other

electrolytes. Chitosan may also affect the morphogenesis of the cell wall by direct

interfering on the activity of enzymes responsible for the growth of fungi [144]. Chitosan

of low molecular weight being effective antibacterial and antifungal agent can be

proposed for use in pharmaceutics.

4.2. Surface characteristics of membranes

Infrared (IR) spectroscopy was performed to investigate the chemical changes and

formation of any new bond in gamma irradiated chitosan and its blend membranes. The

IR spectra of the irradiated chitosan recorded in solid state, showed the overlap stretching

of hydrogen bonded -OH and -NH2, band ranging from 3070-3450cm-1

, which

corresponded to the pure chitosan [125, 156]. The enhanced band intensity of hydrogen

bonded -OH in chitosan CHI50 might be due to the contribution of additional -OH group

formation, possibly, during irradiation process. The stretching of C-H bond appearing at

2877cm-1

also represented chitosan, as likewise reported by Liang et al. [125]. In

addition, the band appeared at 870cm-1

, which represented the pyranose ring confirming

the existing chitosan moiety, also stated by Costa-Junior et al. [157] and Islam and Yasin

[109]. Small bands appearing at 1050 and 1384cm-1

can be attributed to the C-O-C and

methyl groups, respectively which were also reported by Kang et al. [158]. So, irradiation

does not destroy the actual structure of chitosan moieties. The IR spectra of the chitosan

membranes revealed peaks at 1044-1100cm-1

, which indicated the presence of siloxane

linkage (Si-O-) resulted by the reactions of TEOS with chitosan and PVA [109, 128]. The

enhanced band intensity at 3290cm-1

showed an increased interaction between -NH2 (in

chitosan) and the -OH (in PVA and glycerol) groups [125, 156].

4. Discussion

90

Apparently these membranes were smooth but actual structure of chitosan

membranes was evaluated for use in particular applications. Scanning electron

microscopy (SEM) and atomic force microscopy (AFM) were used to study the

morphological features of the membranes in detail. The scanning electron micrographs

and atomic force microscope images revealed a crosslinked network structure with a

variety of pore sizes. The porosity of the membranes is an important factor in their

swelling characteristics [109]. The micrograph images showed that the pore size of the

membranes decreased as chain length of the polymers decreased. The contact angle is

also very important property to determine the hydrophilicity of the materials. The

reported contact angle value of pure chitosan is less than 90o [159, 160],

presenting

chitosan as hydrophilic polymer. But, our chitosan membranes showed a decrease in

contact angle (60.2o to 67.3

o), which might be due to the incorporation of -OH groups

from PVA and glycerol. This indicated that availability of polar groups (-OH) from PVA

and glycerol also contributed towards hydrophilicity of the membranes. Based on

hydrophilicity and water absorption capacity, these hydrophilic membranes can be

applied for the dehydration of laboratory scale organic solvents [161-163].

4.3. Water absorption response of membranes in various media

4.3.1. Water absorption capacity of membranes in distilled water

It is considered that the water uptake properties mainly depend upon the structure and

chemistry of the materials. Blends of chitosan and PVA are fairly miscible and the

polymer chains, possibly, interact via crosslinkers and form macroporous structure,

which made it suitable for water uptake [164, 165]. The macroporous nature of the

membranes was confirmed by scanning electron microscopic examination. Besides, some

of the water molecules might form association through hydrogen bonds with available

hydrophilic groups on polymer chains which also supported the absorption of water. The

Mem25, Mem50 and Mem75 showed less water absorption capacity (WAC) as compared

to MemC, which might be due to their compact structures. It was observed, that higher

absorbed dose of gamma radiations led to the decrease in molecular weight of chitosan.

4. Discussion

91

Thus, chitosan with smaller chains furnished compact structure with smaller pores, which

allowed lesser water molecules to penetrate inside the membranes. Therefore the

absorption capacity was decreased in the blends in the order Mem25: 662%, Mem50:

557% and Mem75: 444%.

4.3.2. Temperature dependent water absorption capacity of membranes

The effect of temperature on swelling capacity of chitosan membranes was observed in

distilled water at 28oC, 37

oC and 50

oC. There was an increase in the water absorption

capacity of the membranes with increase in temperature of the water absorption medium.

This temperature responsive behavior might be due to the increased association and

dissociation of intra- and intermolecular hydrogen bonding [166]. This increase in

absorption capacity was exhibited by all the membranes. Some other studies also reported

similar type of behavior with chitosan/PVA membranes [166, 167]. Low molecular

weight chitosan membrane showed less water absorption capacity due to its complex

structure. The majority of temperature responsive membranes showed a separation

behavior from solution and solidification above a certain temperature [168]. Above the

low critical solution temperature, membranes turned into gels. While, membranes that

formed through the cooling of a polymer solution had an upper critical solution

temperature. Such types of chitosan membranes, with volume phase transition

characteristics, have been studied mainly for drug delivery systems [168]. These

membranes were reported to be swollen by absorbing more water at higher temperatures

while shrunk at lower temperatures. This temperature responsive behavior is considered

to be very important for biomedical applications and is currently the center of attention in

research due to its potential applications [128, 168].

4.3.3. Effect of pH on water absorption capacity of membranes

As chitosan contain ionizable functional groups, so, its blends with other polymers are

ionic in nature and exhibit different absorption capacities in different pH solutions. The

low pKa value of chitosan appeared to be the major controlling factor. At low pH, the

4. Discussion

92

amino groups (-NH2) present on the chitosan got protonated to ammonium ion (-NH3+).

The mobile counter ion (Cl-) present in the media moved and neutralized the -NH3

+ ion.

As a result, free movement of -NH3+ groups might be restricted on polymer chains.

Therefore, the charge on membrane was neutral which, in turn, increased the inside

osmotic pressure and decreased the absorption of water molecules [16, 109]. However, at

neutral pH, polymer chains were not ionized and the hydrophilic groups were free to

attract water molecules, revealing high water absorption. Intra- and intermolecular

hydrogen bonding present within the polymer network stabilized the swollen structure.

As a result, the chitosan membranes absorbed relatively more water. On the other hand,

complete deprotonation of amino groups took place at higher pH and the degree of

ionization of the chitosan membranes was decreased. This in turn increased the intra- and

intermolecular hydrogen bonding and lowered the absorption capacity. The pH-sensitive

water absorption characteristics would be desirable for the biomedical applications such

as biomedical separation systems and controlled drug release systems etc. [16, 109, 128].

Keeping in mind the membranes response to pH, we used insulin as model drug for its

controlled release at intestine pH value of 6.8. In our case, as per initial studies these

membranes-insulin composites were not as suitable as anticipated for its controlled

release. However, other drugs and membranes composites can be tried for their controlled

release in future studies.

4.3.4. Effect of ionic concentration on water absorption capacity of

membranes

Water absorption capacity of chitosan membranes was investigated in NaCl and BaCl2

solutions at different concentrations. Both the electrolytes have same anion radical (Cl-)

but different nature of cations and charge (Na+, Ba

2+). In both the cases, decrease in

absorption capacity of water was observed with increase in concentration of electrolytes.

This particular behavior in water absorption capacity might be due to increased ionic

strength of external solution, which reduced osmotic pressure difference between the

internal environment and external electrolyte solution [16, 109]. Further, at higher

concentration of electrolyte, more ions were available to surround the position where

4. Discussion

93

water molecules resided on polymer chains. Thus solvent diffusion into the structure was

decreased, which drastically reduced the swelling capacity [128]. At all concentrations,

higher water absorption capacity was observed in NaCl solution as compared to BaCl2

solution which might be due to greater charge density of Ba2+

ion as compared to Na+

ion, providing strong coordination between chitosan and multivalent cation through ionic

crosslinking [128].

4.4. Mechanical analysis of membranes

For biomedical applications, good tensile strength (TS) and elongation at break (EB) are

considerable factors. In our studies, high TS and EB values could be attributed to the

formation of more polar groups (-OH) during irradiation of chitosan at 50 kGy, which

might produce strong hydrogen bonding and consequently higher TS and EB values

(Table 3-5). However, further decrease in molecular weight had reduced the TS and EB

of the membranes. It is generally accepted in polymer sciences that decrease in

mechanical properties can be ascribed to decrease in molecular weight of polymer and

reduced TS value [169]. Also, low molecular weight chitosan produced more compact

structure, which might reduce the elasticity of the complexes. As a result, tensile strength

and elongation of the membranes were also reduced. Besides, viscosity and nature of the

blending polymers might influence the TS and EB of chitosan/PVA blends [53, 118].

Additionally, incorporation of glycerol added hydrogen bonding, which may resist the

breaking of bonds. Srinivasa et al. [111] also reported comparable trends with similar

type of blends. So, generally we can conclude that high TS of the membranes might be

due to associated facts, including crystalline nature of chitosan, film forming property of

PVA and crosslinking by TEOS.

4. Discussion

94

4.5. Water transmission analysis of membranes

The water vapor transmission rate (WVTR) and water vapor permeability (WVP) of

membranes are important factors in diverse applications, particularly for fruit coatings

and packaging [97]. The water barrier characteristics of packages are important factors to

avoid the early deterioration of food components [118]. Chitosan is therefore used as a

food additive and in some cases as an antibacterial agent [59, 131, 170]. These properties

not only help to retard microbial growth in fruit, but also improve the quality and shelf

life of fruits and other foodstuff [97]. The WVTR and WVP of MemC and irradiated

chitosan membranes had been investigated. The values for WVTR and WVP of

membranes (Mem25, Mem50, Mem75) were 164.04, 144.44, 97.32 (x10-3

) g.h-1

.m-2

and

1.87, 1.67, 1.09 (x10-6

) g.Pa-1

.h-1

.m-2

respectively. There was an inverse relationship

between irradiation dose and WVTR/WVP values. This decrease in WVTR and WVP of

chitosan membranes indicates that lowering the molecular weight of chitosan leads to

more intermolecular interaction among polymer chains, resulting in tightening of the

polymer network and lower permeability. This property has been found extremely

effective in preventing food contamination and spoilage [19].

4. Discussion

95

Conclusions

Gamma irradiation is another and better way to develop and improve the antimicrobial

activity of the chitosan. Chitosan from crab shells was modified into variable molecular

weight membranes using gamma radiation. Increasing the dose of radiation resulted in a

decrease in the molecular weight of chitosan. The chitosan was also blended with PVA to

form membranes. The increased radiation dose shortened the polymer chains, which

formed compact structure when cross-linked with PVA. Consequently, the properties of

chitosan and chitosan blends were greatly influenced by the variation in molecular

weight. The water absorption capacity of the membranes was decreased with increasing

radiation dose due to more compact structures and a decrease in pore size of membranes

was confirmed by SEM micrographs. The water barrier properties of membranes were

better with low molecular weight chitosan (CHI75), whereas mechanical properties were

better for high molecular weight chitosan (CHI25). Thus chitosan with improved

properties can be prepared by varying the radiation dose. In present work, the

antimicrobial properties of the irradiated chitosan were studied. In case of irradiated

chitosan powder, significant growth inhibition of the tested bacteria was observed. The

antibacterial studies showed that chitosan has effective antibacterial properties, which

were enhanced by a decrease in molecular weight after treatments. So, low molecular

weight chitosan and chitosan blends with other polymers, particularly PVA, can be used

as antimicrobial agents in wound dressing, food packaging and food processing industry.

Such antibacterial membranes with suitable mechanical and water vapor transmission

properties can find numerous applications in the field of biomedicine. Keeping in mind

this study, it is possible to give general recommendation for chitosan and its membranes

use for burn wound dressings, antimicrobial coatings and packaging applications. Further

studies for various drugs for their controlled release can be evaluated in future using

chitosan and its blends to enhance the sphere of biomedical applications.

4. Discussion

96

Recommendations/Future work

Future prospects regarding chitosan and its membranes prepared in this study can be as

under:

The antimicrobial chitosan membranes prepared and characterized during

our study can be used for wound dressings after further characterization

including biocompatibility and biodegradability tests for chitosan and its

membranes. Moreover, assessing their healing effects along with

antibacterial properties using model animals can further be elucidated.

As irradiated chitosans proved to be virtuous antibacterial and antifungal

agents, they can be used for protective films/coatings and packaging

materials to extend the shelf life of food materials.

Characterization and evaluation of chitosan membranes for controlled

release of some more drugs (in vitro, in vivo )

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International Journal of Biological Macromolecules 65 (2014) 81– 88

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

jo ur nal homep age: www.elsev ier .com/ locate / i jb iomac

haracterization and potential applications of gamma irradiatedhitosan and its blends with poly(vinyl alcohol)

jaz Banoa,b, Muhammad Afzal Ghauria,∗, Tariq Yasinb,∗∗,ingrong Huangc, Annie D’Souza Palaparthi c

Industrial Biotechnology Division, National Institute for Biotechnology, Genetic Engineering, P.O. Box 577, Faisalabad, PakistanDepartment of Metallurgy and Materials Engineering, Pakistan Institute of Engineering and Applied Sciences, P.O. Nilore, Islamabad 45650, PakistanDepartment of Food Science, Rutgers University, 65 Dudley Road, New Brunswick, NJ 08901, USA

r t i c l e i n f o

rticle history:eceived 22 October 2013eceived in revised form0 December 2013ccepted 5 January 2014vailable online 10 January 2014

a b s t r a c t

Naturally available chitosan (CHI), of high molecular weight, results in reduced efficiency of these poly-mers for antibacterial activity. In this regard, irradiation is a widely used method for achieving reductionin molecular weight of polymers, which may improve some of its characteristics. Chitosan was extractedfrom crab shells and degraded by gamma radiations. Effect of radiation dose on chitosan was analyzedby Fourier transform infrared (FTIR) spectroscopy. Furthermore, the irradiated chitosan was blended

eywords:hitosanadiationsolecular weight

ntibacterial activity

with poly(vinyl alcohol) (PVA) and crosslinked with tetraethylorthosilicate (TEOS) into membranes. Themembranes were found to be smooth, transparent and macroporous in structure, exhibiting high tensilestrength (TS: 27-47 MPa) and elongation at break (EB: 292.6-407.3%). The effect of molecular weight ofchitosan and chitosan blends on antibacterial activity was determined. Irradiated low molecular weightchitosan and membranes showed strong antibacterial activity against Escherichia coli and Bacillus subtilis.

. Introduction

Poly [beta (1,4)-2-amino-2-deoxy-D-glucose], known as chi-osan, is obtained from chitin by a deacetylation process [1]. Chitins the principal structural polysaccharide of the shells of crus-aceans and is the second most abundant, naturally occurringolysaccharide after cellulose [1,2]. Crosslinked chitosan mem-ranes are three dimensional networks that can absorb and retain

large amount of water while maintaining their structures. Chi-osan membranes have various applications in the field of medicinesuch as tissue engineering, dressings for burns and controlled drugelease systems) and food packaging [3–8]. The molecular weightMW) and degree of deacetylation (DDA) of chitosan varies greatlyepending on the source of chitin and the method of deacetyla-ion. The DDA of chitosan is typically over 70%, making it soluble incidic aqueous solutions. Due to its biodegradability, biocompati-
ility and lack of toxicity, much attention is being paid to chitosan,articularly for biomedical applications [9–11].
∗ Corresponding author. Tel.: +92 41 2550814; fax: +92 41 2651472.∗∗ Corresponding author. Tel.: +92 51 2207380; fax: +92 51 2208070.

E-mail addresses: [email protected] (M.A. Ghauri), [email protected]. Yasin).

141-8130/$ – see front matter © 2014 Published by Elsevier B.V.ttp://dx.doi.org/10.1016/j.ijbiomac.2014.01.015

© 2014 Published by Elsevier B.V.

The antibacterial activity of chitosan is affected by molecularweight and degree of deacetylation. Low molecular weight chi-tosan has strong antibacterial properties and is also harmless tohuman body [12–16]. Being a cationic polymer, most chitosanblends have the ability to respond to external stimuli such as tem-perature, pH, and electric fields [13]. Recently the antibacterialand antifungal activities of chitosan have attracted much inter-est [14,17,18]. For the food packaging industry, food quality andsafety to human health are the two major concerns as consumersprefer fresh and minimally processed products. Particularly, bacte-rial contamination of ready to eat products constitute is of concern[12,19]. Chitosan has proven a useful antimicrobial agent in foodprocessing, particularly for improving the shelf life of food materi-als [20,21].

As a blending agent, polyvinyl alcohol (PVA) has excellentmiscibility and film forming properties. It contributes better ten-sile strength and flexibility to the blends. It is synthetic andinexpensive polymer used to produce membranes. PVA blendedchitosan has been employed in many biomedical applicationsdue to its biodegradable, non-carcinogenic and biocompatiblenature [3,4,21–23]. The antibacterial properties of numerous
chitosan blends, including cellulose/chitosan [15], PVA/chitosan[22,23], starch/chitosan [24,25], banana flour/chitosan [26],gelatin/chitosan [27], pullulans/chitosan [28], guar gum/chitosan[29], have been investigated.
dx.doi.org/10.1016/j.ijbiomac.2014.01.015

http://www.sciencedirect.com/science/journal/01418130

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mailto:[email protected]

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dx.doi.org/10.1016/j.ijbiomac.2014.01.015

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In this study, molecular weight of chitosan was reduced byamma radiations at different doses. The chitosan of variableolecular weight was mixed with PVA and crosslinked with

etraethylorthosilicate (TEOS) as biocompatible and non-toxicrosslinker to form membranes. The mechanical, water barriernd contact angle values for chitosan and chitosan blends wereecorded and effects of change in molecular weight on these prop-rties were investigated. Furthermore, the antibacterial activity ofhitosan and membranes was studied against Escherichia coli andacillus subtilis.

. Materials and methods

.1. Materials

Chitosan was obtained from crab shells having 75% DDA.VA (degree of hydrolysis 98.0-98.8 mol%, degree of polymeriza-ion ∼4300, MW 195 kDa) was obtained from Aldrich (Germany).etraethylorthosilicate (TEOS) (d = 0.933 g/ml) was obtained fromluka (Germany) and glycerol (98% reagent grade) from Fisher Sci-ntific (Pittsburg, PA, USA). All other chemicals (NaCl, BaCl2, NaOH,Cl, CH3COOH etc.) were of analytical grade.

.2. Degradation of chitosan by irradiation

A chitosan solution (2%, w/v in acetic acid) was mixed withydrogen peroxide (1%, v/v) and irradiated under cobalt-60 gammaadiations at dose rate of 1.02 kGy/h up to maximum of 75 kGy.he irradiated chitosan was precipitated, filtered and washedn distilled water until a neutral pH was achieved. The precip-tates were vacuum dried at 50 ◦C and used for further studies.n-irradiated chitosan and chitosan irradiated at 25 kGy, 50 kGynd 75 kGy will be referred to as CHIC, CHI25, CHI50 and CHI75,espectively. The molecular weights of extracted chitosan wereetermined using an Ubbelohde viscometer [30]. The viscosityverage molecular weight (Mv) of irradiated chitosan samples isiven in Table 1.

.3. Preparation of membranes

Each of the chitosan samples was dissolved in acetic acid (0.5 M)o yield a 2% (w/v) chitosan solution. PVA was separately dissolvedn deionized water at 80 ◦C to make 4% (w/v) solution. The twoolutions were mixed and an appropriate amount of glycerol wasdded. The mixture was stirred thoroughly for 30 min at 55–60 ◦Cnd crosslinker (TEOS) was then added drop wise. After half anour, the resulting mixture was poured into plastic dishes and dried

n oven at 40 ◦C. Membranes will be referred to as MemC throughem75, depending upon the amount of radiation used on the chi-

osan, as detailed in Table 1. All formulations contained fixed massatio of CHI:PVA – 5:95.

.4. Infrared spectroscopy (IR)

The IR spectra of samples were determined in the range of000 to 400 cm−1 using a Fourier transform infrared (FTIR) spec-rophotometer (Thermo Electron Corp., Nicolet 6700, Waltham,

assachusetts, USA) at room temperature. Spectra were recordedith 200 scans at 6.0 cm−1 resolution using an attenuated total

eflectance (ATR) technique with a diamond crystal tip.

.5. Scanning electron microscopy (SEM)

The surface morphology of membranes was examined using model JSM-7500F field emission scanning electron microscope

gical Macromolecules 65 (2014) 81– 88

(Jeol, Japan). The membranes were prepared by freeze-drying sam-ples after swelling in distilled water. The images were examined atdifferent magnifications.

2.6. Water absorption studies

Small pieces of membranes were weighed and immersed indeionized water (30 mL), until equilibrium was reached. Theswollen membranes were taken out and extra water was care-fully removed. The water absorption capacity (WAC) was calculatedusing the equation

WAC (%) =[

Ws − Wd

Wd

]× 100

where “Wd” is the weight of the dry membrane and “Ws” is theweight of swollen membrane at equilibrium [31]. The results werereported as the average of three readings.

The swelling response of membranes was also measured at dif-ferent temperatures, pH and in ionic solutions.

2.7. Mechanical properties

The tensile strength (TS) and elongation at break (EB) of themembranes were measured on a TA.XT2 Texture Analyzer (TextureTechnologies, New York, USA) at a speed of 0.1 mm/s. The appropri-ate sized membranes (1.0 × 10.0 cm) were cut using a razor bladeand the gauge length was set at 10.0 mm. For each sample, themeasurements were replicated four times.

2.8. Water vapor transmission rate (WVTR) and water vaporpermeability (WVP)

The WVTR and WVP of the membranes were analyzed accordingto the ASTM Method E96/E96 M. Circular test cups were filled withcalcium chloride (10.0 ± 0.5 g) as desiccant at 0% relative humidity(RH), and sealed with the test membranes. The membranes weretightly attached and the initial weights of the cups were recorded.The cups were placed in an environmental desiccator set at 25 ◦Cand 53% RH. After reaching equilibrium state in the desiccators,cups were weighed daily for 14 days. The WVTR was calculated asthe slope of the regression line drawn between elapsed time andthe weight change of the test cups. The actual WVTR and WVP ofthe membranes were calculated using to the following equations

WVTR = G/t

A

WVP = WVTR�P

= WVTRS(R1 − R2)

where “G” is weight change (g), “t” is time (h), “A” is the test area(m2), and “�P” is vapor pressure difference (Pa).

2.9. Contact angle measurements

Static water contact angles of membranes were determined atroom temperature by the drop method using a VCA Optima XEDynamic Contact Angle Analyzer (AST Products Inc., Billerica, MA).The rate of change of surface wettability was taken at different
points on the membranes. A CCD camera was used to record images,immediately after the water drop was deposited onto the mem-brane surface. The measurements were repeated 10 times for eachmembrane with the results presented as a mean of these readings.

I. Bano et al. / International Journal of Biological Macromolecules 65 (2014) 81– 88 83

Table 1Molecular weight of chitosan, and physical properties of chitosan membranes.

Chitosan Dose Mva (g/mol) Membranesb TSc (MPa) EBd (%)

CHIC 0 kGy 220,000 MemC 28.54 407.3CHI25 25 kGy 801 Mem25 35.04 359.3CHI50 50 kGy 473 Mem50 47.19 359.4CHI75 75 kGy 259 Mem75 27.07 292.6

a Viscosity average molecular weight.

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tosan and PVA are fairly miscible with each other and the polymerchains possibly interact via crosslinks to form macroporous struc-tures which make them suitable for absorbing water [36,37]. Thepore structures of the membranes were further confirmed by SEM

b CHI:PVA = 5:95.c Tensile strength.d Elongation at break.

.10. Assessment of the antibacterial properties of irradiatedhitosan and chitosan membranes by agar well diffusion andptical density (OD) methods

Antibacterial activity was assessed against the Gram’s negativeacterium E. coli and the Gram’s positive bacterium B. subtilis using

modified agar well diffusion method [27]. Chitosan solutions (2%,/v) were prepared in acetic acid (0.5 M), adjusted to pH 5.0-5.5

nd 100 �L pipetted into wells (7 mm diameter) on agar plates,hich were previously seeded with 20 �L of bacterial inoculum

ontaining approximately 105-106 CFU/mL. The plates were thenncubated at 37 ◦C overnight and the width of inhibition zones

easured.The antibacterial activity of the chitosan membranes

50 ± 0.2 mg) was performed according to the method describedy Zhai et al. [25] with slight modifications. Chitosan membranesere initially washed in sterile distilled water, weighed and placed

n test tubes containing nutrient broth with bacterial culture0.5%) containing approximately 105–106 CFU/mL. The cultureubes were incubated overnight at 37 ◦C with shaking (150 rpm).uring incubation, the turbidity of the medium was measuredt 600 nm at regular time intervals until a constant reading wasbtained. All experiments were conducted in triplicate underseptic conditions. Moreover, in order to determine the effect ofacterial growth phase on the antibacterial activity of the chitosanembranes, the bacterial cultures were evaluated at two growth

hases i.e., stationary phase and log phase.

. Results and discussion

.1. FTIR characterization

Infrared (IR) spectroscopy was performed to investigate thehemical changes and possible formation of new bonds in chitosannd chitosan membranes. The IR spectra of un-irradiated and irradi-ted chitosan recorded in solid state, showed the overlap stretchingf hydrogen bonded OH and NH2 band ranging from 3070 to450 cm−1 (Fig. 1), which corresponds to pure chitosan [31,32]. An

ncrease in the peak intensity around 3300 cm−1 has previouslyeen reported to be due to the introduction of additional OHroups [32]. The enhanced band intensity of hydrogen bonded OHn chitosan CHI50 might be due to the formation of additional OHroups, possibly as a result of irradiation. The stretching of C Honds appearing at 2877 cm−1 also represents chitosan, as reportedy Liang et al. [1]. In addition, the band appeared at 870 cm−1, whichepresents the pyranose ring, confirms the presence of the chitosanoiety, as previously reported by Costa-Junior et al. [33] and Islam

t al. [3] Small bands at 1050 and 1384 cm−1 can be attributed tohe C O C and methyl groups, respectively (Fig. 1) [34].

The IR spectra of the chitosan membranes revealed peaks at044-1100 cm−1, which indicate the presence of siloxane linkagesSi O ) resulting from the reaction of TEOS with chitosan and PVAFig. 1) [3,35]. The enhanced band intensity at 3290 cm−1 showed

an increased interaction between the NH2 (in chitosan) and theOH (in PVA and glycerol) groups [31,32].

3.2. Scanning electron microscopy (SEM)

Scanning electron microscopy was used to study the mor-phological features of the membranes in detail. The membranesappeared to be smooth and transparent but the micrograph images(Fig. 2.) revealed a crosslinked network structure with a variety ofpore sizes. The porosity of the membranes is an important factorin their swelling characteristics [3]. The micrograph images showthat the pore size of the membranes decreased as chain length ofthe polymers decreased (Fig. 2).

3.3. Water absorption studies

3.3.1. Time dependent water absorption capacityThe time dependent water absorption capacity (WAC) of the

membranes was investigated in distilled water. The membranesreached equilibrium state within 195 min at room temperature. Ofthe four membranes, MemC, prepared from un-irradiated chitosanhad highest water absorption capacity (1209%; Fig. 3a), whereasthe membranes prepared from irradiated chitosan had lower WACto 50%.The water uptake properties of materials depend mainlyupon the structure and chemistry of the material. Blends of chi-

Fig. 1. Infrared spectra (4000-500 cm−1) of chitosan, PVA and Mem25.

84 I. Bano et al. / International Journal of Biological Macromolecules 65 (2014) 81– 88

Fig. 2. SEM analysis of chitosan membranes at different magnifications. Shown are SEM micrographs of MemC (panels A1 and A2), Mem25 (Panels B1 and B2), Mem50(panels C1 and C2) and Mem75 (panels D1 and D2). In each case the first panel shows micrographs taken at 1990× magnification and the second at 3300× magnification.

Fig. 3. Water absorption capacity of Mem25, Mem50 and Mem75 over time (a), different temperatures (b), pH (c) and in different concentrations of NaCl (solid symbols)and BaCl2 (hollow symbols) (d).

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Fig. 2). Some of the water molecules might form hydrogen bondsith hydrophilic groups on polymer chains to increase absorption

f water. The lower WAC of Mem50 and Mem75 might be due toheir compact structures. Increasing the dose of gamma radiationsed to a decrease in molecular weight (Table 1) of chitosan. Thushitosan with of lower molecular weight yielded more compacttructures with smaller pores resulting in lower WAC. Thereforehe absorption capacity was decreased.

.3.2. Temperature dependent water absorption capacityThe effect of temperature on the WAC of chitosan membranes

as assessed (Fig. 3b). There was an increase in the water absorp-ion capacity with an increase in temperature (28-50 ◦C) for all the

embranes. This might be due to the increased association andissociation of intra- and intermolecular hydrogen bonding [38].imilar results have been reported previously for chitosan/PVAembranes [38,39]. The low molecular weight chitosan membrane

howed less WAC due to its complex structure. The majority of tem-erature responsive membranes show a separation behavior fromolution and solidification above a certain temperature, defined ashe lower critical solution temperature [40]. Above the lower criti-al solution temperature, membranes turned into gels. In contrast,embranes that were formed through the cooling of a polymer

olution had an upper critical solution temperature. Such chitosanembranes, with volume phase transition characteristics, are of

nterest mainly for use drug delivery systems. They are reportedo be swollen at higher temperatures and shrunken at lower tem-eratures. This temperature responsive behavior is considered toe very important for biomedical applications and for this rea-on these membranes are the focus of much research at this time35,40].

.3.3. Effect of pH on water absorption capacityThe swelling behavior of the chitosan membranes was greatly

nfluenced by the pH of the medium. The pH dependent swellingquilibrium of the membranes was determined in solutions of var-ous pH values (2.0 to 8.0; Fig. 3c). Since chitosan contains ionizableunctional groups, blends with other polymers are ionic in naturend exhibit pH-dependent absorption capacities. The absorptionapacity with increased with increasing pH and reached a max-mum (MemC, 963%; Mem25, 797%; Mem50, 718%; and Mem75,24%) at neutral pH and decreased at higher pH values. The lowKa value of chitosan appeared to be the major controlling fac-or. At low pH, the amino groups ( NH2) present on the chitosanre protonated to ammonium ion ( NH3

+). The mobile counter ionCl−) present in the media neutralized the NH3

+ ion. As a result,he free movement of NH3

+ groups might be restricted on poly-er chains. Therefore, the charge on membrane was neutral which,

n turn, increased the inside osmotic pressure leading to a decreasen the absorption of water molecules [3,4]. However, at neutral pH,olymer chains are not ionized and the hydrophilic groups are freeo attract water molecules, leading to high water absorption. Intra-nd intermolecular hydrogen bonding present within the polymeretwork stabilized the swollen structure. As a result, the chitosanembranes absorbed relatively more water. On the other hand,

omplete deprotonation of amino groups occurs at higher pH andhe degree of ionization of the chitosan membranes was decreased.his in turn increased the intra- and intermolecular hydrogen bond-ng and lowered the absorption capacity. The pH-sensitive waterbsorption would be desirable characteristic for biomedical appli-ations such as separation and controlled drug release systems3,4,35].

.3.4. Effect of ionic concentration on water absorption capacityThe influence of electrolytes (NaCl and BaCl2) and their con-

entrations on the absorption capacity of chitosan membranes was

gical Macromolecules 65 (2014) 81– 88 85

determined (Fig. 3d). The electrolytes used have same anionic rad-ical (Cl−) but differing cations and with differing charges (Na+,Ba2+). For both increasing electrolyte concentrations resulted in adecrease in WAC. This might be due to increased ionic strengthof the external solution, reducing the osmotic pressure differencebetween the internal environment and external electrolyte solu-tion [3,4]. Further, at higher concentrations of electrolyte, moreions are available to surround the position where water moleculesresided on polymer chains. Thus solvent diffusion into the struc-ture might be decreased, resulting in a reduced swelling capacity[35]. At all concentrations a higher WAC was observed for NaCl thanBaCl2. This might be due to the greater charge density of Ba2+, pro-viding strong coordination between chitosan and the multivalentcation through ionic crosslinking [35].

3.4. Mechanical properties

For biomedical applications high TS and EB are desirable. Theeffects of chitosan molecular weight on the mechanical the prop-erties of chitosan/PVA membranes were investigated (Table 1). Allmembranes showed fairly good mechanical properties, althoughthe membrane containing CHI50 had slightly higher TS (47 MPa)and EB (359.42%) values. These high TS and EB values may be dueto the formation of more polar groups ( OH) during irradiation ofchitosan at 50 kGy, which might produce strong hydrogen bond-ing and consequently higher TS and EB values. Enhanced polaritywas also verified by the low contact angle value for Mem50. Fur-ther, FTIR analysis supported the formation of some additional OHgroups during irradiation at 50 kGy. Similar findings were reportedby Liang et al. [1] and Khan et al. [41]. However, a further decreasein molecular weight reduced the TS and EB of the membranes. It isgenerally accepted in polymer sciences that a decrease in mechan-ical properties can be ascribed to decrease in the molecular weightof a polymer and reduced TS value [42]. As a result, TS and EB ofthe membranes were also reduced. The viscosity and nature of theblending polymers might influence the TS and EB of chitosan/PVAblends [12,26]. Additionally, incorporation of glycerol added hydro-gen bonding, which may resist the breaking of bonds. Srinivasa et al.[22] reported similar results for comparable blends. So, generallywe can conclude that the high TS of the membranes might be dueto the crystalline nature of chitosan, the film forming properties ofPVA and crosslinking by TEOS.

3.5. Water vapor transmission rate (WVTR) and water vaporpermeability (WVP)

The WVTR and WVP of membranes are important factors indiverse applications, particularly for fruit coatings and packaging[43]. The water barrier characteristics of packages are importantfactors to avoid the early deterioration of food [26]. Chitosan isalso used as a food additive and in some cases as an antibacterialagent [21,44,45]. These properties not only help to retard micro-bial growth in fruit, but also improve the quality and shelf-life offruit and other foodstuffs [43]. The WVTR and WVP of MemC (datanot shown) and irradiated chitosan membranes had been inves-tigated. The values for WVTR and WVP of membranes (Mem25,Mem50, Mem75) were 164.04, 144.44, 97.32 (×10−3) g h−1 m−2

and 1.87, 1.67, 1.09 (×10−6) g Pa−1 h−1 m−2, respectively. There wasin inverse relationship between irradiation dose and WVTR/WVPvalues. This decrease in WVTR and WVP of chitosan membranesindicates that lowering the molecular weight of chitosan leads to
more intermolecular interaction among polymer chains, resultingin tightening of the polymer network and lower permeability. Thisproperty has been found extremely effective in preventing foodcontamination and spoilage [7].

86 I. Bano et al. / International Journal of Biolo

Fig. 4. Rate of decrease of contact angle for chitosan membranes.

Fig. 5. Agar well diffusion analysis of the antibacterial act


3.6. Contact angle measurements

The contact angle is very important property in determining thehydrophilicity of materials. Contact angle is conventionally mea-sured through the liquid. The rate of decrease in contact angle wasmeasured using water to determine the hydrophilicity of the chi-tosan membranes. A decrease in contact angle was observed overtime for all the membranes (Fig. 4). The contact angle value ofpure chitosan is less than 90◦ [46,47], indicating that chitosan isa hydrophilic polymer. However, the low molecular weight chi-tosan membranes (Mem25, Mem50, Mem75) showed a decreasein contact angle (60.2–67.3◦), which might be due to the incorpo-ration of OH groups from PVA and glycerol The hydrophilicity andWAC properties of these hydrophilic membranes indicate that theycan be used for the dehydration of laboratory scale organic solvents[48–50].

3.7. Assessment of the antibacterial properties of irradiatedchitosan and chitosan membranes by agar well diffusion and
optical density (OD) methods
It is important for materials used in medical applications tobe sterile. The antibacterial activity of some biomaterials is the

ivity of chitosan against E. coli (a) and B. subtilis (b).

I. Bano et al. / International Journal of Biological Macromolecules 65 (2014) 81– 88 87

Fig. 6. Antibacterial activity of chitosan membranes against E. coli (from stationaryp

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pure and membrane form, had a strong antibacterial effect. How-ever, the effect was attenuated when in membrane form, possiblyreduced availability. The results also showed that the antibacterialeffects of chitosan were enhanced at lower molecular weights. This

hase).

ost valuable property and has been employed in many biomedi-al applications. The antibacterial property of chitosan has attracteduch interest [8,17]. Here the antimicrobial properties of the un-

rradiated chitosan, irradiated chitosan and their blend membranesere studied against E. coli and B. subtilis. For un-irradiated and

rradiated chitosan, a significant growth inhibition of the testedacteria was observed (Fig. 5). However, the inhibitory effects dif-ered for the two bacteria and depended upon the molecular weightf the chitosan. Low molecular weight chitosan showed a strongerntibacterial effect against the Gram’s negative bacterium thangainst the Gram’s positive bacterium, as previously reported byhung et al. [51]. However, this conflicts with the result of an earliertudy [14]. The results obtained here showed that E. coli, a Gram’segative bacterium, was more sensitive to all types of gamma

rradiated chitosan. In addition, chitosan having a low moleculareight displayed a larger zone of inhibition in comparison to higholecular weight chitosan (CHIC) (Fig. 5). It is reported that chi-

osan’s antibacterial activity is influenced by a number of factorsncluding chitosan itself and the tested bacterium, which may actn orderly and/or independent way [13]. The results obtained herendicate that the cationic nature of the chitosan may play a majorole in antibacterial activity by interacting with polyanions on theurface of bacterial cells. The higher negative charge on the cell sur-ace of the Gram’s negative bacteria may lead to the absorption of

ore gamma irradiated chitosan and thus higher inhibitory effectgainst E. coli.

For chitosan membranes, antibacterial studies were carried outsing optical density (OD) method because these membranes wereot suitable for use in the agar well/disc diffusion method since theembranes curled when in contact with the agar medium. Gener-

lly, bacteria have differing sensitivities at different culture stagesowards variable molecular weights of chitosan [52]. Therefore,he antibacterial activities of the chitosan membranes were testedy using the bacterial culture harvested from log and stationaryhases. It was observed that membranes produced from gamma

rradiated chitosan were very effective in inhibiting the growth of. coli cultures taken at the stationary phase (Fig. 6). In anotherxperiment, bacterial culture was used from log phase to study the
ntibacterial property of chitosan membranes against E. coli and. subtilis. The results showed that, in general, all membranes pre-ared from irradiated chitosan had stronger antibacterial activitygainst E. coli (Fig. 7) than membranes prepared from un-irradiated
Fig. 7. Antibacterial activity of chitosan membranes against E. coli (from log phase).

chitosan. However, chitosan membranes showed lower antibacte-rial activity against B. subtilis (Fig. 8). Possibly this is due to the lackof availability of chitosan when in membrane form and thick wallof peptidoglycan layers in Gram’s positive bacteria.

The results obtained indicate that cell sensitivity is influencedby the growth phase of the microbes and mode of availabilityof chitosan and its molecular weight. The possible mechanismsfor antibacterial activity were outlined by Zheng et al. [16]. Theantibacterial activity of chitosan is primarily due to its polycationicnature. Formation of impermeable membrane as a result of inter-action between cationic groups on chitosan and anionic groups onthe surface of cells inhibit the transport of essential nutrients. Chi-tosan also has chelating property due to its cationic nature. Thischelating effect can also contribute towards antibacterial mech-anism. Low molecular weight chitosan can enter bacterial cells bypervasion and disrupt their metabolism [13,16]. These mechanismsmay work simultaneously. In our case, irradiated chitosan, in both

Fig. 8. Antibacterial activity of chitosan membranes against B. subtilis (from logphase).

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ay be due to the fact that the low molecular weight chitosan canork in two ways i.e., destroying the microbial cell membranes andisrupting the metabolism of the microorganisms.

. Conclusions

Chitosan from crab shells was modified into variable molec-lar weight membranes using gamma radiation. Increasing theose of radiation resulted in a decrease of the molecular weightf chitosan. The chitosan was also blended with PVA to formembranes. The increased radiation dose shortened the polymer

hains, which formed compact structure when cross-linked withVA. Consequently, the properties of chitosan and chitosan blendsere greatly influenced by the variation in molecular weight. TheAC of the membranes was decreased with increasing radiation

ose due to more compact structures and a decrease in pore sizef membranes was confirmed by SEM micrographs. The waterarrier properties of membranes were better with low molecu-

ar weight chitosan (CHI75), whereas mechanical properties wereetter for high molecular weight chitosan (CHI25). Thus chitosanith improved properties can be prepared by varying the radiationose. The antibacterial studies showed that chitosan has effec-ive antibacterial properties, which were enhanced by a decreasen molecular weight. So, low molecular weight chitosan and chi-osan blends with other polymers, particularly PVA, can be used asntibacterial. Such antibacterial membranes with suitable mechan-cal and water vapor transmission properties can find numerouspplications in the field of biomedicine.

cknowledgements

Ijaz Bano gratefully acknowledges the Higher Education Com-ission of Pakistan for the scholarship support to carry out this

tudy. Authors are grateful to Mr. Atif Islam for his technical sug-estions whenever required. Authors are highly thankful to Ms.yesha Ihsan for SEM images. We are grateful to Dr. R. W. Briddon

or assisting with improving the language in the manuscript. Ms.asrin Akhtar is acknowledged for proof reading of the manuscript.

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