Advances in Physicochemical Properties of Biopolymers: Part 2

Table of Contents

Welcome

Table of COntents

Title Page

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Disclaimer:

Limitation of Liability:

General:

PREFACE

REFERENCES

ACKNOWLEDEGEMENT

List of Contributors

SECTION A. SPECIALS CASES: POLYSACCHARIDES

Chitosan and its Modifications as Biologically Active Compounds in Different Applications

Abstract

1. INTRODUCTION

2. CHITOSAN HISTORY AND ORIGIN

3. PHYSICOCHEMICAL CHARACTERIZATIONS OF CHITOSAN

3.1. Chemical Structure

3.2. Degree of N-acetylation (DA)

3.3. Molecular Weight (MW)

3.4. Solubility and Solution Properties

3.5. Chemical Reactivity

4. BIOLOGICAL ACTIVITIES OF CHITOSAN AND ITS DERIVATIVES IN DIFFERENT AREAS

4.1. Chitosan and Its Derivatives in Agricultural Applications

4.1.1. Control of Plant Bacteria

4.1.2. Control of Plant Fungi

4.1.3. Control of Plant Viruses

4.1.4. Seed-Coating and Plant Growth Promotion

4.1.5. Control of Agrochemicals Release

4.2. Chitosan and Its Derivatives in Food and Nutrition

4.2.1. Nutritional and Hypocholesterolemic Effect

4.2.2. Food Packaging and Antimicrobial Activity

4.2.3. Antioxidant Activity

4.3. Chitosan and Its Derivatives in Biomedical Applications

4.3.1. Drug Delivery Systems and Pharmaceutical Industries

4.3.2. Gene Delivery

4.3.3. Wound Healing and Dressing

4.3.4. Tissue Engineering

4.3.5. Anticoagulant Activity

4.3.6. Ophthalmology

4.4. Chitosan and Its Derivatives in Biotechnological Applications

4.4.1. Enzyme Immobilization and Biosensors

4.4.2. Cell Immobilization

4.4.3. Protein Purification

4.4.4. Biodegradable Membranes

5. POSSIBLE SIDE EFFECTS OF CHITOSAN

CONCLUDING REMARKS

CONFLICT OF INTEREST

ACKNOWLEDGEMENT

REFERENCES

Biopolymers of Microbial Origin

Abstract

INTRODUCTION

Biopolymers: Towards a Comprehensive and Appropriate Definition

Polyesters (Polyhydroxy Acids)

Aromatic (Co)Polyesters

Polyamides

Proteins

Polyurethanes (Bio-Based PU)

Polysaccharides

Lipids (Triglycerides)

Microbial Polysaccharides

General Properties of Microbial Polysaccharides

Chemical Composition

Macromolecular Conformation

Gel Forming Ability

Structural Analysis of Polysaccharides

Physical Properties

Molecular Mass

Rheological Behavior

Chemical Structure and Physical Properties

Exopolysaccharides of Industrial or Commercial Interest

Acetan (Xylinan)

Applications

Alginates

Microbial Production

General Properties

Applications

Chitin and Chitosan

Chitin

Chitosan

General Properties of Chitin and Chitosan

Applications

Curdlan

Microbial Production

General Properties

Applications

Dextran

General Properties

Applications

Polyhydroxyalkanoates, (PHA)s

General Properties

Applications

Pullulan

General Properties

Applications

Scleroglucan

General Properties

Applications

Sphingans

Gellan Gum

General Properties

Applications

Welan Gum

Applications

Rhamsan, Diutan and Other Sphingan Gums

Xanthan Gum

General Properties

Applications

CONFLICT OF INTEREST

ACNOWLEDGEMENTS

REFERENCES

Analysis of Biopolymers from Brazilian Seeds

Abstract

INTRODUCTION

Structural Features of Xyloglucan Seeds

Structural Features of Galactomannan Seeds

Galactomannan Family and the Distribution of Galactose

Galactomannan and Xyloglucan Properties in Solution

Aqueous Binary System: Galactomannan or Xyloglucan

Galactomannan and Xyloglucan Applications

CONCLUDING REMARKS

CONFLICT OF INTEREST

ACNOWLEDGEMENTS

REFERENCES

Chitosan, From Residue to Industry

Abstract

INTRODUCTION

RAW MATERIAL

MOLECULAR CHARACTERISTICS

Molecular Structure

Deacetylation Degree and Molecular Weight

Solubility in Aqueous Media

MOLECULAR PROPERTIES

Toxicity and Biocompatibility

Biodegradability

Antimicrobial Activity

Filmogenic Properties

Complex Formation with Metals

PRODUCTION METHODS

Chitin Isolation

Chitosan Production Process

CHARACTERIZATION

Deacetylation Degree

Molecular Weight

REGULATORY STATUS

MARKET AND APPLICATIONS

RESEARCH ON CHITOSAN AT INTI

CONFLICT OF INTEREST

ACKNOWLEDGEMENTS

REFERENCES

SECTION B. APPLICATIONS

Amphiphilic Chitosan-Polymer Derivatives and Albumin-Based Formulations for Drug Delivery Applications

Abstract

INTRODUCTION

1. CHITOSAN AND AMPHIPHILIC DERIVATIVES

1.1. Biological Properties and Biopharmaceutical Profile

1.2. Chemical Conjugation between Chitosan and Hydrophobic Polymers

1.2.1. Hydrophobic Interactions

1.3. Chitosan-derivatives in Drug Delivery

1.3.1. Anti-cancer Drugs

1.3.2. Proteins/Peptides

1.3.3. Growth Factors

1.3.4. Antibiotics

1.3.5. Anti-inflammatory Drugs

1.3.6. Vaccines Delivery

2. ALBUMIN

2.1. Albumin in Drug Delivery

2.1.1. Types of Albumin

Ovalbumin (OVA)

Bovine Serum Albumin (BSA)

Human Serum Albumin (HSA)

2.2. Ligand-binding Capacity

2.3. Albumin Receptors, Half-life and Interaction with FcRn

2.4. Albumin-based Drug Delivery Systems

2.4.1. Nano- and Microparticles

2.4.2. Drug-albumin Conjugates and Derivatives

2.4.3. Albumin Microbubbles

2.4.4. Albumin Hydrogels

2.5. Update on Clinical Applications

CONCLUDING REMARKS

CONFLICT OF INTEREST

ACKNOWLEDGEMENT

REFERENCES

Biopolymers as Microencapsulation Materials in the Food Industry

Abstract

INTRODUCTION

Microencapsulation

Applications of Microencapsulation

Microencapsulated Substances

Microencapsulation Materials

Microencapsulation with Gums

Carrageenan

Alginates

Gum Arabic

Microencapsulation with Carbohydrates

Starch

Chitosan

Microencapsulation with celluloses

Cellulose Acetate Phthalate

Microencapsulation with Proteins

CONCLUDING REMARKS

CONFLICT OF INTEREST

ACKNOWLEDGEMENT

REFERENCES

Gels in Biomedical Applications: An Overview on Wound Healing and Tissue Engineering

Abstract

INTRODUCTION

HYDROGELS: SYNTHESIS AND PHYSICOCHEMICAL PROPERTIES

Chemical Structures of Collagen, Gelatin and Hyaluronic Acid

Hydrogels: Classification and Synthesis Methodology

PROPERTIES AND CHARACTERIZATION OF HYDROGELS

HYDROGELS FOR WOUND HEALING

Collagen

Gelatin

Hyaluronic Acid

HYDROGELS FOR TISSUE ENGINEERING

Collagen

Gelatin

Hyaluronic Acid

Commercially Available Hydrogels for Wound Healing and Tissue Engineering

CONCLUDING REMARKS AND FUTURE INSIGHTS

CONFLICT OF INTEREST

ACKNOWLEDGEMENTS

REFERENCES

Approaches for Improving the Mechanical Properties of Collagen Biomaterials

Abstract

INTRODUCTION

Biological Importance of Collagen and its Structure

Properties of in vitro Reconstituted Collagen Matrices

Effects of Metalloproteinases on Collagen´s Remodeling

Effects of Architecture in Dense Collagen

The Contribution of Cells

Collagen Cross-linking Methods: Enzymatic

Collagen Cross-linking Methods: Synthetic Methods

CONCLUDING REMARKS

CONFLICT OF INTEREST

ACKNOWLEDGEMENT

REFERENCES

Protein Plastic Foams

Abstract

INTRODUCTION

PROTEINS AND THEIR SOURCES

THERMOSET PROTEIN-BASED FOAMS

PROTEINS INCORPORATED INTO POLYURETHANE FOAMS

THERMOPLASTIC FOAMING PRINCIPLES

FACTORS WHICH AFFECT THERMOPLASTIC FOAMING

Crystallinity and Thermal Transitions in Polymers and Proteins

Solubility and Diffusivity

Viscoelastic Behavior and Melt Rheology in Foaming

Strain Hardening

BATCH FOAMING

Gas Absorption

Foaming

EXAMPLES OF BATCH FOAMED PROTEINS

FOAM EXTRUSION

THERMOPLASTIC PROTEIN EXTRUSION FOAMING

CONCLUSION

CONFLICT OF INTEREST

ACKNOWLEDGEMENTS

REFERENCES

Biopolymer Electrospinning

Abstract

INTRODUCTION

History of Electrospinning

Electrospinning Theory

General Electrospinning Device

Collector Types and Properties

Oriented Collector

Double Ground Collector

Collector Composition/Substrates

Dimension of the Collector

Configurations of Nozzle in Electrospinning

Electrospinning Materials

Electrospinning Parameters

Solution Parameters

Viscosity

Surface Tension

Polymer Solution Conductivity/Surface Charge Density

Dielectric Constant

Vapour Pressure

Process Parameters

Voltage

Tip to Collector Distance

Feed-rate

Collector Material

Electrospinning Setup

Diameter of Needle Orifice

Environmental (ambient) Parameters

Temperature

Humidity

Type of Atmosphere

Pressure

Co-electrospinning

Process Conditions Required

Electrospun Nanofibers and Their Applications

CONCLUDING REMARKS

CONFLICT OF INTEREST

ACKNOWLEDGEMENTS

REFERENCES

Biopackaging: Tara Gum Films

Abstract

1. INTRODUCTION

2. experimental

2.1. Film Preparation

2.2. Solubility Tests

2.3. X-Ray Diffraction

2.4. Thermal Analysis

2.5. SEM Images and EDAX Analysis

2.6. Water Vapor Permeation (WVP)

2.7. Gas Permeation

2.8. Mechanical Properties

3. results

3.1. Solubility Test

3.2. XRD Analysis

3.3. Thermogravimetric Analysis

3.4. SEM Images

3.5. Water Vapor Permeation (WVP)

3.6. Gas Permeation

3.7. Mechanical Properties

conclusions

CONFLICT OF INTEREST

ACKNOWLEDGEMENT

REFERENCES

CONCLUSIONS

Advances in Physicochemical

Properties of Biopolymers

(Part 2)

Edited by:

Martin Masuelli & Denis Renard

Instituto de Física Aplicada-CONICET,Universidad Nacional

de San Luis,Chacabuco 917,CP 5700,San Luis, Argentina

BENTHAM SCIENCE PUBLISHERS LTD.

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PREFACE

The objective of this ebook is to provide to the readers the most recent state-of-the-art on physicochemical properties of biopolymers and their related end-uses applications. Biopolymers are usually described as polymers produced in a natural way by living species. Their molecular backbones are composed of repeating units of saccharide, nucleic acids, or amino acids and sometimes various additional side chains contributing also to their functionalities.

If the largest part of biopolymers is extracted from biomass, such as polysaccharides from cellulose and proteins from collagen, milk or wheat, biopolymers can also be produced from monomers using conventional chemical processes as polylactic acid, or directly in microorganisms or genetically modified organisms, as polyhydroxyalkanoates. The genetic manipulation of microorganisms brings a tremendous potentiality for the biotechnological production of biopolymers with tailored properties quite suitable for high-value medical applications such as tissue engineering and drug delivery.

Biopolymers from renewable sources, on the contrary display structural complexity and natural variability that need to be deeply studied and characterized before probing into the structure-function relationships for further applications. Research on natural polymers has focused on developing more environmentally friendly applications to reduce pollution caused by non-biodegradable material. Historically, biopolymers were mainly used by mankind as food, or for making clothing and furniture. Since the industrial time, fossil fuels such as oil are the greatest source in the development and manufacture of almost every commercial product, such as the plastic, which is currently used at a very large scale. But these fuels are not unlimited resources, and environemental concerns over all aspects of using fossil fuels for production and energy must be taken into account. We must act in a sustainable manner, which means that the resources must be consumed at a rate such that they can be restored by natural cycles of our planet [1].

Therefore, in recent years, the renewable nature of biopolymers leads them to a renaissance and a new considerable interest by industry due to the unique properties, including biodegradability, biocompatibility and nontoxicity, of biopolymers. To fulfil all these different functions, biopolymers must exhibit rather diverse properties. They must very specifically interact with a large variety of different substances, components and materials, and often they must have extraordinarily high affinities to them. Finally, they must have a high strength. Some of these properties are utilized directly or indirectly for various applications. This and the possibility to produce them from renewable resources, as living matter mostly does, make biopolymers interesting candidates to industry [2]. As a consequence of their properties, these biopolymers derived from natural products have found a place of choice in areas as diverse as effluent treatment, papermaking, chemical, food, cosmetic, pharmaceutical, petroleum and textile industries, as well as in analytical chemistry (biosensors) and molecular biology. However, biopolymers have to compete with polymers derived from fossil fuel not only because of their functional properties but also in terms of cost. In this respect, biopolymers are competitive when the price of oil is high and the price of feedstocks, such as starch from corn, is low.1 The continuing development of new and existing biopolymers will enable these materials to help supplement the increasing global demand for biopolymers-based products and to develop new markets with their niche applications.

The most common biopolymers used for industrial applications and thoroughly considered in this ebook are polysaccharides from plant, algal, microbial and animal origins such as starch, cellulose, lignin, arabinoxylans, sulfated polysaccharides from seaweeds, galactomannans and xyloglucans from brazilian seeds, chitin and its derivative chitosan. Natural gums such as mesquite, tara and arabic gums are also widely used in food and non-food industry and are dicussed in this ebook. Animal and plant proteins such as collagen, gelatin, albumin, dairy proteins and wheat, corn and soy proteins are also considered as sources of proteins for biomedical, microencapsulation and plastic foams applications. Nucleic acids such as DNA and RNA and their related applications in genetic engeenering for instance are not considered in this ebook.

This ebook presents a comprehensive review and compile information on biopolymers in 27 chapters covering from isolation and production, properties and applications, modification, and relevant analytical methods to reveal the structure and properties of some biopolymers.

Authors write this ebook from Argentina, France, Mexico, Spain, Iran, Brazil, Egypt, Turkey, Venezuela, India, Russia, Portugal, New Zealand and Malaysia. This ebook has tried to arrange the ebook chapters in a subject order to make it easier for the readers to find what they need. However, the reader can still find information on the same subject in more than one Section.

Section A (Part 1), which includes one chapter, is mainly an introduction to biopolymers. It includes concepts and molecular weight determination.

Section B (Part 1), which includes twelve chapters, refers to some physical chemistry determinations of biopolymers.

Section C (Part 1), which consists of two chapters, deals with studies on hydrodynamic properties of biopolymers.

Section D (Part 1), which consists of one chapter, refers to theoretical models for biopolymers.

Section A (Part 2), which includes four chapters, refers to special cases of polysaccharides separation and purification.

Section B (Part 2), which includes seven chapters, deals with applications of biopolymers/hydrogels in drug delivery systems, biomaterials, biothermoplastics, bio(nano)composites, bionanostructures, biocapsules, bioadsorbents, bioelectrospinning and biopackaging. This section deserves a special attention because it forms a fascinating interdisciplinary area that brings together biology, chemistry, materials science and (nano)-technology.

This ebook is expected to be of help to many graduate and post-graduate students, professors, scientists, pharmacists, engineers and other experts in a variety of disciplines, both academic and industrial, dedicated to the determination of polymers and biopolymers properties. This ebook may not only support research and development, but also be suitable for teaching. The audience will benefit with an excellent review offering advanced knowledge about technical determinations and physicochemical properties of macromolecules, a thorough knowledge of hydrodynamics and different methods of characterization. Readers will find in this ebook a triple deal, including educational, scientific and industrial applications.

The first main objective of this e-book is therefore to highlight the progress in different techniques of molecular weight determinations and physicochemical properties of biopolymers. The last two decades have seen a number of significant advances in the methodology for evaluating the molecular weight distributions of polydisperse macromolecular systems in solution at the molecular level. These advances have centered on the coupling of chromatographic or membrane based fractionation procedures with multiple detectors on line such as multi-angle laser light scattering, refractive index, UV-Vis absorbance and intrinsic viscosity detection systems. Recent advances in SEC-MALLS (size exclusion chromatography coupled to multi-angle laser light scattering) and FFF-MALLS (field flow fractionation coupled on line to MALLS) applied to complex polymers from renewable resources are therefore presented in this e-book. Beyond molecular charcaterization using HPSEC-A4F-MALLS technique, tremendous efforts were made these last years to elucidate the structural variability and complexity of polysaccharides using matrix-assisted laser-desorption ionization (MALDI) and electrospray ionization (ESI) mass spectrometry coupled or not to nuclear magnetic resonance (NMR) spectroscopy. One chapter of this ebook in section B considers the sequence, interresidue linkage position and substitution pattern of sulfated polysaccharides after enzymatic hydrolyses.

The most widely used method for the dynamic characterization of macromolecules in solution is the capillary viscometry, as it is a simple and economic method. Although in literature there is much information on hydrodynamic measurements from intrinsic viscosity determinations, very few of them evaluate the conformation of different biopolymers. The importance of this type of study lies in the analysis of the polysaccharides or proteins behaviour in industrial processes and product quality control after extraction and purification. These physicochemical studies help to elucidate the chemical structure, macromolecular conformation and the ability biopolymers have to form gels, films, agglomerates, etc. A particular attention is paid in this ebook on the intrinsic viscosity determination of proteins and strong synthetic polyelectrolytes for which theoretical models always need to be implemented in order to get reliable dynamic structural informations.

The ebook also focuses on the structural analyses at the mesoscopic scale using mechanical analyses, microscopy, small angle scattering and free volume measurements and different applications related to biopolymers such as biomaterials, microcapsules, biothermoplastics, nanostructured biocomposites, super-absorbents, bioelectrospinning, biopolymers-based dermal and transdermal drug delivery systems, and biopackaging. All these applications using biopolymers aim to provide a means to reduce dependence on fossil fuels, and decrease the environmental impact of non-biodegradable materials. The main challenge to overcome with biopolymers-based materials is the control of biopolymer-biopolymer interactions, a challenge always present and discussed throughout the ebook by authors.

To conclude, the content of this ebook will bring its readers a basic understanding of the physical chemistry of biopolymers, but also the latest findings about new macromolecules recently discovered and published. Theoretical aspects of computational structural description of biopolymers are also thoroughly described. Therefore, this ebook will appeal to different readers as a great source of knowledge about the science of biopolymers.

Biopolymers Audience

Separation, purification, characterization of biopolymers

Hydrodynamic, molecular weight, size, shape, conformation

Macromolecular assembly

Molecular design and bio-nanotechnology

Biopolymer processing and degradation

Experimental and theoretical studies of biopolymer structures

Three-dimensional structures of biopolymers determined by X-ray, neutrons, NMR

Interactions and thermodynamics

Food biocolloids

Structure and function

Preparation and characterization of novel biomaterials

Capsules and microcapsules

Biocatalysis

Biopolymers for bioremediation

Thin films, membranes & packaging

REFERENCES

Denis Renard

Unité de Recherches Biopolymères,

Interactions, Assemblages,

INRA, Rue de la Géraudière,

France

Email: drenard@nantes.inra.fr

ACKNOWLEDEGEMENT

We first would like to thanks all the authors contributing to the realization of this ebook.

We would like to thank the Agronomic Research National Institute, France and the Área de Química Física - Departamento de Química - Facultad de Química Bioquímica y Farmacia - Universidad Nacional de San Luis – Instituto de Física Aplicada – CONICET, Argentina for giving us the time and opportunity for the writing and edition work of this ebook.

We are grateful to the editing team at Bentham eBooks for their help, especially Humaira Hashmi and Salma Sarfaraz.

Finally, we wish to express our gratitude to our spouses, Maria Gabriela and Marie-Christine, for their patience and support during the long hours of writing, reviewing, commenting, editing of this ebook.

List of Contributors

SECTION A. SPECIALS CASES: POLYSACCHARIDES

Chitosan and its Modifications as Biologically Active Compounds in Different Applications

Mohamed E. I. Badawy¹, *, Entsar I. Rabea²

¹ Department of Pesticide Chemistry and Technology, Faculty of Agriculture, 21545-El-Shatby, Alexandria University, Alexandria, Egypt.

² Department of Plant Protection, Faculty of Agriculture, Damanhour University, 22516-Damanhour, Egypt.

Abstract

A chitosan biopolymer is a reactive functional polymer, which gives possibilities for chemical modifications to generate new properties and functions. Biocompatibility, biodegradability, non-toxicity to mammals, and potential biological activities make this compound with its derivatives advantageous for many applications in different fields, such as agriculture, food and nutrition, biomedicine, pharmaceutics, and biotechnology. In this chapter, we provide collaborative studies of the biological activity of chitosan and its major derivatives in different applications. In addition, the chapter provides the latest technological applications and prospects of products based on chitosan molecule.

Keywords: Biological activity, Chemical modification, Chitosan, Physico-chemical properties, Technological applications.

* Corresponding author Mohamed E. I. Badawy: Department of Pesticide Chemistry and Technology, Faculty of Agriculture, 21545-El-Shatby, Alexandria University, Alexandria, Egypt; Tel: +2039575269; Fax: +2035972780; E-mail: m_eltaher@yahoo.com

1. INTRODUCTION

Chitosan is a linear biopolymer which consists of higher than 70% of β-(1-4)- 2-deoxy-β-D-glucopyranose (GlcN) and lower than 30% of β-(1-4)-2-acetamido-2-deoxy-β-D-glucose (GlcNAc) units linked by β-1,4-glucosidic bonds. It can be obtained through a deacetylation process of purified chitin, a naturally abundant polysaccharide and the supporting material of crustaceans, insects, yeast, and fungi [1-4]. The main difference between chitin and chitosan is the degree of N-acetylation (DA) [5, 6]. Chitosan is of commercial interest due to its high percentage of nitrogen (~ 6.0-7.0%) compared to synthetically substituted

cellulose (1.25%) that makes it a good chelating agent for metal ions [7]. It has unique characteristics such as biocompatibility, biodegradability, low toxicity to mammals, and possesses reactive functional groups that make it useful in different areas of application related to (i) agriculture (as a biopesticide against plant diseases and pests, seed-coating, postharvest, and controlled release agrochemicals) [8-15], (ii) food industry and nutrition (anticholesterolemic dietary products and antimicrobial coatings for fruit and vegetables) [3, 16, 17], (iii) biotechnology (spinning, a dye-binder for textiles, strengthening additive in paper, enzyme and cell immobilization, protein separation, chromatography column matrix, and cosmetic functional ingredients) [18-20]; (iv) biomedicine (drug and gene delivery, blood coagulation, wound healing, bone regeneration, immunoadjuvant activity, pharmaceutics, and ophthalmology) [21, 22], and (v) combinations of chitosan with other natural or synthetic polymers (grafting, polyelectrolyte complexation, blends, and coatings) [23]. However, chitosan exhibits a limitation in its solubility and reactivity therefore, many studies have paid attention to modify its chemical structure [12, 24-26]. Although several works have been reported to obtain functional derivatives of chitosan by chemical modification, very few attained solubility in general organic solvents [27, 28] and some binary solvent systems [29, 30]. Other several studies modified the structure of chitosan by chemical or enzymatic methods to obtain high biologically active compounds and improve solubility in general organic solvents and aqueous solutions at wide range of the pH [25, 31-34]. This chapter covers the literatures dealing with the physicochemical properties of chitosan, the biological activities of chitosan and its derivatives of major importance and applications in different areas.

2. CHITOSAN HISTORY AND ORIGIN

The term chitosan (pronounced Kite-O-San) was given to deacetylated chitin by Hoppe-Seiler [35]. The name chitin is derived from Greek, meaning tunic or envelope and it was first discovered in mushrooms in 1811 by Henri Braconnot [36]. In 1823, Odier found the same material in insects and plants and named it chitin [37]. The concept of chitin was further known by Lassaigne, who demonstrated the presence of nitrogen in its structure when purified the coleopteran elytra and Bombyx mori exuviae, and then treated the residues with potassium at warm temperature thus obtaining potassium cyanide that confirmed the presence of nitrogen in chitin [38]. In 1859, Rouget treated chitin with strong alkali, which resulted in a substance that could, unlike chitin itself, be dissolved in acidic aqueous solutions, named as chitosan [39].

Therefore, up to date, the chitin and chitosan are mainly produced commercially from the waste products of crustaceous exoskeletons of aquatic organisms, fungi and insects. Chitin is found in the outer skeleton of insects (20-38%), crabs, and shrimps and lobsters (69-70%). It was also found that chitin is a major structural component of fungal cell wall (5-20%), which is being considered as an alternative source of chitosan and which varies between different species of fungi from 2 to 60% of dry mycelia [40-42]. Zygomycetes, especially Mucor rouxii, M. mucedo, M. circinelloides, Rhizomucor miehei, Rhizopus oryzae, Phycomyces blakesleeanus, and Cunninghamella elegans have high content of chitin compared to other fungi [43-45]. Moreover, Ascomycetes and Basidiomycetes contain significant quantities of chitin and acidic polysaccharides (26-65%) as cell wall components [46]. However, mushrooms including Lentinus edodes, Lycophyllum shimeji, Pleurotus sajor-caju, and Volvariella volvacea, contain minor content of chitinous components [47] and fungi of Oomycetes and the fission yeasts considered to lack chitin [48]. In contrast, chitosan occurs as a major component in walls of Zygomycetes, but probably is a minor component in many other fungi [49].

The isolation of chitin from shells waste consists of three chemical steps: deproteinization (DP), demineralization (DM), and decolorization (DC); whereby the order of the first two steps is generally considered irrelevant if protein or pigment recovery is not an objective [50]. Purified chitin is then converted to chitosan by hydrolysis of acetamide groups of chitin as a deacetylation process. This process is carried out by two main procedures: the Broussignac process [51], in which chitin is treated with a mixture of solid potassium hydroxide (50% w/w) in 96% ethanol and monoethylene glycol (1:1), and the Kurita process [52], according to which chitin is treated with hot aqueous sodium hydroxide solution (50% w/v). However, the latter is preferred for industrial purposes. In some cases, the deacetylation reaction is carried out in the presence of thiophenol [53, 54] as a scavenger of oxygen or under N2 atmosphere to prevent chain degradation that invariably occurs due to peeling reaction under strong alkaline conditions. Here, DA took place using a calculated four times excess of NaOH for the total N-deacetylation of all amino groups in chitin with reaction time of 1 h at 100°C. These chemical methods are widely used in industrial scale however, the products obtained may suffer some inconsistencies such as protein contamination, inconsistent levels of DA, and hydrolysis of the polymer. In addition, there are some additional problems, such as environmental issues due to the large amount of waste concentrated alkaline solution [43, 55]. Therefore, alternatives to the chemical processes for production of chitin and chitosan are the fermentation and enzymatic techniques which are widely used on an industrial scale [56-61]. While chitin remained an unused natural resource for a long time, interest in chitosan polymer and its derivatives has increased in recent years due to their distinctive properties.

3. PHYSICOCHEMICAL CHARACTERIZATIONS OF CHITOSAN

3.1. Chemical Structure

Chitosan is composed of GlcN and remaining of GlcNAc units which indicate high content of free amino groups compared to chitin (Fig. 1) [6, 62]. The polymer chains appear as linear in structure, which undergoes one full twist, every 10.1 to 10.5 Å along the polymer chain axis. A separate left and right direction can be occurred to each polymer chain as the presence of each chiral glycosidic unit in the polymer chain, and all units are connected by an oxygen atom that links C-1 of one glycosidic unit to C-4 of a neighboring unit. After heating, chitosan decomposes prior to melting, thus it has no melt point and its crystalline structure is mainly characterized by spectroscopic techniques such as X-ray, UV, FT-IR, and NMR [63-69]. The IR spectrum showed main absorption band at 3432 cm-1 due to the partially overlapped of the amine and hydroxyl (N-H, O-H, and NH2) stretching vibrations. Weak absorption bands at 2868 and 2919 cm-1 represent a group -CH- and -OH stretching vibration, respectively. The absorption bands at a wave number of 1656 and 1380 cm-1 correspond to the C=O stretching of CO and amide group, respectively. The absorption band at wave number of 1597 cm-1 was due to the deformation NH of amino groups. The absorption band at 1325 cm-1 corresponds to amid III band. While the absorption band at a wave number of 1151, 1092 and 1024 cm-1 corresponds to the symmetric stretching of the C-O-C and symmetrical skeletal vibration involved in CO and CN stretching, respectively [70, 71]. In addition, the ¹H-NMR spectrum of chitosan is shown in Fig. (2A). The peak at δ 2.06-2.13 ppm is assigned to the proton of residual CH3 in acetyl group. The peak at δ 3.10-3.28 ppm is attributed to H-2 of GlcN residue. The broad multiplet peak at δ 3.53-4.12 ppm is assigned for H-3,4,5,6 of GlcN unit and H-2,3,4,5,6 of GlcNAc unit. The intense band at 5.03-5.17 ppm is related to OH groups and D2O (solvent). In this region, as observed more clearly from an extended spectrum, some different anomeric protons (H-1 of GlcN and GlcNAc units) are appeared at 4.80-4.91 ppm [72, 73].

Further evidence for confirmation: the chemical structure was obtained from ¹³C-NMR spectroscopy. Strong and intense peaks for carbon atoms are found at δ 25.95 (NH(CO)CH3), 56.37 (C-2), 60.99 (C-6), 70.74 (C-3), 75.44 (C-5), 77.80 (C-4), 98.21 (C-1), and 178.24 (C(O)CH3).

Fig. (1))

Chemical structure and illustration of the possible reaction sites in chitosan (GlcN and remaining of GlcNAc units). DA is a degree of acetylation.

Fig. (2))

¹H-NMR (A) and ¹³C-NMR (B) spectra of chitosan dissolved in 1% CD3COOD/D2O at 25°C.

3.2. Degree of N-acetylation (DA)

Chitosan is the fully or partially N-deacetylated derivative of chitin with a DA of less than 30%. This ratio has an important effect on the physicochemical and biological properties of chitosan. Determination of the DA of chitosan products has been achieved by several techniques including IR and UV spectroscopy, conductometric and potentiometric titration, elemental analysis, gel permeation chromatography (GPC), ¹H-, ¹³C- and ¹⁵N-NMR [63-66, 74-77]. The IR method is limited and not accurate because the result interpretation is correlated to the complexity in adopting a suitable base line [71]. However, the method of ¹H-NMR seems to be the most convenient technique to get the correct DA and degree of deacetylation (DDA) of the soluble samples [78]. The DA is calculated from the integral ratio between proton of acetyl group of GlcNAc at 2.06-2.13 ppm and the protons of GlcN at δ 3.10-4.12 ppm. Additionally, ¹³C-NMR is also well-situated for DA determination in the case of pure chitin up to fully deacetylated chitosan with a good conformity between measurements in liquid phase and solid state. The most efficient UV method for determination of the DA is based on the use of dual standards of GlcN and GlcNAc [79]. In this method, several solutions with different concentrations of GlcN hydrochloride and GlcNAc are prepared and their UV absorbencies at λ201 nm are measured by UV-Spectrophotometer. Reading the absorption of the solutions at 201 nm, the DA is calculated by the following equation:

Where m is the weight of chitosan in mg; A is the absorbance, and V is the volume of solution in liters.

3.3. Molecular Weight (MW)

The MW influences on the viscosity of aqueous solution of chitosan and has a significant role in the biological and industrial applications in many fields. In order to determine the MW of chitosan, different techniques methods have been widely used such as viscometry [80], gel permeation chromatographic (GPC) [81], and light scattering (LS) [82, 83] techniques. However, the LS method gives absolute values of the MW, but the technique is more difficult and sometimes the data are not easy to interpret [82, 83]. The viscometry is the most relevant technique in determination of the average MW of chitosan products which depends on the Mark-Houwink equation ([η] = KMa), where the parameters of this equation are known under conditions used [84, 85]. [η] is the intrinsic viscosity, K and a are viscometric parameters and depend on the solvent type. For a chitosan dissolved in 0.5 M CH3COOH/0.2 M CH3COONa buffer, K and a are found to be 3.5 × 10-4 and 0.76, respectively [86, 87]. The viscosity of chitosan solutions could be measured by using Ubbelohde Viscometer immersed in a constant temperature bath at 25 °C. The running times of the solutions and solvent through the viscometer were used to calculate the relative viscosity (ηrel = tch/tsol), specific viscosity (ηsp = ηrel -1), and reduced viscosity (ηred = ηsp/c). Where tch is the running time of the chitosan solution, tsol is the running time of the solvent, and c is the chitosan concentration in g/dl. The [η] value is obtained graphically by extrapolating the η red versus concentration (g/dL) data to zero concentration ([η] = C(ηred)c=0) and the intercept on the ordinate is the intrinsic viscosity [84, 85, 88, 89]. Finally, the average MW is calculated from the Mark-Houwink equation.

3.4. Solubility and Solution Properties

Chitosan obtained by deacetylation process of chitin becomes soluble in all aqueous acidic media when the average DA is lower than 0.5. Formic and acetic acids are the most commonly used for solubilization of chitosan [90, 91]. The water-soluble ionic complex of chitosan and acetic acid, chitosonium acetate, can be handled like any other soluble linear polymer. The solubilization occurs by protonation of the amino group on the C-2 position of the GlcN unit, whereby the polysaccharide is converted to a polyelectrolyte in acidic media. After disolutition of chitosan, it can be cast into films, spun into fibers, precipitated into particles from suitable solvents, which revert the ionized ammonium groups back into amine functionality, and crosslink to produce chitosan fibers [27]. However, the poor solubility of unmodified chitosan in both water and organic solvents make its utilization limited in the biological and industrial applications. Being chitosan soluble in aqueous solutions, it is largely used in different applications as solutions, gels, or films and fibers. The physical properties of chitosan in a solution depend on the ionic concentration, the pH, the nature of the acid used for protonation, the distribution of acetyl groups along the polymer chain, the conditions of the production of the polymer, and the MW [88, 92]. Fully deacetylated chitosan can be reacetylated in homogeneous phase in order to get samples soluble in acidic conditions up to DA~0.6 in relation with a random distribution of the acetyl groups [93]. Under these conditions, the presence of free amino groups at C-2 position of D-glucosamine units allows performing specific reactions in homogeneous conditions [2, 91, 94, 95].

It was also reported that the solubility of chitosan in neutral water can be obtained by chemical modification such as quaternization that forms quaternary ammonium chitosan salts with permanent cationic charges on the polysaccharide backbone. These derivatives have attracted much attention and are most widely used in different areas of applications. Quaternization of chitosan can be obtained using two different ways: by addition of a substituent which contains a quaternary ammonium group [96] or by quaternization of amino groups of chitosan by means of a suitable alkylating agent [97]. The first possibility uses quaternary ammonium epoxides like glycidyltrimethylammonium chloride. The second one, quaternization of chitosan itself, utilizes alkylchlorides or dialkylsulfates such as dimethyl- or diethylsulfate.

3.5. Chemical Reactivity

Chitosan has three types of reactive functional groups including two hydroxyl groups on C-3 and C-6 in each repeating unit, and the amino group on C-2 in each deacetylated unit and the possible reaction sites are shown in Fig. (1). The three functional groups allow the conjugation of many substituents resulting in new modified derivatives with good physicochemical and biological properties. The presence of nucleophilic amino groups allows several reactions such as acetylation, quaternization, Schiff’s bases, reductive amination, acylation, phosphorylation and chelation of metals [34, 98-107]. Reductive amination reaction is the most reliable procedure for preparation of Schiff bases of chitosan and N-alkyl or -aryl chitosan derivatives, wherein the amino group is converted to an imine (Schiff base) with an aldehyde or ketone, and subsequently was reduced by sodium borohydride or sodium cyanoborohydride to form N-alkyl or -aryl derivatives [32, 108-110]. However, the hydroxyl functional groups give various reactions such as O-acetylation, H-bonding with polar atoms, selective etherification and esterification, cross-linking, and graft copolymerization [24, 111]. Selective O-substitution can be performed by protecting the amino groups during the reaction [78, 101].

Several reactions occurring on the amino or hydroxyl groups or both of them have been studied and produced many chitosan derivatives with high solubility in water and wide range of the pH such as carboxymethyl chitosans [34, 112-114], carboxyalkyl and aryl chitosans [73, 115-117], acyl and carboxyacyl chitosans [31, 73, 78, 101, 118-120], hydroxyalkyl chitosans [121, 122], quaternary ammonium chitosan salts [32, 96, 109, 123-125], chitosan sulfate [126-128], phosphorylated chitosan and N-methylene phosphonic chitosan products [129-136], succinyl chitosan [137], thiolated chitosan products [138-141] and sugar-modified chitosan products [142-145]. Chitosan can also be cleaved by hydrolyzing agents due to the presence of rather unstable glycosidic bonds similar to all polysaccharides [146]. Cleavage of glycosidic bonds leads to production of low MW chitosan oligomers such as β (1→4) linked homo- or heterooligomers of GlcNAc and/or GlcN named as chitoologosaccharides (COS) with variation in the degree of polymerization (DP) as well as number and sequence of GlcN and GlcNAc [147, 148].

4. BIOLOGICAL ACTIVITIES OF CHITOSAN AND ITS DERIVATIVES IN DIFFERENT AREAS

4.1. Chitosan and Its Derivatives in Agricultural Applications

4.1.1. Control of Plant Bacteria

Chitosan is widely-used as a biocide either alone or blended with other natural products against plant pathogenic bacteria that induce decay and harmful effects of agricultural crops during the growing season and postharvest phase [11, 13, 149-153]. The minimum inhibitory concentration (MIC) ranged from 10 to 5000 mg/L, depending on the MW, the pH, chemical modification, DA, degree of substitution (DS), and the type of microorganism [11, 123, 151, 154]. Most of the antimicrobial activities have been investigated against bacteria of Agrobacterium tumefaciens, Corynebacterium fascians, Erwinia amylovolora, E. carotovora, Pseudomonas solanacearum, Pseudomonas syringae, Sarcina lutea, and Xanthomonas axonopodishowever, nearly all of these studies were conducted in vitro (Table 1) [31, 155, 156].

Chitosan, at 200 mg/L, showed strong in vitro antibacterial activity against nine bacterial strains of Xanthomonas that cause leaf spot disease of E. pulcherrima [156]. This obtained result was consistent with the results of Wang and co-authors, who reported that the same type of chitosan, at 100 mg/L, had strong activity against twelve bacteria of Xanthomonas strains up to 61.88% after 24 h of incubation compared to the control [155]. Two kinds of chitosan with different MW and DDA possess a strong antibacterial activity against rice bacterial leaf blight and leaf streak X. oryzae and significantly reduced the incidence and severity of disease compared to control under greenhouse conditions [173].

In addition, chitosan at 100, 200, and 400 mg/L inhibited the in vitro growth of Acidovorax avenae subsp. avenae that causes rice bacterial brown stripe disease [157]. Coqueiro and Di Piero added that the chitosan of different MWs, at 1000 and 3000 mg/L, reduced tomato leaf spot severity caused by X. gardneri under greenhouse conditions by 70%, when applied up to 3 days before inoculation [172].

Table 1 List of scientific publications reporting the antimicrobial activity of chitosan and its derivatives against bacteria attack plants.

Chitosan with different MWs (360, 611, and 953 kDa) had a direct effect on bacterial cells of A. tumefaciens, C. fascians, E. amylovolora, E. carotovora, P. solanacearum, and S. lutea and the activity increased with the increase of the MW up to MIC 500 mg/L for C. fascians [175]. In an other study, chitosan of 50, 37, and 57 kDa indicated that the lowest MW (50 kDa) exhibited the highest activity against A. tumefaciens (MIC = 2600 mg/L), while chitosan 37 kDa was the most active against E. carotovora (MIC = 950 mg/L) [163].

It was reported that the antibacterial activity was increased as the MW decreased [148, 176]. However, it is difficult to find a clear correlation between the MW and the antibacterial activity [151].

Grafting the alkyl chains or aryl moieties into chitosan backbone significantly increased the activity against plant pathogenic bacteria [161, 162, 165, 177]. However, this activity is limited to acidic conditions due to its poor solubility above pH 6.5, where chitosan start to lose its cationic nature. Therefore, many studies have focused on the preparation of water-soluble chitosan derivatives such as quaternary ammonium chitosans as antibacterial agents against plant pathogens [31, 164]. A series of N-(benzyl) and quaternary N-(benzyl) chitosan derivatives was synthesized and their ctivities were evaluated in vitro against A. tumefaciens and E. carotovora [160]. MICs of N-(benzyl) chitosan derivatives were ranged from 800 to 1780 mg/L against A. tumefaciens and 700-1550 mg/L against E. carotovora. However, the MICs of quaternary N-(benzyl) chitosan derivatives were ranged from 500 to 875 mg/L against A. tumefaciens and from 600 to 975 mg/L against E. carotovora. These compounds also inhibited some of exocellular enzymes including polygalacturonase and pectin-lyase at 1000 mg/L. N- (4-Carboxybutyroyl) chitosans at different DS (0.10-0.53) were also prepared and exhibited antibacterial activity with MIC ranging from 800 to 1200 mg/L against A. tumefaciens and 725 to 1100 mg/L against E. carotovora and the inhibitory effects increased with the DS increase [31]. These data confirmed that grafting of benzyl moiety or quaternization of the derivatives onto chitosan molecule was successful to inhibit bacterial growth. Moreover, increasing the water solubility of compounds by quaternization significantly increased the activity.

4.1.2. Control of Plant Fungi

The antifungal activity of chitosan and its derivatives against various species of plant pathogenic fungi has been reviewed and approved [11, 13, 149-152, 178]. The list of scientific publications reporting the antimicrobial activity of chitosan and its derivatives against fungi attack plants is shown in Table 2. The MICs varied between 10 and 5000 mg/L [31, 162-165, 175, 177, 179, 180]. The inhibition activity was observed on different stages of fungal growth such as mycelia, sporulation, spore viability and germination, and production of fungal virulence factors. It has generally been recognized that the antifungal activity depends on the concentration applied, MW, DA, the pH of the chitosan solution, the viscosity and the target organism. In general, chitosan applied at 1000 mg/L is capable of reducing the growth of several fungi, except that there contain a chitosan molecule in their cell walls such as Zygomycetes [181]. For example, El Ghaouth and others reported that chitosan at concentrations ranging from 750 to 6000 mg/L, was high potent against spore germination and germ tube elongation of B. cinerea and R. stolonifer [179]. Chitosan caused 25-90.5% mycelial growth inhibition in Penicillium digitatum, P. italicum, Botrydiplodia lecanidionand B. cinerea depending on the type and the concentration [182]. Benhamou and co-authors found that concentrations of 500 and 1000 mg/L of chitosan were completely effective in reducing the disease incidence caused by F. oxysporum in tomato plants [183]. At the same time, El Ghaouth and co-authors revealed that the mycelial growth of P. aphanidermatum was inhibited by 400 mg/L of chitosan [184]. Mycelial growth of A. alternata, B. cinerea, C. gloeosporioides and R. stolonifer was significantly reduced at the range from 750-6000 mg/L [175]. Other studies showed a linear decrease of R. solani growth as the concentration of chitosan increased gradually from 500 to 6000 mg/L [185]. However, complete inhibition was recorded against F. oxysporum, R. stolonifer, P. digitatum and C. gloeosporioides at 30000 mg/L [186, 187].

The MW of chitosan has also a significant effect on the antifungal activity and several studies reported that the activity was increased as the MW decreased however, it is difficult to find a clear correlation between the MW and the antifungal activity [148, 163, 175, 180, 188]. COS are effective in inhibition of spore germination, germ tube elongation and mycelial growth of fungal phytopathogens such as Alternaria kikuchiana, A. solani, B. cinerea, F. grami-nearum, F. oxysporum, R. stolonifer, Penicillium, Physalospora piricola, P. cap-sici, Pyricularia oryzae, and V.dahliae [180, 189-191]. Moreover, chemical modification of chitosan with different substituents effectively enhanced the antifungal activity. The derivatives include N-(benzyl)chitosan derivatives against B. cinerea, Botryodiplodia theobromae, F. oxysporum, P. oryzae, and P. infestans [106, 110, 165, 192, 193], N,O-acyl chitosans against B. cinerea [78], heterocyclic chitosan derivatives against P. debaryanum and F. oxysporum [177], N-(cinnamyl) chitosan derivatives [162], ammonium dithiocarbamate chitosan and triethylenediamine dithiocarbamate chitosan [194], Schiff bases of phenyl and hydroxyphenyl chitosans against B. cinerea [195], (1,2,3-Triazol-4-yl) methyl nicotinate chitosan against R. solani, Stemphylium solani, and A. porri [196], diethyl dithiocarbamate chitosan against A. porri, Gloeosporium theae-sinensis, and S. solani [197], α-aminophosphonate chitosan derivatives against P. asparagi and F. oxysporum [198], acyl chitosan derivatives against R. solani and S. rolfsii [199], thiadiazole chitosan derivatives against C. lagenarium, P. asparagi and Monilinia fructicola [200], thiosemicarbazone chitosan derivatives against S. sol-ani, R. solani, A. solani, and P. asparagi [201], and quaternary ammonium chitosan derivatives against B. cinerea, B. theobromae, Cladosporium cucumerinum, C. lagenarium, F. oxysporum, M. fructicola, P. debaryanum, and Ph. infestans [106, 160, 164, 202, 203].

Table 2 List of scientific publications reporting the antimicrobial activity of chitosan and its derivatives against fungi attack plants.