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Chitin and Chitosan
Wiley Series
in
Renewable Resources
Series Editor:
Christian V. Stevens, Faculty of Bioscience Engineering, Ghent University, Belgium
Titles in the Series:

Wood Modification: Chemical, Thermal and Other Processes
Callum A. S. Hill
Renewables‐Based Technology: Sustainability Assessment

Jo Dewulf, Herman Van Langenhove
Biofuels
Wim Soetaert, Erik Vandamme
Handbook of Natural Colorants

Thomas Bechtold, Rita Mussak
Surfactants from Renewable Resources

Mikael Kjellin, Ingegärd Johansson
Industrial Applications of Natural Fibres: Structure, Properties and Technical Applications

Jörg Müssig
Thermochemical Processing of Biomass: Conversion into Fuels, Chemicals and Power

Robert C. Brown
Biorefinery Co‐Products: Phytochemicals, Primary Metabolites and Value‐Added Biomass

Processing
Chantal Bergeron, Danielle Julie Carrier, Shri Ramaswamy
Aqueous Pretreatment of Plant Biomass for Biological and Chemical Conversion to Fuels and
Chemicals
Charles E. Wyman
Bio‐Based Plastics: Materials and Applications

Stephan Kabasci
Introduction to Wood and Natural Fiber Composites

Douglas D. Stokke, Qinglin Wu, Guangping Han
Cellulosic Energy Cropping Systems

Douglas L. Karlen
Introduction to Chemicals from Biomass, 2nd Edition

James H. Clark, Fabien Deswarte
Lignin and Lignans as Renewable Raw Materials: Chemistry, Technology and Applications
Francisco G. Calvo‐Flores, Jose A. Dobado, Joaquín Isac‐García, Francisco J. Martín‐Martínez
Sustainability Assessment of Renewables‐Based Products: Methods and Case Studies
Jo Dewulf, Steven De Meester, Rodrigo A. F. Alvarenga
Cellulose Nanocrystals: Properties, Production and Applications

Wadood Hamad
Fuels, Chemicals and Materials from the Oceans and Aquatic Sources
Francesca M. Kerton, Ning Yan
Bio‐Based Solvents
François Jérôme and Rafael Luque
Nanoporous Catalysts for Biomass Conversion

Feng-Shou Xiao and Liang Wang
Thermochemical Processing of Biomass: Conversion into Fuels, Chemicals and Power,

2nd Edition
Robert C. Brown
Forthcoming Titles:
The Chemical Biology of Plant Biostimulants
Danny Geelen, Lin Xu
Biorefinery of Inorganics: Recovering Mineral Nutrients from Biomass and Organic Waste
Erik Meers, Gerard Velthof
Waste Valorization: Waste Streams in a Circular Economy

Sze Ki Lin, Chong Li, Guneet Kaur, Xiaofeng Yang
Process Systems Engineering for Biofuels Development

Adrián Bonilla-Petriciolet, Gade Pandu Rangaiah
Biobased Packaging: Material, Environmental and Economic Aspects

Mohd Sapuan Salit, Rushdan Ahmad Ilyas
Chitin and Chitosan:
Properties and
Applications
Edited by
LAMBERTUS A.M. VAN DEN BROEK

Wageningen Food & Biobased Research
Wageningen
The Netherlands
CARMEN G. BOERIU
Wageningen Food & Biobased Research
Wageningen
The Netherlands
This edition first published 2020
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Contents
List of Contributors xvii

Series Preface xxi
Prefacexxiii
1 Sources of Chitin and Chitosan and their Isolation 1

Leen Bastiaens, Lise Soetemans, Els D’Hondt, and Kathy Elst
1.1 Chitin and Chitosan 2
1.1.1 Chemical Structure 2
1.1.2 Different Crystalline Forms of Chitin 2
1.2 Sources of Chitin and Chitosan 5
1.2.1 Sources of Chitin 5
1.2.2 Sources for Chitosan 10
1.3 Isolation of Chitin 11
1.3.1 Technology Principles 11
1.3.2 Isolation of Chitin from Crustaceans 13
1.3.3 Isolation of Chitin from Insects 16
1.3.4 Isolation of Chitin from Other Biomass Types 16
1.4 Production of Chitosan 19
1.4.1 Conversion of Chitin to Chitosan 19
1.4.2 Chitosan Extracted from Fungi 24
1.5 Towards Commercial Applications 25
1.6 Outlook 28
References28
2 Methods of Isolating Chitin from Sponges (Porifera) 35

Sonia Ż ółtowska, Christine Klinger, Iaroslav Petrenko, Marcin Wysokowski,
Yvonne Joseph, Teofil Jesionowski, and Hermann Ehrlich
2.1 Introduction 35
2.2 Brief Overview of Classical Methods of Isolating Chitin from
Invertebrates38
viii Contents
2.3 The Modern Approach to Chitin Isolation from Sponges 40

2.3.1 Methods of Isolating Chitin from Glass
Sponges (Hexactinellida) 41
2.3.2 Methods of Isolating Chitin from
Demosponges (Demospongiae) 43
2.4 Prospective Applications of Poriferan Chitin 49
2.4.1 Poriferan Chitin and Modern Bioinspired Materials Science 49
2.4.2 Chitinous 3D Scaffolds of Sponge Origin for
Tissue Engineering 51
2.5 Outlook 54
Acknowledgment54
References54
3 Physicochemical Properties of Chitosan and its Degradation Products 61

Karolina Gzyra‐Jagieła, Bożenna Pęczek, Maria Wis ́niewska‐Wrona, and
Natalia Gutowska
3.1 Physicochemical Properties of Chitosan 62
3.1.1 Determination of Molar Mass 62
3.1.2 Determination of the Deacetylation Degree 67
3.1.3 Determination of Dynamic Viscosity 70
3.1.4 Determination of Nitrogen 70
3.1.5 Determination of Ash Content 71
3.1.6 Determination of Heavy Metal Content 71
3.1.7 Determination of Water Retention Value (WRV) 72
3.1.8 Determination of Solubility in Hydrochloric Acid 72
3.1.9 Determination of Water Content 72
3.1.10 Determination of Protein Content 73
3.1.11 Quantitative Determination of Chitosan by Ninhydrin 73
3.2 Products of Degradation and their Application 74
3.3 Outlook 77
References77
4 New Developments in the Analysis of Partially Acetylated Chitosan

Polymers and Oligomers81
Stefan Cord‐Landwehr, Anna Niehues, Jasper Wattjes, and
Bruno M. Moerschbacher
4.1 Introduction 82
4.2 Chitosan Oligomers 83
4.2.1 Degree of Polymerisation (DP), Fraction and Pattern of
Acetylation (FA and PA)83
4.3 Chitosan Polymers 86
4.3.1 Molecular Weight (MW) / Degree of Polymerisation (DP)
and its Dispersity (ÐMW / ÐDP)86
4.3.2 Fraction of Acetylation (FA) and its Dispersity (ÐFA)87
4.3.3 Pattern of Acetylation (PA)89
4.4 Outlook 91
References92
Contents ix
5 Chitosan‐Based Hydrogels 97
Zhengke Wang, Ling Yang, and Wen Fang
5.1 Introduction 97
5.2 Chitosan‐Based Multilayered Hydrogels 98
5.2.1 Periodic Precipitation 99
5.2.2 Alternating Process 100
5.2.3 Induced by Electrical Signals 100
5.2.4 Layer‐by‐Layer (LbL) Assembly 101
5.2.5 Sequential Curing 101
5.3 Chitin/Chitosan Physical Hydrogels Based on Alkali/Urea Solvent System 103
5.3.1 Chitin Hydrogels Based on Alkali/Urea Solvent System 104
5.3.2 Chitosan Hydrogels Based on Alkali/Urea Solvent System 104
5.4 Chitosan‐Based Injectable Hydrogels 108
5.4.1 Physical Association Networks 108
5.4.2 Chemical Association Networks 110
5.4.3 Double‐Network Hydrogels 116
5.5 Chitosan‐Based Self‐Healing Hydrogels 119
5.5.1 Physical Interactions 119
5.5.2 Dynamic Chemical Bonds 121
5.6 Chitosan‐Based Shape Memory Hydrogels 125
5.6.1 Water‐/Solvent‐Triggered Shape Recovery 126
5.6.2 pH‐triggered Shape Recovery 126
5.6.3 Ultrasound Triggered Shape Recovery 126
5.6.4 Self‐Actuated Shape Memory Hydrogels 127
5.6.5 Chitosan‐Based Hydrogels with Triple Shape Memory Effect 127
5.7 Superabsorbent Chitosan‐Based Hydrogels 131
5.7.1 Cross‐Linked Chitosan‐Based Hydrogels 132
5.7.2 Hydrogels by Graft Copolymerization 133
5.7.3 Chitosan‐Based Composite Hydrogels 134
5.7.4 Pure Chitosan‐Based Materials 135
5.8 Outlook 136
References136
6 Beneficial Health Effects of Chitin and Chitosan 145

Liyou Dong, Harry J. Wichers, and Coen Govers
6.1 Immunomodulatory Effects of Chitin and Chitosan as Demonstrated
with In Vitro Studies 146
6.2 Beneficial Health Effects Mediated by Chitin and Chitosan as
Demonstrated with Animal Studies 149
6.2.1 Immune Modulation 149
6.2.2 Anti‐Pathogenic Effects 155
6.2.3 Anti‐Tumour Effects 157
6.3 Beneficial Health Effects Mediated by Chitin and Chitosan as
Demonstrated with Clinical Trials 158
6.3.1 Cholesterol Reduction and CVD Preventive Effects 158
6.3.2 Other Health Effects 160
6.4 Requirements to forward the Field of Study Towards the Beneficial
Health Effects of Chitin and Chitosan 163
x Contents
6.5 Outlook 164

Acknowledgement164
References164
7 Antimicrobial Properties of Chitin and Chitosan 169

Magdalena Kucharska, Monika Sikora, Kinga Brzoza‐Malczewska, and
Monika Owczarek
7.1 Microbiological Activity of Chitosan – The Mechanism of its Antibacterial
and Antifungal Activity 169
7.2 The use of Chitin/Chitosan’s Microbiological Activity in Medicine
and Pharmacy171
7.3 Microbiological Activity of Chitosan in the Food Industry 174
7.4 Microbiological Activity of Chitosan in Paper and Textile Industries 176
7.5 Microbiological Activity of Chitosan in Agriculture 177
7.6 Outlook 181
References181
8 Enzymes for Modification of Chitin and Chitosan 189

Gustav Vaaje‐Kolstad, Tina Rise Tuveng, Sophanit Mekasha, and
Vincent G.H. Eijsink
8.1 CAZymes in Chitin Degradation and Modification 190
8.1.1 Chitinases 191
8.1.2 β‐N‐acetylhexosaminidases195
8.1.3 Exo‐β‐glucosaminidases195
8.1.4 Chitosanases 197
8.1.5 Lytic Polysaccharide Monooxygenases 199
8.1.6 Carbohydrate Esterases 200
8.1.7 Carbohydrate‐Binding Modules 204
8.2 Modular Diversity in Chitinases, Chitosanases and LPMOs204
8.3 Biological Roles of Chitin‐Active Enzymes 205
8.4 Microbial Degradation and Utilisation of Chitin 208
8.4.1 Chitin Degradation by Serratia marcescens209
8.4.2 Chitin Degradation by Bacteria in the Bacteroidetes
Phylum211
8.4.3 Chitin Degradation by Thermococcus Kodakarensis211
8.4.4 Chitin Degradation by Fungi 212
8.5 Biotechnological Perspectives 213
8.6 Biorefining of Chitin‐Rich Biomass 214
8.7 Outlook 216
References216
9 Chitin and Chitosan as Sources of Bio‐Based Building Blocks

and Chemicals229
Malgorzata Kaisler, Lambertus A.M. van den Broek, and
Carmen G. Boeriu
9.1 Introduction 230
9.2 Chitin Conversion into Chitosan, Chitooligosaccharides and
Monosaccharides232
Contents xi
9.2.1 Chitosan Production 232

9.2.2 Production of Chitooligosaccharides 234
9.2.3 Production of GlcNAc and GlcN from Chitin 235
9.3 Building Blocks for Polymers from Chitin and its Derivatives 238
9.3.1 Furan‐Based Monomers 238
9.3.2 Amino Alcohol and Amino Acid Building Blocks 239
9.4 Outlook 239
Acknowledgement240
References240
10 Chemical and Enzymatic Modification of Chitosan to Produce New

Functional Materials with Improved Properties 245
Carmen G. Boeriu and Lambertus A.M. van den Broek
10.2 Functional Chitosan Derivatives by Chemical and Enzymatic
Modification246
10.2.1 Anionic Chitosan Derivatives 248
10.2.2 Hydroxyalkylchitosans 250
10.2.3 Quaternised and Highly Cationic Chitosan Derivatives 250
10.2.4 Hydroxyaryl Chitosan Derivatives 250
10.2.5 Carbohydrate‐Modified Chitosan 251
10.3 Graft Co‐Polymers of Chitosan 251
10.4 Cross‐Linked Chitosan and Chitosan Polymer Networks 254
10.5 Outlook 254
References255
11 Chitosan‐Based Drug Delivery Systems 259

Cristian Peptu, Andra Cristina Humelnicu, Razvan Rotaru,
Maria Emiliana Fortuna, Xenia Patras, Mirela Teodorescu,
Bogdan Ionel Tamba, and Valeria Harabagiu
11.2 Beneficial Effects of Chitosan 261
11.2.1 Interaction with Anionic Drugs 263
11.2.2 Mucoadhesive Properties 263
11.2.3 Transfection Activity 263
11.2.4 Efflux Pump Inhibitory Properties 265
11.2.5 Permeation‐Enhancing Properties 265
11.3 Chitosan—an Active Polymer for Bypassing Biological Barriers 265
11.3.1 Skin Barrier 266
11.3.2 Mucosa Barrier 267
11.3.3 Ophthalmic Barrier 269
11.3.4 Blood–Brain Barrier 270
11.4 Chitosan‐Based DDS Formulations 271
11.4.1 Hydrogels 275
11.4.2 Micro/NPs 275
11.4.3 Nanofibers 275
11.4.4 Scaffolds and Membranes 275
xii Contents
11.5 Outlook 276

Acknowledgment276
References276
12 The Application of Chitin and its Derivatives for the Design of Advanced
Medical Devices 291
Marcin H. Struszczyk, Longina Madej‐Kiełbik, and Dorota Zielińska
12.1 Selection of the Raw Sources: Safety Criteria 291
12.1.1 Aspect of Animal Tissue‐Originated Derivatives 292
12.1.2 General Requirements for Chitinous Biopolymers Applied
in Designing Medical Devices 292
12.1.3 Characterisation of the Biopolymer for Application in Wound
Dressing Designing293
12.1.4 Aspect of the Sterilization of the Final Wound Dressing 295
12.2 Types of Wound Dressings Consisting of Chitin‐Derived Biopolymers
Available in the Market297
12.3 Performance and Safety Assessment 297
12.4 New Ideas and Concepts 301
12.5 Risk Acceptance and Design Process Aspects 306
12.6 Outlook 308
Acknowledgements308
References308
13 Food Applications of Chitosan and its Derivatives 315

Suse Botelho da Silva, Daiana de Souza, and Liziane Dantas Lacerda
13.2 Chitosan and its Derivatives as Food Additive 316
13.2.1 Antioxidant 318
13.2.2 Antimicrobial 319
13.2.3 Stabilizer and Thickener 319
13.3 Functional Ingredient and Health Beneficial Effects 320
13.4 Active Packaging 321
13.5 Enzyme Immobilization 331
13.6 Encapsulation and Delivering of Bioactive Ingredients 332
13.7 Adsorption and Chelation of Toxic and Undesirable Compounds 334
13.8 Outlook 339
References340
14 Potential of Chitosans in the Development of Edible Food Packaging 349

Véronique Coma and Artur Bartkowiak
14.1 Potential Limitations for Real Introduction into the Market 350
14.1.1 Generally Recognized as Safe (GRAS)351
14.1.2 Solubility 351
14.1.3 Source—Origin 352
14.1.4 Structure Variability 352
14.2 Films and Coatings for Food Preservation 353
Contents xiii
14.2.1 Definitions and Interests 353

14.2.2 Main Relevant Chitosan‐Based Material Properties 353
14.3 Specific Case of Chitosan Nanoparticles (CSNPs)357
14.3.1 CSNPs 357
14.3.2 CSNPs in Various Edible Films 358
14.3.3 Antimicrobial Activities of CSNPs in Edible Films 359
14.3.4 Toxicity Studies of CSNPs360
14.4 Applications to Sensitive Real Food Products 360
14.4.1 Fruits and Vegetables 361
14.4.2 Meat and Meat Products 362
14.4.3 Fish and Seafood Products 362
14.5 Conclusions 364
References364
15 The Use of Chitosan‐Based Nanoformulations for Controlling Fungi

During Storage of Horticultural Commodities 371
Silvia Bautista‐Baños, Zormy Nacary Correa‐Pacheco, and Rosa Isela
Ventura‐Aguilar
15.2 Importance of Fruits and Vegetables 372
15.3 Storage Disorders and Diseases of Horticultural Products 374
15.4 Plant Fungi Inhibition by Chitosan Application 375
15.5 Chitosan Integrated with Other Alternative Methods for Controlling
Postharvest Fungi 376
15.6 Chitosan‐Based Formulations 376
15.7 Physiological Response and Quality Retention of Horticultural
Commodities to Chitosan Coating Application 376
15.8 Influence of Chitosan Coatings on the Shelf Life of
Horticultural Products 378
15.9 Effects of Chitosan Coatings with Additional Compounds on Quality
and Microorganisms Development 379
15.10 Integration of Chitosan Nanoparticles into Coating Formulations
and their Effects on the Quality of Horticultural Commodities
and Development of Microorganisms 384
15.11 Outlook 387
Acknowledgments387
References387
16 Chitosan Application in Textile Processing and Fabric Coating 395

Thomas Hahn, Leonie Bossog, Tom Hager, Werner Wunderlich, Rudi Breier,
Thomas Stegmaier, and Susanne Zibek
16.1 Chitosan in the Textile Industry 396
16.2 Textile Production 398
16.3 General Test Methods 400
16.4 Fibres and Yarns from Chitin and Chitosan 401
16.4.1 Chitin and Chitosan Solubilisation for Spinning Purposes 402
xiv Contents
16.4.2 Chitosan Spinning Processes 402

16.4.3 Mechanical Properties of Chitosan Fibres/Yarns 404
16.5 Sizing with Chitosan 406
16.5.1 Miscibility of Chitosan with Other Sizing Agents 407
16.5.2 Viscosity of Chitosan‐Containing Sizing Agents 408
16.5.3 Adhesion and Wetting 410
16.5.4 Mechanical–Physical Properties of Chitosan Films 411
16.5.5 Removal and Processing of Chitosan Sizing after Weaving 412
16.6 Chitosan as a Finishing Agent or Coating 414
16.6.1 Chitosan as a Carrier and Linker 415
16.6.2 Formation of a Durable Finish with Chitosan 416
16.6.3 Chitosan as an Active Agent 417
16.7 Outlook 419
Nomenclature420
References421
17 Chitin and Chitosan for Water Purification 429

Petrisor Samoila, Andra Cristina Humelnicu, Maria Ignat,
Corneliu Cojocaru, and Valeria Harabagiu
17.2 Wastewater Treatment by Adsorption 432
17.2.1 Principle of the Adsorption Process 432
17.2.2 Adsorption of Organic Compounds 434
17.2.3 Adsorption of Heavy Metals 437
17.3 Wastewater Treatment by Coagulation/Flocculation 440
17.4 Wastewater Treatment by Membrane Separation 446
17.4.1 Principle of Ultrafiltration Process 446
17.4.2 Fabrication of Ultrafiltration Blend Membranes 448
17.4.3 Chitosan‐Enhanced Ultrafiltration 450
17.5 Outlook 452
Acknowledgement452
References453
18 Chitosan for Sensors and Electrochemical Applications 461

Suse Botelho da Silva, Guilherme Lopes Batista, and
Cristiane Krause Santin
18.2 Chitosan: A Biopolymer with Unique Properties 462
18.3 Modification and Preparation of Chitosan‐Based Materials
for Electrochemical Applications 463
18.4 The Proton Conductivity of Chitosan 465
18.5 Selected Applications 467
18.5.1 Electrochemical Sensors 467
18.5.2 Spectroscopic Sensors 470
18.5.3 Other Electrochemical Devices 471
18.6 Outlook 472
References473
Contents xv
19 Marketing and Regulations of Chitin and Chitosan from Insects 477

Nathalie Berezina and Antoine Hubert
19.1 Historical Outline 477
19.2 Natural Origins of Chitin 478
19.3 Specificities of Chitin Biopolymer 479
19.4 Differences Among Chitins from Insects and Other Sources 479
19.4.1 Differences of Chemical Compositions of the Cuticles 479
19.4.2 Differences of Physical Assemblies of Chains and Molecules 480
19.5 Extraction and Purification Specificities of Chitins from Insects 480
19.5.1 Different Cuticle Structures and Contents of Insects 480
19.5.2 Chemical Extraction 480
19.5.3 Biological Extraction 481
19.5.4 Characterization and Transformation into Chitosan 481
19.6 Market Opportunities and its Regulations 482
19.6.1 Agriculture Applications 482
19.6.2 Water Treatment Applications 483
19.6.3 Material Applications 483
19.6.4 Biomedical Applications 484
19.7 Outlook 485
References485
Index491
List of Contributors
Artur Bartkowiak Center of Bioimmobilisation and Innovative Packaging Materials,

Faculty of Food Sciences and Fisheries, West Pomeranian University of Technology,
Szczecin, Poland
Leen Bastiaens VITO (Flemish Institute for Technological Research), Mol, Belgium
Silvia Bautista‐Baños Centro de Desarrollo de Productos Bióticos (CEPROBI), Instituto
Politécnico Nacional (IPN), Yautepec, Morelos, Mexico
Nathalie Berezina Ynsect, Évry, France
Carmen G. Boeriu Wageningen Food & Biobased Research, Wageningen, The
Netherlands
Leonie Bossog Textilchemie Dr. Petry GmbH, Reutlingen, Germany
Suse Botelho da Silva Food and Chemical Engineering, Polytechnic School, Unisinos
University, São Leopoldo, RS, Brazil
Rudi Breier Textilchemie Dr. Petry GmbH, Reutlingen, Germany
Lambertus A.M. van den Broek Wageningen Food & Biobased Research, Wageningen,
The Netherlands
Kinga Brzoza‐Malczewska Institute of Biopolymers and Chemical Fibres, Lodz, Poland
Corneliu Cojocaru ‘Petru Poni’ Institute of Macromolecular Chemistry, Romanian
Academy, Iași, Romania
Véronique Coma University of Bordeaux, LCPO, UMR 5629, Centre National de la
Recherche Scientifique (CNRS), Pessac, France
Stefan Cord‐Landwehr University of Münster, Institute for Biology and Biotechnology
of Plants, Münster, Germany
Zormy Nacary Correa‐Pacheco CONACYT-CEPROBI, Instituto Politécnico Nacional,
Yautepec, Morelos, Mexico
xviii List of Contributors
Els D’Hondt VITO (Flemish Institute for Technological Research), Mol, Belgium
Liyou Dong Food & Health Research, Wageningen Food & Biobased Research,
Wageningen, The Netherlands; Food Chemistry, Wageningen University, Wageningen, The
Netherlands
Hermann Ehrlich Institute of Electronics and Sensor Materials, TU Bergakademie‐
Freiberg, Freiberg, Germany
Vincent G.H. Eijsink Faculty of Chemistry, Biotechnology, and Food Science, The
Norwegian University of Life Sciences (NMBU), Ås, Norway
Kathy Elst VITO (Flemish Institute for Technological Research), Mol, Belgium
Wen Fang Institute of Biomedical Macromolecules, Department of Polymer Science
and Engineering, Zhejiang University, Hangzhou, China
Maria Emiliana Fortuna ‘Petru Poni’ Institute of Macromolecular Chemistry,
Romanian Academy, Iași, Romania
Coen Govers Food & Health Research, Wageningen Food & Biobased Research,
Wageningen, The Netherlands
Natalia Gutowska Institute of Biopolymers and Chemical Fibres, Lodz, Poland
Karolina Gzyra‐Jagieła Institute of Biopolymers and Chemical Fibres, Lodz, Poland
Tom Hager German Institutes of Textile and Fiber Research, Denkendorf, Germany
Thomas Hahn Fraunhofer Institute of Interfacial Engineering and Biotechnology,
Stuttgart, Germany
Valeria Harabagiu ‘Petru Poni’ Institute of Macromolecular Chemistry, Romanian
Antoine Hubert Ynsect, Évry, France
Andra Cristina Humelnicu ‘Petru Poni’ Institute of Macromolecular Chemistry,
Romanian Academy, Iași, Romania
Maria Ignat ‘Petru Poni’ Institute of Macromolecular Chemistry, Romanian Academy,
Iași, Romania
Teofil Jesionowski Institute of Chemical Technology and Engineering, Faculty of
Chemical Technology, Poznan University of Technology, Poznan, Poland
Yvonne Joseph Institute of Electronics and Sensor Materials, TU Bergakademie‐
Freiberg, Freiberg, Germany
Malgorzata Kaisler Bioprocess Engineering Group, Wageningen University,
Wageningen, The Netherlands; Wageningen Food & Biobased Research, Wageningen, The
Netherlands
Christine Klinger Institute of Physical Chemistry, TU Bergakademie‐Freiberg,
Freiberg, Germany
List of Contributors xix
Cristiane Krause Santin Food and Chemical Engineering, Polytechnic School, Unisinos
University, São Leopoldo, RS, Brazil; itt CHIP – Unisinos Semiconductor Institute, São
Leopoldo, RS, Brazil
Magdalena Kucharska Institute of Biopolymers and Chemical Fibres, Lodz, Poland
Liziane Dantas Lacerda Food and Chemical Engineering, Polytechnic School, Unisinos
Guilherme Lopes Batista itt CHIP – Unisinos Semiconductor Institute, São Leopoldo,
RS, Brazil
Longina Madej‐Kiełbik The Institute of Security Technologies “MORATEX”, Lodz,
Poland
Sophanit Mekasha Faculty of Chemistry, Biotechnology, and Food Science, The
Bruno M. Moerschbacher University of Münster, Institute for Biology and
Biotechnology of Plants, Münster, Germany
Anna Niehues University of Münster, Institute for Biology and Biotechnology of Plants,
Münster, Germany
Monika Owczarek Institute of Biopolymers and Chemical Fibres, Lodz, Poland
Xenia Patras ‘Petru Poni’ Institute of Macromolecular Chemistry, Romanian Academy,
Iași, Romania
Bożenna Pe ̨czek Institute of Biopolymers and Chemical Fibres, Lodz, Poland
Cristian Peptu ‘Petru Poni’ Institute of Macromolecular Chemistry, Romanian Academy,
Iași, Romania
Iaroslav Petrenko Institute of Experimental Physics, TU Bergakademie‐Freiberg,
Freiberg, Germany
Razvan Rotaru ‘Petru Poni’ Institute of Macromolecular Chemistry, Romanian
Petrisor Samoila ‘Petru Poni’ Institute of Macromolecular Chemistry, Romanian
Monika Sikora Institute of Biopolymers and Chemical Fibres, Lodz, Poland
Lise Soetemans VITO (Flemish Institute for Technological Research), Mol, Belgium
Daiana de Souza Food and Chemical Engineering, Polytechnic School, Unisinos
Thomas Stegmaier German Institutes of Textile and Fiber Research, Denkendorf,
Germany
Marcin H. Struszczyk The Institute of Security Technologies “MORATEX”, Lodz,
Poland
xx List of Contributors
Bogdan Ionel Tamba A&B Pharm Corporation, Roman, Neamt ̦, Romania

Mirela Teodorescu ‘Petru Poni’ Institute of Macromolecular Chemistry, Romanian
Tina Rise Tuveng Faculty of Chemistry, Biotechnology, and Food Science, The
Gustav Vaaje‐Kolstad Faculty of Chemistry, Biotechnology, and Food Science, The
Rosa Isela Ventura‐Aguilar CONACYT-CEPROBI, Instituto Politécnico Nacional,
Yautepec, Morelos, Mexico
Zhengke Wang Institute of Biomedical Macromolecules, Department of Polymer
Science and Engineering, Zhejiang University, Hangzhou, China
Jasper Wattjes University of Münster, Institute for Biology and Biotechnology of
Plants, Münster, Germany
Harry J. Wichers Food & Health Research, Wageningen Food & Biobased Research,
Wageningen, The Netherlands; Food Chemistry, Wageningen University, Wageningen, The
Netherlands
Maria Wis ń iewska‐Wrona Institute of Biopolymers and Chemical Fibres, Lodz, Poland
Werner Wunderlich German Institutes of Textile and Fiber Research, Denkendorf,
Germany
Marcin Wysokowski Institute of Chemical Technology and Engineering, Faculty of
Chemical Technology, Poznan University of Technology, Poznan, Poland; Institute of
Electronics and Sensor Materials, TU Bergakademie‐Freiberg, Freiberg, Germany
Ling Yang Institute of Biomedical Macromolecules, Department of Polymer Science
and Engineering, Zhejiang University, Hangzhou, China
Susanne Zibek Fraunhofer Institute of Interfacial Engineering and Biotechnology,
Stuttgart, Germany
Dorota Zielińska The Institute of Security Technologies “MORATEX”, Lodz, Poland
Sonia Ż ółtowska Institute of Chemical Technology and Engineering, Faculty of
Chemical Technology, Poznan University of Technology, Poznan, Poland; Institute of
Electronics and Sensor Materials, TU Bergakademie‐Freiberg, Freiberg, Germany
Series Preface
Renewable resources, their use and modification are involved in a multitude of important
processes with a major influence on our everyday lives. Applications can be found in the
energy sector; paints and coatings; and the chemical, pharmaceutical, and textile industry,
to name but a few.
The area interconnects several scientific disciplines (agriculture, biochemistry, chemis-
try, technology, environmental sciences, forestry), which makes it very difficult to have an
expert view on the complicated interaction. Therefore, the idea to create a series of scien-
tific books, focusing on specific topics concerning renewable resources, has been very
opportune and can help to clarify some of the underlying connections in this area.
In a very fast‐changing world, trends are not only characteristic of fashion and political
standpoints; science too is not free from hypes and buzzwords. The use of renewable
resources is again more important nowadays; however, it is not part of a hype or a fashion.
As the lively discussions among scientists continue about how many years we will still be
able to use fossil fuels – opinions ranging from 50 to 500 years – they do agree that the
reserve is limited, and that it is essential not only to search for new energy carriers but also
for new material sources.
In this respect, the field of renewable resources is a crucial area in the search for alterna-
tives for fossil‐based raw materials and energy. In the field of energy supply, biomass‐ and
renewables‐based resources will be part of the solution alongside other alternatives such as
solar energy, wind energy, hydraulic power, hydrogen technology and nuclear energy. In
the field of material sciences, the impact of renewable resources will probably be even big-
ger. Integral utilisation of crops and the use of waste streams in certain industries will grow
in importance, leading to a more sustainable way of producing materials. Although our
society was much more (almost exclusively) based on renewable resources centuries ago,
this disappeared in the Western world in the nineteenth century. Now it is time to focus
again on this field of research. However, it should not mean a ‘retour à la nature’, but
should be a multidisciplinary effort on a highly technological level to perform research
towards new opportunities, to develop new crops and products from renewable resources.
This will be essential to guarantee an acceptable level of comfort for the growing number
of people living on our planet. It is ‘the’ challenge for the coming generations of scientists
to develop more sustainable ways to create prosperity and to fight poverty and hunger in
the world. A global approach is certainly favoured.
xxii Series Preface
This challenge can only be dealt with if scientists are attracted to this area and are recog-
nised for their efforts in this interdisciplinary field. It is, therefore, also essential that con-
sumers recognise the fate of renewable resources in a number of products.
Furthermore, scientists do need to communicate and discuss the relevance of their work.
The use and modification of renewable resources may not follow the path of the genetic
engineering concept in view of consumer acceptance in Europe. Related to this aspect, the
series will certainly help to increase the visibility of the importance of renewable resources.
Being convinced of the value of the renewables approach for the industrial world, as well
as for developing countries, I was myself delighted to collaborate on this series of books
focusing on the different aspects of renewable resources. I hope that readers become aware
of the complexity, the interaction and interconnections, and the challenges of this field, and
that they will help to communicate on the importance of renewable resources.
I certainly want to thank the people of Wiley’s Chichester office, especially David
Hughes, Jenny Cossham and Lyn Roberts, in seeing the need for such a series of books on
renewable resources, for initiating and supporting it, and for helping to carry the project to
the end.
Last, but not least, I want to thank my family, especially my wife Hilde and children
Paulien and Pieter‐Jan, for their patience, and for giving me the time to work on the series
when other activities seemed to be more inviting.
Christian V. Stevens,
Faculty of Bioscience Engineering
Ghent University, Belgium
Series Editor, ‘Renewable Resources’
June 2005
Preface
Chitin was reported for the first time about 200 years ago, in extracts of mushrooms and
insects. About 40 years later, chitosan was obtained from chitin by acid treatment. These
polysaccharides are among the most abundant natural biopolymers in the world. They are,
for example, present in crustaceans, insects and fungi. Just before World War II, there was
a huge interest in the applications of these polysaccharides as a bioplastic. However, the
simultaneous upcoming of synthetic polymers and the exponential increase in high‐
performance synthetic polymers, which outperformed their natural counterparts, resulted
in a decrease of interest in chitin/chitosan materials. In the 1970s, large‐scale production
of chitin and chitosan from the shells of marine organisms started, owing to the develop-
ment of aquaculture and the enactment of severe environmental regulations to decrease
the amount of shellfish dumping in the oceans. Nowadays there is a need to be less
dependent on fossil resources. The transition to a biobased economy and the increasing
societal demand for more green and environmentally friendly products urge us to look for
chemicals, materials and fuels based on renewable resources. The enormous potential of
chitin and chitosan on account of their abundance, unique properties and numerous appli-
cations makes them interesting biomass resources. This book, Chitin and Chitosan:
Properties and Applications, shows the state‐of‐the‐art and future perspectives of chitin
and chitosan materials and applications. The book presents the most recent developments
in the science and technology of all related fields, from extraction and characterisation to
modification, material synthesis and end‐user applications. This book comprises 19 chapters
that deal with most topics related to chitin and chitosan polymers and materials.
In Chapters 1–4, the sources of chitin and chitosan are described and how these biopoly-
mers can be isolated. Next to the isolation, the analysis of the biopolymers is described.
The different sources and/or isolation methods can result in different structures and proper-
ties. In Chapter 5–7, hydrogels, health effects and the anti‐microbial effects of chitin and
chitosan are discussed. To improve or to modify the properties, enzymes and chemical
reactions can be applied to customise these biopolymers, as shown in Chapters 8–10. The
applications of chitin and chitosan in drug delivery, medical devices, agriculture, food,
packaging, horticulture, textile, water purification and sensors are discussed in more detail
in Chapters 11–18. And finally, Chapter 19 is devoted to the market and regulation of chitin
and chitosan.
xxiv Preface
These topics have never been addressed previously in a single book. Books, book chap-
ters and reviews have been dedicated to the specific fields of application of chitin and chi-
tosan materials. This book presents an overview of the latest scientific and technological
advances in almost all areas of application, and show the great potential of chitin and chi-
tosan as materials of the future. We hope that the reader will be inspired by the examples
given of these biopolymers in different areas. We are confident that chitin and chitosan will
become major renewable resources in the biobased circular economy.
This book should be useful for scholars and those in academia, such as undergraduate
and postgraduate students in the areas of agriculture, polymer and material sciences,
biobased economy and life sciences. In addition, we hope this book will aid researchers
and specialists from industry in the field of (bio)polymers, packaging, biomedical applica-
tions, water treatment, textiles, sensors, and agriculture and food – as well as regional and
national policy‐makers.
The input is from well‐known experts from all over the world. We would like to express
our great gratitude to all chapter authors of this book, who have made excellent contribu-
tions. In addition, we would like to thank Sarah Higginbotham, Emma Strickland and
Lesley Jebaraj from Wiley for all their help.
Lambertus A.M. van den Broek and Carmen G. Boeriu

Wageningen 2019
1
Sources of Chitin and Chitosan
and their Isolation
Leen Bastiaens, Lise Soetemans, Els D’Hondt, and Kathy Elst
VITO – (Flemish Institute for Technological Research), Mol, Belgium
Chitin is a natural biomolecule that was reported for the first time in 1811 by the French
professor Henri Braconnot as fungine [1] and in 1823 by Antoine Odier as chitin. Chitin
consists of large, crystalline nitrogen‐containing polysaccharides made of chains of a mod-
ified glucose monosaccharide, being N‐acetylglucosamine. It is ubiquitously present in the
world and has even been reported to be one of the most abundant biomolecules on earth,
with an estimated annual production of 1011–1014 tons [2, 3]. Chitin serves as template for
biomineralization such as calcification and silicification, providing preferential sites for
nucleation, and controlling the location and orientation of mineral phases [4, 5]. This phe-
nomenon explains the presence of chitin in solid structures in a variety of biomass such as
cell walls of fungi and diatoms and in exoskeletons of Crustaceans. Chitin is present in
diverse structures in at least 19 animal phyla besides its presence in bacteria, fungi, and
algae [5].
Chitosan is mainly known as a partially deacetylated derivative of chitin that is more
water soluble than chitin, and as such is easier to process. For this reason, chitosan—and,
in some cases, even more preferably, the relatively small sized (1–10 kDa) chitosan
oligomers—are the molecules that are envisioned for multiple applications such as agricul-
ture; water and wastewater treatment; food and beverages; chemicals; feed; cosmetics; and
personal care [6, 7]. In addition, chitosan oligomers have been reported as being bioactive
[8], offering potential for application in, for instance, wound dressing and cosmetics.
Although chitin and chitosan are versatile and promising biomaterials [9], the extraction
Chitin and Chitosan: Properties and Applications, First Edition.

Edited by Lambertus A.M. van den Broek and Carmen G. Boeriu.
© 2020 John Wiley & Sons Ltd. Published 2020 by John Wiley & Sons Ltd.
2 Chitin and Chitosan: Properties and Applications
and purification of chitin and its conversion to chitosan (oligomers) require several process
steps, and these have been mentioned as bottlenecks that hinder the wider use of the under-
spent chitin in the world.
This chapter intends to provide more information related to (1) the structure of chitin, (2)
sources of chitin and chitosan, and (3) their extraction and purification, as well as (4) the
conversion of chitin into chitosan. The further conversion of chitosan to chitosan oligomers
is the subject of Chapter 3.
1.1 Chitin and Chitosan

1.1.1 Chemical Structure
Chitin, and its derivate chitosan, are natural polysaccharides consisting of 2 monosaccha-
rides, N‐acetyl‐D‐glucosamine and D‐glucosamine, connected by β‐1,4‐ glycoside bonds.
Depending on the frequency of the latter monosaccharides, the molecule is defined as
chitin or chitosan. Chitin contains mainly N‐acetyl‐D‐glucosamine and can be transformed
to chitosan by partial deacetylation of the monomer N‐acetyl‐D‐glucosamine to D‐glucosa-
mine (see Figure 1.1) [7]. Diverse definitions of chitin and chitosan circulate in literature.
Most sources mention a deacetylation degree of at least 50% [7, 10] as a criterion to define
the molecule as chitosan. Others report a deacetylation degree of at least 60% or 75% for
chitosan, implying that, respectively, more than 60% or 75% of the monosaccharides are
D‐glucosamine moieties [11–13]. Chitin in its natural appearance is usually already a het-
eropolymer, with a deacetylation degree ranging between 5% and 20% [14]. The structure
of chitin is very similar to that of cellulose and shares generally the same function of pro-
viding structure integrity and protection of the organism.
1.1.2 Different Crystalline Forms of Chitin

Chitin usually functions as a supporting material and is composed of layers of polysaccha-
ride sheets. Each individual sheet consists of multiple parallel‐positioned chitin chains
[17], as schematically presented in Figure 1.2. Highly crystalline fibers are formed when
the polymer sheets are placed next to each other and form interactions [12]. Depending on
their orientation, three crystalline forms have been reported (α, β, and γ).
The most abundant form is α‐chitin, which is present in almost all crustaceans, insects,
fungi, and yeast cell walls [7]. In this formation, the chitin sheets (three sheets as example
in Figure 1.2a), consisting of parallel chitin chains (for each sheet, two chains are pre-
sented in Figure 1.2a), are positioned in an anti‐parallel way, allowing a maximum forma-
tion of hydrogen bonding. More specifically, two intramolecular and two intermolecular
bondings are formed: a first intermolecular bonding with a vertical neighbor chain (in the
same sheet), and another with a horizontal neighbor chain form a different sheet [15].
These hydrogen bounds create a remarkably high crystallinity, resulting in a more stiff
and stable material. Therefore, α‐chitin is characterized as a non‐reactive and insoluble
product [16]. Since this form is the most common polymorphic, α‐chitin has been exten-
sively studied [12].
On the other hand, in β‐chitin, the chitin sheets are ordered in parallel (Figure 1.2b)
with weaker intermolecular forces. This results in a softer molecule with a higher affinity
Insects
Algae Crustaceans
Chitin
OH OH OH OH
H O H O H H O H H O H
H O H H O H
H H H H
OH OH OH OH
O H
O O
H HN H H NH H NH
NH2
O O O
H3C H3C H3C
n
Deacetylation
Chitosan
Fungi OH OH Mollusks
OH OH
H O H O H H O H H O H
H O H H O H
H H H H
OH OH OH OH
O H
O O
H NH2 H H NH H NH2
NH2
O
H 3C
n
Figure 1.1 Chemical structure of chitin and chitosan and some examples of species that contain chitin.
(a) (b)
Sheet 1
H C
H C H C
H C
H C H C O
H C O O
H C
HO O O
H C O O
H C HO
O O O O
O O
OH O
O NH OCH
O
O NH OCH NH OCH O
O O O HO
O OH
HO O NH CH
OH NH OCH NH CH HO
NH OCH O NH CH
O H C NH HO HO
HO
OH NH CH HO HO OH
NH CH H CO NH O OH OH
HO OH
HO OH
OH O
O OH OH
O OH O
OH
O O
O O O
O O O
OH O O O O
O O O
O O O
O NH OCH
O
OH NH OCH NH OCH O
OH NH OCH O O HO
NH OCH H C O HO NH CH
O NH HO NH CH HO
OH NH CH NH CH
HO NH CH O HO
H CO NH HO OH
HO OH HO
HO O OH OH
OH O OH
O O OH OH OH
OH O
O O O O O
O
OH O
O
O O O O
O O O O
O OH O O
O OH NH OCH O NH OCH
NH OCH O NH OCH NH OCH O
O H C NH HO O HO
OH NH CH HO O NH CH
HO NH O NH CH HO
CH H CO NH NH CH
HO HO
HO OH HO OH
OH O
O OH HO
O
OH
OH OH
OH OH
O O OH O
O O
OH O O
O O O
O OH O O O O
O OH O O O
O NH OCH O O
O NH OCH H C NH HO NH OCH
OH NH OCH O
HO NH CH O O NH OCH HO
NH CH H CO NH O HO NH
HO HO CH
HO OH NH CH NH CH
OH O HO
O HO OH
O OH OH HO
OH HO
OH
HO O OH
CH OH OH HO
HO HO
HO CH HO
Chain 1 n HO HO
HO
n
n n
Figure 1.2 Schematic representation of (a) α‐form and (b) β‐form of chitin.
Sources of Chitin and Chitosan and their Isolation 5
for solvents and a higher reactivity. It is proven to be soluble in formic acid and can be
swollen in water [15]. This chitin form is present in the squid pen, in the tubes of pogo-
nophoran and vestimentiferan worms, and in monocrystalline spines excreted by diatoms
such as Thalassiosira fluviatilis [7]. Although squid and tubes of Tevnia jerichonana both
contain β‐chitin, their crystallinity differs. This implies that the crystallinity also depends
on the source. Chitin obtained from squid pens is semi‐crystalline, and chitin from T. jeri-
chonana is almost complete crystalline [7, 8, 16].
The third formation, γ‐chitin, is less common. It is considered to be a mixture or inter-
mediate form of α‐ and β‐chitin with both parallel and antiparallel arrangements [16]. More
specifically, every third chitin chain has the opposite direction to the two preceding chitin
sheets [13, 15]. Very few studies have been carried out on γ‐chitin, and it has been s uggested
that γ‐chitin may be a distorted version of the other two instead of a true third polymorphic
form.
1.2 Sources of Chitin and Chitosan

1.2.1 Sources of Chitin
For more than a century, scientists reported chitin to be present in a variety of organ-
isms. Initially, zoologists named all hard yellow–brownish structures chitin, without
chemical analysis, sometimes generating misleading data. Later on, it was accepted that
the presence of chitin could only be demonstrated after chemical tests. Hymann (1958),
for instance, used an iodine‐based color test to study the presence of chitin in different
sea animals. Later on, more sophisticated techniques such as Fourier transform infrared
spectroscopy (FTIR), nuclear magnetic resonance (NMR), mass spectroscopy (MS),
X‐ray diffraction (XRD), and Raman spectroscopy were used [18]. Quantification of
chitin is challenging and only reported in more recent publications. Currently, quantita-
tive data on chitin contents are still incomplete, and available numbers need to be inter-
preted with care. Not only are different quantification methods used, but also varying
parts of the biomass are considered (whole organism versus chitin‐rich part of the
organisms).
Nowadays, it is estimated that a large portion of chitin produced in the biosphere is
present in the oceans [19, 20]. It can be found in aquatic species belonging to phyla
such as Cnidaria (corals [21, 22]), Entoprocta [23], Phoronida (horseshoe worms [18]),
Ectoprocta [18], Brachiopoda (lamp shells [18]), Bryozoa [19], Porifera (sponges [5,
24]), and Mollusca (squid [8, 23], cuttlefish [26], and clams [8]). Further, chitin has
also been detected in fungi (mushrooms and yeasts [1]), algae (diatoms [27], coralline
algae [28], green algae [29, 30]), Onychophora (velvet worms), and protozoa [31]. The
most easily accessible sources of chitin, however, are the exoskeletons of Arthropoda,
which includes insects [32–35], arachnids (spiders [36] and scorpions [37]), myria-
pods (millipedes and centipedes [38]), as well as Crustaceans (shrimp, krill, crab, and
lobster [8, 9, 18, 37]).
Table 1.1 lists examples of chitin‐containing sources, along with available compositional
data. The amount of chitin varies with species type, the biomass part considered, and even
with seasons and growth stages [40]. Values ranging from <1% to 72% (w/w) on dry matter
basis on the biomass type have been reported.
Table 1.1 Sources of chitin.
% (w/w) chitin in biomass

Origin Species Biomass type N (%) CaCO3 (%) % protein type of dry weight Ref.
Crustaceans (Arthropoda phylum)
Crab cuticle 40–50 15–30* [8]
Blue swimming crab (male) Portunus pelagicus Shells 68.87 10.33 20.8 [26]
Blue swimming crab (female) Portunus pelagicus Shells 65.5 14.36 20.14 [26]
Crabs Shells 66.58 16.68 16.73 [25]
Marbled crab Grapsus marmoratus 10 [41]
Red crab Portunus puber 10 [41]
Spider crab Maja squinado 16 [41]
Cancer crab Cuticula 72.1 [40]
Carcinus crab Whole body 64.2 [40]
King crab Paralithodes Whole body 35 [40]
Shrimp cuticle 20–30 30–40* [8]
Jinga shrimp Metapenaeus affinis Shells 45.66 37.59 16.75 [26]
Brown shrimp Penaeus aztecus Shells 48.97 29.5 21.53 [25]
Pink shrimp Penaeus duorarum Shells 42.26 34.02 23.72 [25]
Shrimp Palaemon Fabricius 22 [41]
Shrimp Penaeus monodon Shells 5.74 10 [33]
Grooved tiger prawn Penaeus semisulcatus Shells 52.03 28.84 19.13 [26]
Scyllarid lobster Thenus orientalis Shells 61.81 16.93 21.26 [26]
Locust lobster Scyllarus arctus 25 [41]
Spiny lobster Palinurus vulgaris 32 [41]
European lobster Homarus vulgaris 17 [41]
Crayfish Procambarus clarkii Shells 63.94 15.56 20.6 [25]
Crayfish Astacus fluviatilis 36 [41]
Krill cuticle 20–25 20–30* [8]
Barnacle Lepas anatifera 7 [41]
Squilla Squilla mantis 24 [41]
Isopoda Oniscus asellus Dried adult 4.7 6–7* [32]
0004461239.INDD 6 10/26/2019 1:16:41 PM

Insects (Arthropoda phylum)
Honey bees Apis mellifera Exoskeletons 5.56 2.5 [33]
Grasshopper Aiolopus simulatrix Fully dried adult 5.3* [42]
Aiolopus strepens Fully dried adult 7.4* [42]
Duroniella fracta Fully dried adult 5.7* [42]
Duroniella laticornis Fully dried adult 6.5* [42]
Oedipoda miniata Fully dried adult 8.1* [42]
Oedipoda caerulescens 8.9* [42]
Pyrgomorpha cognata 6.6* [42]
Schistocerca gregaria Exoskeletons 2.92 12.2 [33]
Black soldier fly Hermetia illucens Fully dried prepupae 2.7–19.7 2 37.7–40.7 5.6–6.7 [34]
Hermetia illucens Fully dried larvae 17.5 2.10 3 [43]
Hermetia illucens Whole larvae 6.4 Own work
Lesser mealworm Alphitobius diaperinus Whole worm 5.6 Own work
Beetle Melolontha melolontha Fully dried adult 6.72 13–14* [35]
Beetle Calosoma rugosa Exoskeletons 5 [33]
Cockroach Blattella 18.4 ^ [40]
Cockroach Periplaneta Cuticle 54.8^ [40]
Blatta lateralis Fully dried nymphs 19.0 0.67 3 [43]
Silkworm Bombyx 44.2^ [40]
Bombyx mori L. Cuticle 23–52 36–62 [44]
Waxworm Galleria 33.7^ [40]
Tebo worms Chilecomadia moorei Fully dried larvae 15.5 1.11 3 [43]
Tobacco hornworm Manduca sexta Exoskeleton of the 60 20 [21]
adult (organic part)
Ladybug Coleoptera 27–35^ [40]
Shield bug Palomena prasina Fully dried adult 10.8* [45]
Butterfly Pieris 2 [40]
Housefly Musca domestica Fully dried adults 19.7 1.19 3 [43]
Mollusks (Mollusca phylum)
Squid (Cephalopoda) Pen Negligible 20–40* [8]
Pen 4.74 46.23 49 [25]
Loligo vulgaris 40 [41]
(Continued )
0004461239.INDD 7 10/26/2019 1:16:41 PM

Table 1.1 (Continued)
% (w/w) chitin in biomass

Origin Species Biomass type N (%) CaCO3 (%) % protein type of dry weight Ref.
Cuttlefish (Cephalopoda) Sepia spp. Shells 91.25 1.35 7.4 [26]

Pens 88.48 6.12 5.4 [25]
Sepia officinalis 20 [41]
Clam/oyster (Bivalvia) Shell 85–90 3–6* [8]
Other animals
Bryozoa Plumatella repens Dried 13.3* [32]
Black coral (Cnidaria) Antipathella fiordensis Skeleton (organic 70 10 [21]
part)
Horseshoe worms Tube [18]
(Phoronida)
Sponges (Porifera) [28]
Spiders (Arachnids) Geolycosa vultuosa 6.42 8–8.5 [36]
Hogna radiata 6.41 5.5–7 [36]
Fungi
Basidiomycota (yeast) Fomes fomentarius 2.4* [45]
Lactarius vellereus 19 [40]
Full biomass 11 [46]
Basidiomycota (mushroom) Agaricus bisporus Cell wall 43.8 [47]
Zygomycota Mucor rouxii Cell wall 50.1 [48]
44.5 [40]
Rhizopus oryzae Full biomass 14.6 [49]
Ascomycota (yeast) Aspergillus niger Cell wall 42 [40, 48]
Penicillium chrysogenum Cell wall 20.1 [40]
Penicillium notatum Cell wall 18.5 [40]
Saccharomyces cerevisiae Cell wall 2.9 [40]
Algae
Diatoms Thalassiosira fluviatilis Ropes [50]
Green algae Pithophora oedogonia Cell wall [30]
Chlorella vulgaris Cell wall [29]
Note: *not provided how it is measured; ^based on the weight of the organic cuticle, others were measured based on weight differences of the raw materials and that of the sample
obtained after acid and alkaline treatments2, crude ash3 based on acid detergent fiber, minus present amino acids.
0004461239.INDD 8 10/26/2019 1:16:41 PM

1.2.1.1 Crustaceans (Part of Arthropoda)

Chitin is located in the exoskeletons of Arthropoda. The skeleton is a tough and hard mate-
rial designed for mechanical support to the body and functions as an armor against preda-
tors. These characteristics can be dedicated to the presence of highly crystalline α‐chitin
that, combined with proteins, forms a hybrid material with high stiffness (at least 150 GPa).
These chitin–protein complexes, together with minerals for strength, form a fibrous struc-
ture that is among the most resistant organic materials [38, 52]. The shells of crustaceans
mainly contain chitin (20–30%), proteins (20–40%), minerals (30–60%), pigments, and
sometimes also lipids (0–14%) [8, 38]. Based on Table 1.1, it can be said that the chitin
content ranges from 6% to 72% in crustacean shells. This large variation can be explained
by the origin of the biomass (e.g., differences in species such as gray shrimp versus pink
shrimp, or in growth phase), the part of the biomass considered for chitin analysis (e.g.,
shells as such or stripped of remaining flesh), or the varying pretreatment or analysis
method used. Crabs and shrimp are mostly used at an industrial level and contain 10–72%
chitin. As mentioned previously, the differences in species impacts the chitin content—for
example, the Cancer crab contains 72% chitin as compared to 64% chitin in the Carcinus
crab and 35% chitin in the king crab.
1.2.1.2 Insects (Part of Arthropoda)

Chitin is found in the exoskeleton of insects, but also in internal structures such as the inner
cuticular linings of the alimentary canal and the tracheal system. In contrast with the exo-
skeletons of crustaceans, insect cuticles contain also catecholamines besides chitin, pro-
teins, lipids, and minerals. The catecholamines are cross‐linked by o‐quinones with proteins
and possibly also with chitin [14]. α‐Chitin in the exoskeleton of insects serves the same
function as for crustaceans. It increases the strength of the skeleton, gives structure, pre-
vents physical and chemical damages, and protects against infectious diseases [14]. The
chitin content differs significantly between different insect species. In addition, Kaya et al.
found that the chitin content is also significantly dependent on the life stage of the insect.
The larvae of Vespa crabro (wasp) had a chitin content of 2.2% dry weight (DW) in com-
parison with 6.2% for pupa and 10.3% of the adult. This phenomenon can be explained by
the different chitin functions in different body parts during different stages [52]. Similar
results were obtained by Kaya et al. with the larvae of the potato beetle (7% chitin) and the
adult beetle (20% chitin) [53].
1.2.1.3 Other Sources

Spiders (also part of the Arthropoda) contain α‐chitin (5–8.5%) with a high acetylation
degree. Kaya et al. characterized physicochemically the chitin structure of two spider spe-
cies (Geolycosa vultuosa and Hogna radiata), for which acetylation degrees of 97% and
99% were found, respectively [36]. Within the Mollusca, squids receive major attention
because they are the prototype for β‐chitin. In addition, high chitin content (up to 49%) in
the pen have been reported [25], which may be the basis to conclude that squid pen can
become increasingly common as another potentially important chitin source [54]. However,
it should be kept in mind that these high percentages are related to the composition of the
chitin‐rich pen that represents a very minor fraction of the whole squid. Chitin may be
involved in the formation of skeletons in calcifying marine sponges [28]. Sponges are
described more in detail in Chapter 4. Within the Cnidarian taxa, skeletons often contain,
besides chitin, calcium‐based minerals. Black corals form an exception and have a unique
halogenated scleroprotein named antipathin associated with chitin [22]. Lophophorates
(marine and freshwater Octopoda, Phoronida, Brachiopoda) have exoskeletons, named
tubes, that consists of chitin [18]. Chitin is the most important ultrastructural compound of
fungal cell walls, where it is embedded in the amorphous matrix and provides the frame-
work of the cell wall morphology [55]. It exists in the spores, mycelia, and stalks, and has
only been detected as α‐chitin [55]. Its amount ranges from 2% to 50% (w/w) on dry cell
wall base, whereby the lowest value corresponds to yeasts [56] and the highest to
Euascomycetes [55]. Depending on the class of fungi, the cell wall can also contain glu-
cans, mannans, as well as chitosan. As the cell wall is only a part of the fungal biomass, the
overall chitin (plus chitosan) yield is lower, and values have been reported (glucosamine on
dry matter base) of 8–16% (w/w) for Aspergillus niger and Mucor rouxii mycelia [48] and
12% (w/w) for Agaricus bisporus stalks [47].
In the case of algae, since 1965, diatoms such as Thalassiosira were reported to secrete
β‐chitin ropes that span between two recently divided daughter cells to keep them together,
creating flexible cell chains that float in the water [27, 50]. However, chitin has also been
shown to be present in diatoms in other forms—for instance, in the siliceous shell. In calci-
fied coralline algae such as Clathromorphum compactum, chitin has been reported to be
present that strengthens the skeleton and protects the algae from ocean acidification and
grazing in shallow waters [28]. The presence of chitin was also demonstrated in the cell
walls of the green algae Pithophora oedogonia [30] and Chlorella vulgaris [29]. Quantitative
data on the chitin content in the algae, however, are scarce. The fact that chitin in algae is
plant‐based can be an advantage for some applications.
1.2.2 Sources for Chitosan

Chitosan is mainly known as a partially deacetylated derivative of chitin, but has also
been found to be naturally present in some types of biomass. Some fungi contain chi-
tosan as an important constituent of their cell wall at various stages their life cycle. The
class of Zygomycetes (e.g., the Mucor, Absidia, Benjaminiella, Cunninghamella,
Gongronella, and Rhizopus genera) has especially been recognized as a valuable source
of chitosan [59, 60]. Chitosan content of 1–10% on dry biomass base have been found
with a reported degree of deacetylation of 83–94%. Chitosan is not directly synthesized,
but is rather the result of an efficient conversion of chitin to chitosan by the presence of
a deacetylase enzyme [57]. The deacetylation enzymes are thought to be in close proxim-
ity to the regions where the chitin transverses the plasma membrane [59]. As chitin is
synthesized, the deacetylase enzyme converts it to chitosan [57]. Since chitosan can be
isolated with less extreme procedures, fungi may become an interesting source of chi-
tosan in the future [26].
In addition to fungi, bacteria too have been reported to be able to convert chitin into
chitosan using enzymatic deacetylation. Kaur et al. isolated, from soil, bacteria (Bacillus
sp. and Serritia sp.) that produce chitin deacetylase and release chitosan. Although the
efficiency of this process is limited due the insolubility of chitin [13], it contributes to the
degradation of chitin in nature. Further, these fast‐growing bacteria, or the isolated enzymes,
offer a tool for biological chitosan production [3] (see Section 1.4).
1.3 Isolation of Chitin

Although large quantities of chitin are available and future applications look promising,
chitin is still an underutilized resource. Limited attention has been given to chitin, in con-
trast to the extensive studies on cellulose, because of the relatively laborious isolation pro-
cess. Chitin present in biomass is strongly connected to other biomolecules such as proteins
and minerals. These impurities need to be removed to generate a high‐purity product for
application development [60]. Traditional extraction processes are long, involve high con-
sumption of hazardous chemicals, are energy consuming, and are environmentally pollut-
ing due to the need for high amounts of mineral acids and alkali [8, 40, 62].
1.3.1 Technology Principles

The process steps that have been reported to isolate and purify chitin from biomass are
summarized in Figure 1.3, and explained in more detail in the subsequent subsections. It
concerns biomass pre‐treatment, deproteination, demineralization, decoloration, and post‐
treatment processes. Depending on the biomass type, the individual steps can change order,
or may even not be required. The degree of crystallinity and the deacetylation degree of
chitin depend on the source, and may also alter during the purification process, as was
shown for crustaceans [62].
Chitin
Pre- Post-
containing treatment Deproteination Demineralisation Decoloration treatment Chitin
biomass
Figure 1.3 Processes involved in the isolation and purification of chitin from biomass.
1.3.1.1 Pre‐Treatment
The pretreatment step groups all activities that are required to prepare the biomass for chi-
tin extraction. This may comprise the removal of soft tissues by scraping, boiling, and
pressing. For other organisms, boiling can be a part of a hygienization step. The biomass is
mostly dried and reduced in size. However, for fermentation purposes, for example, drying
is not necessary. Examples of pre‐treatments are given in Table 1.2.
1.3.1.2 Demineralization
When a high amount of minerals is present, demineralization is advisable. For example, the
exoskeleton of crustaceans can contain more than 50% (w/w) of CaCO3 to enhance its
strength [38, 52]. Two approaches have been reported—chemical and biological deminer-
alization. Chemical demineralization uses acids such as HCl, HNO3, H2SO4, CH3COOH,
and HCOOH [60]. Among these acids, HCl is the most commonly used reagent for remov-
ing the mineral constituents [40]. Biological demineralization is based on acid‐producing
biological processes using bacteria [3, 39, 40] or enzymes such as Alcalase® [40]. The acids
Table 1.2 Examples of pre‐treatment steps.
Origin Biomass type Pretreatment step(s) Ref.

Crustaceans Shells Washed with tap water or boiled; drying, mechanical [63]
crushing to reduce size; sieving aiming at 60–120 μm
fraction
Shrimp shells Pressing and washing to remove residual meat and [64]
connective tissue
Shrimp waste Shells were dried at 50°C for 24 h and homogenized in [65]
a laboratory mixer
Shrimp waste Washed with tap water, dehydrated at room [66]
temperature, and shred into pieces smaller than 2 mm
Shrimp shells Scraped to remove loose tissue, washed and dried, and [33]
finally grounded to pass through a 250 μm sieve
Insects Grasshopper Cleaned by washing with distilled water, dried at room [42]
temperature and pulverization
Crickets, waxworms, Starved for 24 hours to clear the gastrointestinal tract of [67]
mealworm, any residual food, killed by freezing
silkworms, beetles
Beetles Starved for 48 hours to clear the gastrointestinal tract of [68]
any residual food, washed with water, and killed by
freezing. Thawed at room temperature and air‐dried at
50°C for 2 days. The dried beetles were milled to a
powder to pass through a 20‐mesh screen.
Mollusks Squid pen The squid pens were washed with water, dried, and cut [69]
in small pieces (sieved from 2 to 5 mm).
Squid pen Samples were ground into about 18 mesh (ASTM) size [70]
formed (such as lactic acid) react with calcium carbonate, resulting in a precipitate that can
be removed by washing. Khanafari et al. stated that fermentation is less likely to change the
physicochemical characteristics of the chitin chain [65]. Table 1.3 summarizes some dem-
ineralization procedures as examples.
1.3.1.3 Deproteination
Chitin is traditionally viewed as a chain that is embedded in a protein‐matrix. During the
purification step, complete removal of proteins is advisable when applications in medical,
food, or feed are envisioned since proteins can be allergenic [10]. The proteins are bound
by multiple hydrogen bridges, and the ‘free’ amino groups of the chitin may covalently be
bound to the proteins [8, 51]. Therefore, extreme process parameters are often required for
the deproteination. Chemical deproteination is commonly performed with sodium hydrox-
ide as a preferential reagent. The effectiveness of alkali deproteination depends on the
process temperature, the alkali concentration, as well as the alkali/biomass ratio [26].
During deproteination, chitin may undergo some changes such as partial deacetylation and
hydrolysis of the polymer [10]. Also, biological deproteination using (1) proteases (such as
pepsin, papain, and trypsin) secreted by proteolytic bacteria in the fermentation medium or
(2) isolated proteases (crude or purified) has been reported [39, 40]. As the conditions are
less harsh as compared to chemical deproteination, small peptides and amino acids remain
attached to the chitin chain after enzymatic hydrolysis [10], and the deacetylation degree is
altered less (less increase) [72]. Some examples are summarized in Table 1.4.
Table 1.3 Examples of demineralization steps.
Origin Biomass type Demineralization approach (scale) Ref.

Crustaceans Crustacean shell Chemical: 0.2–2 M HCl; 1–48 hours; 0–100°C [39]
waste
Prawn shells Chemical: 0.5–1% (v/v) HCl; 24 hours; 25°C; 4:1 or 10:1 (v/w) [63]
Shrimp shells Chemical: 0.25 M HCl; 0.25 hours; 25°C; 40:1 (v/w); 2–5 [60]
repeats
Shrimp waste Biological: Lactobacillus plantarum (PTTC 1058), Lactobacillus [65]
acidophilus (PTTC 1643), and Lactobacillus rhamnosus (PTCC
1637) were added (OD600 =0.8–1) to 50 mL water + 5 g of
shrimp waste. Incubation for 7 days at 30°C and 5% CO2
Shrimp shells Biological: Lactobacillus plantarum PTCC 1058—5% w/v [40]
shrimp shell powder—addition of peptone, yeast extract,
meat extract, K2HPO4
Crustaceans/ Barnacles, crab, Chemical: 0.5–2 M HCl; 0.5–1.5 hours; 4–50°C; 10:1 (v/w): [71]
mollusks lobster, crayfish, 1–3 repeats
shrimp, cuttlefish,
squid
Prawn, shrimp, crab, Chemical: 0.25 M HCl; 0.25–3 hours; 40 mL/g [26]
lobster, cuttlefish
Marine shell waste Chemical: 0.55 M HCl; 2 hours; 25°C; 11.25: 1 (v/w) [41]
Crustaceans/ Shrimp shells, Desert Chemical: 1 M HCl; 25°C; 15 mL/g [33]
insects locus, honey bee,
beetles
Insects Palomena prasina Chemical: 2 M HCl; 2 hours; 100°C [45]
Grasshopper Chemical: 4 M HCl; 1 hour; 75°C [42]
Beetle Chemical: 1 M HCl; 30 min; 100°C [68]
1.3.1.4 Decoloration and Other Post‐Treatment Processes

When desired, a decoloration step can be introduced to remove pigments (such as pink
colors for crustaceans). A mild oxidizing treatment with, for example, hydrogen peroxide
[74] or potassium permanganate [75], or an extraction with solvents such as acetone [63],
ethanol, and chloroform [42], have been reported. Finally, some post‐treatment steps such
as neutralization, drying, and milling may be required to finalize the chitin production
process (Table 1.5).
1.3.2 Isolation of Chitin from Crustaceans

Isolation of chitin from crustaceans is well studied and documented. Generally, chitin is
produced from crustacean waste (shells) through washing and crushing of the raw material
in a pretreatment step (Table 1.2). The shells are washed with tap water or boiled before
drying [64]. After drying, the shells are mechanically crushed. Different particle sizes are
obtained and reported, such as, for example, 60–120 μm [63], less than 250 μm [26],
0.5–1 mm [76], or 0.5–10 mm [77].
Acid calcination is selected most often as a demineralization step (Table 1.3), with HCl
as the preferred reagent [40]. A concentration below 1 M is usually applied at room tem-
perature. For crustaceans, the mineral content differs significantly, resulting in the need for
different treatments [25]. As a result, the time and number of repeats can differ, depending
on the biomass. Percot et al. found that, for shrimp shells, the demineralization was
Table 1.4 Examples of deproteination steps.
Origin Biomass type Deproteination approach (scale) Ref.

Crustaceans Marine shell waste Chemical: 30–50% (w/v) NaOH; 1–72 hours; 0–100°C [40]
Shell waste Chemical: 1 M NaOH; 1–72 hours; 65–100°C [39]
Shrimp shells Chemical: 1% (w/v) NaOH; 24 hours; 70°C [60]
Chemical: 0.3 M NaOH; 1 hour; 80–85°C; repeat till [41]
absence of color
Shrimp shells Biological: Protease production of Bacillus cereus SV1 [72]
was used, shells with water (1:2 w/v) were cooked for
20 min and protease was added, incubation for
3 hours at 40°C
Shrimp shells Biological: Bacillus mojavensis A21 and Bacillus subtilis [71]
A26 were used to produce proteases, 15 g shrimp
shells + 45 mL water was mixed with the protease
Crustaceans/ Shrimp waste Chemical: 0.5–1.25 M NaOH; 0.5–24 hours; 60–100°C; [66]
Mollusca 1:10 (w/v)Room temperature
Fish scales Chemical: 0.5 M NaOH; 18 hours; 25°C; repeated till [73]
absence of color
Crustaceans/ Shrimp shells, squid Chemical: 1 M NaOH; 105–110°C; repeated till absence [25]
Mollusca pens, crab shells, of color
lobster shells
Prawns shells Chemical: 5% (w/v) NaOH; 2 hours; 60°C; 8:1 (v/w) [63]
Bryozoan, Chemical: 2 M NaOH; 20 hours; 140°C; [45]
insects,
fungal
Marine shell waste Chemical: 0.3 M NaOH; 1 hour; 80–85°C; 2–7 repeats [74]
Insects Grasshopper Chemical: 2 M NaOH; 18 hours; 175°C [42]
Beetle Chemical: 1 M NaOH; 24 hours; 80°C [68]
c omplete within 15 min at ambient temperature [60]. Younes et al. performed a two‐level
fractional factorial design and concluded that the number of batches and the concentration
of HCl are the most influential parameters for the demineralization efficiency. As tempera-
ture was not found to be a significant parameter, lower temperatures are preferred to reduce
chain degradation and chitin deacetylation. The best demineralization for shrimp was
found when using lower temperatures and longer reaction times [71]. Tolaimate et al. con-
firmed that the number of repeats (a multi process) had a positive influence, but did not find
a relation between higher acid concentration and longer reaction time in their settings [74].
Biological demineralization by the use of fermentation, for instance, solid‐state fermenta-
tion with organic acid–producing bacteria, received more attention recently as being a
more eco‐friendly, safer, and more technologically flexible alternative to chemical demin-
eralization. Arbia et al. and Kaur et al. gave an overview of the most common bacteria
utilized [39, 40]. Lactic acid–producing bacteria were usually selected, for example,
Lactobacillus spp. B2 or L. plantorum A3. Other acid‐producing microorganisms that were
tested include Bacillus subtilis, Pseudomonas aeruginosa K‐187, and Serratia marcescens
FS‐3. Since the acid also lowered the pH, which activated proteases [10], demineralization
and deproteination occurred partially simultaneously. Khanafari et al. proved that deminer-
alization using fermentation could be evenly effective if not better than chemical deminer-
alization. They also stated that fermentation was less likely to change the physicochemical
characteristics of the chitin chain [65].
Table 1.5 Examples of decoloration and post‐treatment steps.
Origin Biomass type Post treatments approach Ref.

Crustaceans Shrimp waste Sample filtrated under vacuum and washed with [66]
deionized water
Decoloration: hydrogen peroxide (35% (v/v)) overnight
at room temperature
Post‐treatment: filtered, washed, and dried at 50°C for 3
hours
Shrimp waste Post‐treatment: filtered, washed to neutrality, freeze‐ [72]
dried
Shrimp shells, Sample washed with deionized water to neutrality and [25]
crab shells, lobster dried
shells Decoloration: KMnO4 + oxalic acid + H2SO4 or refluxing
in ethanol for 6 hours
Post‐treatment: filtered, washed, and dried at 50°C for 3
hours
Prawn, shrimp, crab, Post‐treatment: washed to neutrality and washed with [26]
lobster, cuttlefish hot ethanol (10 mL/g), and later boiled in acetone to
remove any impurities; purified chitin then dried
Bryozoa, Decoloration: refluxed with distilled water, methanol, [45]
insect, and chloroform (4:2:1), respectively, room
fungi temperature, 2 hours
Post‐treatment: neutralized and dried (3 days, 50°C)
Insect Grasshopper Decoloration: chloroform, methanol, and distilled water [42]
(1:2:4), rinsed with distilled water
Post‐treatment: dried (60°C, 24 hours)
Beetle Decoloration: 1% (w/v) potassium permanganate [68]
solution, 1 hour, rinsed with distilled water
Post‐treatment: dried (50°C)
In respect to deproteination (Table 1.4), a chemical approach with sodium hydroxide is

often used, even if reaction conditions are very variable. The concentration of NaOH ranges
from 0.1 to 5 M, and the temperature can increase up to 160°C. Chang and Tsai found that
2.5 M NaOH at 75°C and a minimal solution‐to‐solid ratio of 5 mL/g was the most effec-
tive to clean shrimp shells [78]. In contrast, Tokatli and Demirdoven did a Box–Behnken
experimental design and found that 0.95 M NaOH for 75.6 min at 60.5°C were the optimal
conditions to deproteinate a similar biomass [66]. Both studies did not assess possible
changes on the physicochemical properties of the polymer. During deproteination, partial
deacetylation of chitin has often been reported [10]. Nevertheless, Percot et al. found that
temperature and time had no influence on hydrolysis and deacetylation with NaOH con-
centrations below 1 M and temperatures below 70°C [60]. In summary, literature reported
divided conclusions concerning optimal process parameters, which can be attributed to the
different sources (species) used or the different pretreatment applied. For example, Younes
et al. also found that the particle size obtained in the pretreatment step impacted the effi-
ciency of the purification of chitin [71]. Biological deproteination with proteolytic enzymes
and protease‐producing bacteria is an alternative to remove proteins during chitin isolation
[39, 40]. In comparison to chemical deproteination, the efficiency of the enzymatic method
is lower, leaving approximately 5–11% residual protein [10, 72]. In addition, the use of
crude proteolytic extracts obtained from different microorganisms or even from fish v iscera
have been studied [61]. Although the deproteination levels achieved in such cases are gen-
erally lower than those obtained using alkaline treatments, this alternative has the advan-
tage to produce nutritionally valuable protein hydrolysates in addition to chitin.
In conclusion, the intensity of the demineralization and deproteination steps depends on
the source [74]. It is generally accepted that these steps significantly change the physico-
chemical properties of chitin, for example, the molecular weight and acetylation degree
[40, 60]. Both the degree of crystallinity and the acetylation degree of chitin depend on the
source and the method of purification [62]. Most researchers favor performing the demin-
eralization first, followed by deproteination [74]. However, it is considered that the order
of these two phases in interchangeable depending on the source [61, 79].
1.3.3 Isolation of Chitin from Insects

In recent years, the production of proteins from agri‐waste using insects has increased
rapidly, and numerous startup companies have emerged. This has led to a new chitin‐rich
side‐stream composed of spent prepupae shell, residue after molting, and dead insects,
which offers a tremendous potential for chitin and chitosan production as a by‐product.
The procedure to isolate chitin from insect cuticles is very similar to the one used in crus-
taceans. Nevertheless, insects have lower levels of inorganic material, allowing a more
gentle demineralization step [2]. Some examples of chitin extractions from insects are
given in Table 1.6. Only a few articles could be found, indicating that insect chitin is a
relatively new field. For this reason, no standard or engineered procedure for insects was
found.
1.3.4 Isolation of Chitin from Other Biomass Types

As mentioned in Section 1.2.1.3, fungi can contain considerable amounts of chitin, reach-
ing up to 50% of the cell wall on a dry matter base. They are, however, one of the very few
biomass sources in which chitosan can naturally be present in significant amounts, and
many studies target the isolation of chitosan rather than that of chitin (see Section 1.4).
Nevertheless, the isolation of (crude) chitin is regularly targeted on a lab scale, mostly to
investigate its properties [29, 48, 63]. As the characteristics of the fungal biomass differ
from the ones of the traditional and commercial source of chitin (i.e., crustaceans), the
chitin isolation procedure deviates in some aspects from process steps as outlined in
Figure 1.2. During the pretreatment, the filtered and washed fungal biomass is typically
dried, which requires a more extensive dewatering/drying step, given the wet nature of the
biomass. No demineralization step is performed, since the fungal biomass does not contain
appreciable quantities of calcium carbonate and other mineral salts [88]. After pretreat-
ment, a base extraction is applied, which not only aims to remove the proteins but also the
alkali‐soluble polysaccharides, leaving the alkali‐insoluble cell wall behind. This chitin‐
rich cell fraction is characterized and used [89, 90] as such or further purified to remove the
remaining glucans, for instance, by selective enzymatic hydrolysis [91] (or separate chi-
tosan, if present).
Mollusks are generally processed following the standard procedure elaborated for crus-
taceans. Nevertheless, some differences are reported. For instance, it was noticed that the
CO2 emission was stronger in case of cuttlefish than other (crustacean) species [26].
Table 1.6 Examples of chitin extraction procedures applied on insects.
Insect Insect stage Pretreatment Demineralization Deproteination Post‐treatment Ref.

Colorado potato Larvae/adult Drained in 70% (v/v) 20 g + 100 mL 2 M 50 mL 2 M NaOH Filtered and washed, drained in [53]
beetle beetle alcohol, washed, dried at HCl at 65–75°C for at 80–90°C for chloroform (1), methanol (2), and
room temperature, 2 hours 16 hours water (4) for 1 hour, filtered and
grounded, dried at 65°C washed, dried at 60°C
for 3 hours
Crickets: Gryllus Dried crickets, grounded, (3) Oxalic acid, (1) 40 g + 400 mL (2) Drained in ammonium persulfate [80]
bimaculatus washed, and dried at 60°C 3 hours at room 1 M NaOH 95°C solution (50% (w/v)) at 50°C for
temperature for 6 hours 6 hours
(4) Dried at 60°C overnight
Calliptamus Grounded, dried at 70°C for 2 g + 100 mL 1 M HCl 1 M NaOH at Washed and dried at 55°C, drained [81]
barbarus and 30 min at 100°C for 30 min 80–90°C for in chloroform (1), methanol (2),
Otopharynx 21 hours and water (4) for 1 hour
decorus
House fly: Musca Fourth instar Washed with 15% (w/v) (3) 10 mg/mL oxalic (1) 10 g + 100 mL (2) 10 mg/mL potassium [82]
domestica larvae aqueous NaCl, freeze‐ acid aqueous for 1 M NaOH at permanganate aqueous solution
dried, grounded 3 hours 95°C for 6 hours for 4 hours
(4) Neutralized and freeze‐dried
Silkworm Chrysalides Dried by lyophilization for 1 M HCl at 100°C for 1 M NaOH at 80°C Washed with Na2CO3 0.4% (w/v), [83]
12 hours 20 min (10 mL for 24 hours dried at 80°C
HCl/g) (10 mL NaOH/g)
Blowfly: Larvae Washed with NaCl, dried at (3) Oxalic acid (10 mg/ (1) 10 g + 100 mL (2) Decolored with NaCl (0.5%, [84]
Chrysomya 50°C, grounded mL) for 3 hours NaOH 1 M at w/v) for 3 hours
megacephala 95°C for 6 hours (4) Washed to neutral with distilled
water and then freeze‐dried in
vacuum
Black soldier fly: Pupal exuviae Cleaned, dried, grounded 1 M HCl for 1 hour 1 M NaOH at 80°C 1% KMnO4 and excess of KMnO4 [85]
Hermetia and dead for 24 hours was removed by 4% (w/v) oxalic
illucens imago acid
House cricket: 8 weeks old Starved for 48 hours, (2) 200 mL 10 M (1) 20 g + 200 mL 200 mL 1% NaCl (1%, w/v), at room [86]
Brachytrupes frozen‐washed with water, oxalic acid at room 1 M NaOH at temperature for 3 hours, filtered,
portentosus dried at 60°C for 48 hours temperature for 95°C for 6 hours washed to neutral pH, dried at
3 hours 60°C overnight
(Continued)
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Table 1.6 (Continued)
Insect Insect stage Pretreatment Demineralization Deproteination Post‐treatment Ref.

Beetle: Holotrichia Adult beetles Starved for 48 hours, 5 g + 250 mL 1 M HCl 1 M NaOH at 80°C Washed till neutral pH, 1% (w/v) [68]
parallela washed, frozen, defrosted, at 100°C for 30 min for 24 hours potassium permanganate for
dried at 50°C for 48 hours, 1 hour, washed and dried at 5°C
grounded
7 Orthoptera Grasshoppers Washed, dried at room 2 g + 100 mL 4 M HCl 50 mL 2 M NaOH Filtered, washed, drained in [42]
species temperature, grounded at 75°C for 1 hour at 175°C for chloroform (1), methanol (2), and
18 hours water (4), washed with distilled
water, dried at 60°C for 24 hours
Desert locus, Exoskeletons Scraped free from loose 1 M HCl at room 1 M NaOH at Filtered, washed till neutral pH, [33]
beetles, honey tissue, washed, dried, temperature 100°C for washed with hot ethanol and
bee grounded (15 mL/g) 8 hours, repeated boiled in acetone, dried in
several times vacuum oven at 50°C
Melolontha Adults Killed using a killing agent, 2 g + 50 mL 4 M HCl 4 M NaOH at Filtered, washed till neutral pH, [35]
melolontha washed, dried at 60°C for at 75°C for 2 hours 150°C for drained in chloroform (1),
24 hours, grounded 18 hours methanol (2), and water (4) for
20 min, washed, filtered, dried at
60°C for 24 hours
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1.4 Production of Chitosan

Due to the high crystallinity, chitin is insoluble in many common solvents. Even if there are
some exceptions, however, these solvents are usually toxic, corrosive, or degradative, and
thus cannot be used in scaling up procedures for pharmaceutical applications [92]. Due to
this insoluble nature of chitin, attention has been given to convert chitin into more soluble
derivatives. Among the various reactions that can disrupt the intra‐ and intermolecular
bonds causing the high crystallinity, N‐deacetylation is the simplest modification, trans-
forming chitin to chitosan [79]. Chitosan is generally insoluble in organic solvents and
water, but soluble in diluted acid solutions with a pH below 6. This is because the chitosan
amino groups have pKa values around 6.3, implying that, at low pH, the amines will be
protonated and become positively charged. There is a positive correlation between the
degree of deacetylation (DDA) and the number of positive charges potentially present in
the chitosan chain. The charge density, as well as the exact pKa value, and in turn also the
solubility of chitosan, are highly dependent on DDA [7, 87]. Other factors affecting the
charge density of chitosan, such as the ionic strength and the distribution of acetyl groups
along the chain, also affect the solubility of chitosan [7]. Chitosan is chitin’s most impor-
tant derivate in terms of application [7], since the amino group on chitosan makes easy
chemical modification possible [11] and gives the molecule some specific functionalities.
The production of chitosan can be achieved via two approaches that start from a different
biomass type (Figure 1.4). The most common approach is converting extracted and purified
chitin into chitosan via a deacetylation step that may be linked to some pre‐treatment (to
decrease crystallinity) and post‐treatment steps. A second option is to start from chitosan‐
containing fungi biomass and separate it from other biomass components via a cleaning
and chitosan extraction step, potentially linked to some pre‐treatment (drying, etc.) and
post‐treatment steps (drying, milling, etc.). Both approaches are detailed in the following
sections.
Chitin Pretreatment Deacetylation Post-treatment
Chitosan
Fungi Pre- Chitosan Post-

treatment Cleaning step
biomass extraction treatment
Figure 1.4 Processes involved in the production of chitosan.
1.4.1 Conversion of Chitin to Chitosan

1.4.1.1 Deacetylation Reaction Mechanism
Deacetylation is a two‐step nucleophilic substitution reaction (Figure 1.5). The first step
consists of a nucleophilic addition of an hydroxide on the carboxy groups [74], while, in
the second step, an amine is formed (=chitosan) when acetic acid is split off. The reaction
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found, no attempt being made to place it in a special receptacle. The
egg is placed on the ventral surface, well behind the feet, under a
mass of matter in the alimentary canal. Shortly after being hatched
the young destroyer penetrates with its head the skin of the victim,
and in this position commences to feed; it is necessary that it should
obtain its food without killing the Cetonia larva, for it cannot prosper
on decaying food, so that if the Cetonia larva die the Scolia larva
likewise perishes; the latter, accordingly, does not withdraw its head
from the interior of the victim, but remains always in the same
position, as it grows larger extending its head forwards into the front
part of the interior of its victim; the internal organs of the latter are
consumed in a systematic order so as to delay bringing about its
death till the last moment, and thus all the interior of the Cetonia
larva is appropriated till nothing remains but an empty skin. By a
series of experiments, Fabre showed how essential it is that this
apparently revolting operation should be carried on with all details
strictly en règle. If the head of the Scolia larva be taken out from the
victim and applied to another part of the body of the Cetonia, the
result is that it cannot eat; even if it be replaced in the original
situation, after being taken away, it frequently happens that the
Cetonia larva dies, its death involving also that of the destroyer. It is
necessary, too, that the victim should be paralysed, for if an intact
Cetonia larva be taken and bound down in such a position that it
cannot move, and if a small orifice in its skin be made in the proper
spot and a young Scolia larva be placed on it, the little parasite will
avail itself of the opportunity and commence to feed on the larva
provided for it, but the latter will speedily die, and the Scolia
necessarily perishes with it. Thus both the paralysis of the victim and
the special mode of eating are essential to the life of the Scolia. The
operation of stinging the larva so as to produce the necessary
paralysis, or rather insensibility, is a difficult one, and requires great
skill and patience. The Cetonia larva is of large size, and must be
pierced in one particular spot; in order to reach this the Scolia
mounts on its victim, and is frequently dislodged by its struggles;
sooner or later, however, the proper position is obtained by the wasp,
and the larva is then stung in the exact spot necessary to allow the
sting (and the poison introduced by it) to reach the most important of
the nervous ganglia that control the movements of the body, this spot
being, in the case of the Cetonia, the line of demarcation between
the pro- and meso-thorax, on the middle line of the ventral surface of
the body. The Scolia gives but one sting to the victim, and this it will
not administer until it can do so exactly in the proper place. This
practice of devouring the victim slowly, without killing it till all is
eaten, is very widely spread in the Hymenoptera, and it is
satisfactory to find that we may infer from Fabre's observations that it
is not so horrible as it would at first appear; for it is probable that the
stinging prevents decomposition of the victim, not by reason, as
some have supposed, of the poison injected by the wasp having an
antiseptic effect, but rather by means of destroying sensibility, so that
the creature does not die from the pain, as it is believed it did in
certain cases where Fabre induced the young Scolia larva to feed on
a victim that had not been stung. We may here remark that very little
exact information exists as to the operation of stinging. Fabre
attaches great importance to the sting being inflicted on a nerve-
ganglion. Whether a sting that did not reach this part might not have
a sufficient effect appears, however, doubtful.[46]
A remarkable form of Scoliides, with wings of smaller size than usual

and deeply divided, has been described by Saunders under the
name Pseudomeria graeca. Still more remarkable is Komarovia
victoriosa found in Central Asia; in this Insect the male retains the
appearance of a slender, pallid Scolia, but the female differs totally in
form, and has the peculiar wings so reduced in size as to be useless
for flight.
Sub-Fam. 4. Sapygides.—Closely allied to the Scoliides, but

possessing slender legs and antennae; also the first abdominal
segment is less disconnected from the second, so that the
outline is less interrupted; the eyes are deeply emarginate; the
hind body is not spinose at the apex.
Fig. 41.—Sapyga 5-punctata ♀, Britain.
The economy of Sapyga, the only genus, has been the subject of
difference of opinion. The views of Latreille and others that these
species are parasitic upon bees is confirmed by the observations of
Fabre, from which it appears that S. 5-punctata lives in the burrows
of species of the bee-genus Osmia, consuming the store of
provisions, consisting of honey-paste, that the bee has laid up for its
young. According to the same distinguished observer, the Sapyga
larva exhibits hypermetamorphosis (i.e. two consecutive forms), and
in its young state destroys the egg of the bee; but his observations
on this point are incomplete and need repetition. We have two
species of Sapyga in Britain; they differ in colour, and the sexes of S.
5-punctata also differ in this respect; the abdomen, spotted with
white in both sexes is in the female variegate with red. Smith found
our British Sapyga 5-punctata carrying caterpillars.
Sub-Fam. 5. Rhopalosomides.—Antennae elongate,

spinigerous; ocelli very prominent; tarsi of peculiar structure,
their claws bifid.
Fig. 42—Rhopalosoma poeyi. A, female imago; B, front of head. Cuba.

(After Westwood.)
This sub-family has recently been proposed by Ashmead[47] for an
extremely rare American Insect that had previously been placed by
Cresson among parasitic Hymenoptera. Westwood classed
Rhopalosoma among Diploptera, saying of it "animal quoad
affinitates excrucians." We reproduce Westwood's figure, but not
being acquainted with the Insect we can express no opinion as to
whether it is allied to the Scoliidae or to the Sphegidae. The habits
are, we believe, quite unknown.
Fam. 2. Pompilidae.
Pronotum at the sides reaching the tegulae; hind body never

definitely pedicellate, though the first segment is sometimes
elongate and conical; hind legs long; eyes elliptic in form, not
emarginate.
The Pompilidae are perhaps the most extensive and important of the
groups of Fossores, and are distributed over all the lands of the
globe, with the exception of some islands and of the inclement arctic
regions. The sting of the Pompilidae, unlike that of most of the
Fossores, inflicts a burning and painful wound; the creatures
sometimes attain a length of two or three inches, and a sting from
one of these giants may have serious results. Although there is
considerable variety in the external form of the members of the
group, the characters given above will enable a Pompilid to be
recognised with approximate certainty. The elongation of the hind
legs includes all the parts, so that while the femur extends nearly as
far back as the extremity of the body—in dried examples at any rate
—the tibiae and the long tarsi extend far beyond it; thus these
Insects have great powers of running; they are indeed remarkable
for extreme activity and vivacity. They may frequently be seen
running rapidly on the surface of the ground, with quivering wings
and vibrating antennae, and are probably then employed in the
search for prey, or some other of the operations connected with
providing a store of food for their young. Spiders appear to be their
special, if not their only, prey. Several authors have recorded details
as to the various ways in which the prey is attacked. Fabre has
observed the habits of several species, and we select his account of
the modus operandi of species of the genera Pompilus and
Calicurgus, in their attacks on poisonous spiders that inhabit holes in
the ground or in walls. The wasp goes to the mouth of the spider's
burrow, and the latter then dashes to the entry, apparently enraged
at the audacity of its persecutor.
Fig. 43.—Calicurgus hyalinatus ♀. Britain.
The Calicurgus will not actually enter a burrow when there is a spider
in it, because if it did so the spider would speedily dispose of the
aggressor by the aid of its poisonous fangs. The Calicurgus,
therefore, has recourse to strategy with the object of getting the
spider out of its nest; the wasp seizes its redoubtable foe by one foot
and pulls; probably it fails to extract the spider, and in that case
rapidly passes to another burrow to repeat its tactics; sooner or later
a spider is in some moment of inattention or incapacity dragged from
its stronghold, and, being then comparatively helpless, feels itself at
a disadvantage and offers but a feeble resistance to the wasp, which
now pounces on its body and immediately inflicts a sting between
the fangs of the foe, and thus at once paralyses these dangerous
weapons; thereafter it stings the body of the spider near to the
junction of the abdomen and cephalothorax, and so produces
complete inactivity. Having secured its prey, the wasp then seeks a
suitable hole in which to deposit it; probably an empty burrow of a
spider is selected for the purpose, and it may be at a height of
several feet in a wall; the Hymenopteron, walking backwards, drags
its heavy prey up the wall to bring it to the den. When this is
accomplished an egg is deposited on the spider, and the wasp goes
in search of a fragment or two of mortar, with which the mouth of the
burrow is finally blocked. Fabre's accounts refer to the habits of
several species, and give a good insight into some points of the
instincts of both the spider and the wasp. It seems that a sense of
superiority is produced in one or other of the foes, according as it
feels itself in suitable conditions; so that though a spider out of its
burrow and on the ground is speedily vanquished by the Pompilid,
yet if the two be confined together in a vase, both are shy and
inclined to adopt defensive or even evasive tactics, the result
probably being that the wasp will be killed by the spider during the
night, that being the period in which the attacking powers of the
spider are more usually brought into play.
It seems to be the habit of some Pompilus to procure a victim before

they have secured a place for its reception; and Fabre took
advantage of this fact, and made very interesting observations on
some points of the instinct of these wasps. Having found a Pompilus
that, after having caught a spider and paralysed it, was engaged in
making a retreat for its reception, he abstracted the booty, which was
deposited at the top of a small tuft of vegetation near to where the
Pompilus was at work. In this case the burrow in course of
preparation was subterranean, and was formed by the Pompilus
itself, which therefore could not, while it was engaged underground,
see what took place near it. It is the habit of the wasp to leave its
work of excavation from time to time, and to visit the prey as if to
assure itself of the safety of this object, and to enjoy the satisfaction
of touching it with the mouth and palping it. Desirous of testing the
wasp's memory of locality, Fabre took the opportunity, while the
Insect was working at the formation of its burrow, of removing, as we
have said, the booty from the place where it had been deposited,
and putting it in another spot some half-yard off. In a short time the
Pompilus suspended work and went straight to the spot where it had
deposited its property, and finding this absent, entered on a series of
marches, counter-marches, and circles round the spot where it had
left the prey, as if quite sure that this was really the place where the
desired object ought to be. At last convinced that the paralysed prey
was no longer where it had been placed, the Pompilus made
investigations at a greater distance and soon discovered the spider.
Fabre recounts that its movements then appeared to indicate
astonishment at the change of position that it thus ascertained to
have occurred. The wasp, however, soon satisfied itself that this was
really the very object it was seeking, and seizing the spider by the
leg slightly altered its position by placing it on the summit of a small
tuft of vegetation; this latter proceeding being apparently always
carried out by this species of Pompilus. Then it returned to its
excavation, and Fabre again removed the spider to a third spot; the
wasp when it next rested from its work made its way immediately to
the second spot, where it had last left the spider, thus showing that it
possessed an accurate memory for locality; the wasp was very much
surprised at the absence of the valued prize and persisted in seeking
it in the immediate vicinity without once returning to the place where
it had been first located. Fabre repeated this manoeuvre five times,
and the Pompilus invariably returned at once to the spot where it had
last left its prey. The acute memory for localities displayed by this
Insect seems to be more or less general throughout the Aculeate
Hymenoptera, and is of very great importance to them. The power of
finding the object appears to depend on sight, for when Fabre, after
removing the spider to a fresh spot, made a slight depression in the
ground, placed the spider in it and covered it over with a leaf, the
wasp did not find it. At the same time, the Insect's sight must be a
very different sense from our own, for the wasp, when seeking its
lost booty, frequently passed within a couple of inches of it without
perceiving it, though it was not concealed.
Belt gives an example of the habits of the Mexican Pompilus

polistoides. He noticed it, when hunting for spiders, make a dart at a
web in the centre of which a spider was stationed; by this movement
the creature was frightened and fell to the ground, where it was
seized by the wasp and stung. The Pompilus then dragged its
prisoner up a tree and afterwards flew off with it, the burden being
probably too heavy for conveyance to the nest without the vantage of
an elevation to start from.
Several modifications adopted by Pompilidae in their mode of
stinging their spider-victims have been recorded by Ferton; these we
cannot allude to in detail, but will nevertheless mention that one
species stings the body of its spider-prey at random, and that in
other cases it would appear that the paralysis of the spider is
evanescent. In short, there are various degrees of perfection in the
details of the art of stinging.
The most remarkable of the forms of Pompilidae are the numerous

species of Pepsis, a genus peculiar to America, whence upwards of
200 species are already known.[48] Some of them attain a length of
two inches or more, and are able to conquer the largest spiders;
even the formidable Mygale avicularis succumbs to their agility and
skill. Some of these Pepsis have beautifully coloured wings;
according to Cameron, this may be due to scales. P. formosus, Say,
is called in Texas the tarantula-killer; according to Buckley, its mode
of attack on the huge spider is different from that made use of by its
European ally. When it discovers a tarantula it flies "in circles in the
air, around its victim. The spider, as if knowing its fate, stands up and
makes a show of fighting, but the resistance is very feeble and of no
avail. The spider's foe soon discovers a favourable moment and
darts upon the tarantula, whom it wounds with its sting, and again
commences flying in circles." The natural retreat of this huge spider,
Mygale hentzii, is in holes in the ground, and this account does not
inform us whether the spider allows itself to be overcome when in its
nest, or is only attacked when out of its retreat.
The genus Mygnimia includes a very large number of species, and

has a wider geographical distribution than Pepsis, being found in the
tropical regions of both the Old and New Worlds, some of them
rivalling in size and ferocity the larger specimens of the genus
Pepsis. In the Insects of this genus there is usually a more or less
distinct small space of more pallid colour on the middle of each front
wing. Parapompilus is a curious genus consisting of Insects of a
great variety of peculiar coloration, and having the wings short, so as
to be of little use for flight. P. gravesii is an inhabitant of Chili.
Agenia carbonaria and A. hyalipennis are small and feeble Insects
inhabiting the south of Europe. A. carbonaria extends to the south of
England. They construct, as nests for their offspring, small
earthenware vessels, differing in form according to the species,
those of A. hyalipennis being vase-like in shape, while those of A.
carbonaria are contracted near the mouth, something after the
fashion of a wide-mouthed bottle. The Insect is able by some means
—Fabre thinks by the use of saliva—to varnish the interior of the
vessel so that it will not absorb water; the outside of the cells is,
however, not so protected, and speedily crumbles away when
exposed to the action of water; hence the vessel is placed in a
protected situation, such as in a tree-stump, or a hole in a wall, or
even in an empty snail-shell under a heap of stones. The cells are
stored with spiders that have been paralysed by stinging and that
serve as food for the larva of the Agenia. The larva of A. carbonaria
has been described, and some particulars as to its habits have been
given by Verhoeff. It has been stated that this wasp does not
paralyse its prey by stinging, but substitutes a process of biting to
prevent the spider from hurting the larva that is to feed on it; and
Verhoeff's observations seem to show that the legs of the spider are
broken by some proceeding of the kind. The Agenia larva is of
peculiar shape, the head not being inflexed, while the pleurae of
each segment, from the second onwards, are prominent, so as to
give the outline of the body a scalloped appearance. This larva is
much infested by an Ichneumon that devours, it appears, not only
the larva itself, but also the spider that was destined to be food for
the larva. Verhoeff seems to have found some evidence that
Pompilus sericeus may also be a parasite on the Agenia.
The construction of earthenware cells, instead of the burrows usual

in Pompilidae, by the species of this genus is one of the cases
alluded to in our introductory remarks as to allied Fossores exhibiting
different habits. Mr. Pride has recently sent us from Brazil similar
earthen vessels constructed by some Pompilid.
The habits of Pompilids of the genus Ceropales are analogous to
those of the parasitic bees. Pérez has recently given us information
as to a very curious form of parasitism in this genus; he says that
when a Pompilus has obtained a spider as provision for its young, it
is pursued by a Ceropales, which lays an egg on the spider, thus as
it were substituting in advance its own young for that of the
Pompilus. Information as to the subsequent course of events in this
case is not at present forthcoming. In another case a Ceropales was
observed to oviposit on the spider, not while this is being carried in,
but subsequently by entering the nest for the purpose; a habit quite
similar to that of some parasitic bees. Ferton has recently made the
unexpected discovery that some Pompilus act as robbers; one
individual taking away by force the spider that another has captured
and is carrying off.
Lichtenstein described a Pompilid larva, that he afterwards

ascertained to be Calicurgus hyalinatus, as possessing the
extraordinary habit of feeding as an external parasite fixed to the
dorsal surface of a spider; thus repeating, it would appear, the habits
of some of the Ichnemonidae, though the perfect Insect (Fig. 143)
does not differ in structure from its congeners. Emery has given an
account of some Pompilids that do not bury their prey, but after
stinging it and depositing an egg, simply leave the spider on the
spot.
Buller has described the habits of a Pompilid in New Zealand; his

account is interesting because it shows a remarkable similarity in the
proceedings of this antipodean wasp to those of its congeners on our
own side of the world. The species is not scientifically named, but it
appears that it is known in New Zealand as "the Mason-bee." It
forms a nest of yellow clay consisting apparently of about eight cells,
each of which is filled with one or more spiders in a paralysed
condition. The figure given of the larva of this Insect by Buller shows
it to possess a peculiarly formed head.
It is pleasing to find that Pompilidae do not make use of cruel
methods when others will serve their purpose. We are informed that
a large Australian Pompilid—Priocnemis bicolor—may find a Cicada
sucking sap from a hole it has pierced in a tree. The Priocnemis has
not the art of making the puncture necessary to procure sap, so the
wasp seizes the Cicada, and shakes it till it leaves its hold and flies
away, when the Priocnemis takes its place and sips the sap. It is
added that the wasp never hurts the Cicada.
Fam. 3. Sphegidae.
Pronotum free from the tegulae; when the stigmatic lobes extend
as far back as the wing-insertion, they are placed below it and
separated by a space from it.
This large assemblage of Fossores is the one about which the

greatest difference of opinion prevails. It is based entirely on the
prothoracic characters mentioned above, and cannot be looked on
as natural. We shall, however, follow Kohl[49] in treating for the
present as only one family the divisions considered by many as
distinct families. They are ten in number.
Sub-Fam. 1. Sphegides.—Hind body with a slender pedicel of

variable length; two spurs on the middle tibia. The propodeum
usually horizontally elongate.[50]
This group includes a great number of species, about 200 of which

are referred to the genus Sphex.
The habits of one species of this genus have been fully described by
Fabre; he assigns to the species the name of S. flavipennis, but Kohl
considers that it is more probably S. maxillosus. This Insect forms its
nests, in the South of France, in the ground, excavating a main shaft
with which are connected cells intended for the reception of the
provisions for the young. The entrance to the burrow is formed by
piercing a hole in the side of a very slight elevation of the soil. Thus
the entrance to the construction consists of a horizontal gallery,
playing the part of a vestibule, and this is used by the Sphex as a
place of retreat and shelter for itself; at the end of the vestibule,
which may be two or three inches long, the excavation takes an
abrupt turn downwards, extending in this manner another two or
three inches, and terminating in an oval cell the larger diameter of
which is situate in a horizontal plane. When this first cell has been
completed, stored with food, and an egg laid in it, the entrance to it is
blocked up, and another similar cell is formed on one side; a third
and sometimes a fourth are afterwards made and provisioned, then
the Insect commences anew, and a fresh tunnel is formed; ten such
constructions being the number usually prepared by each wasp. The
Insect works with extreme energy, and as the period of its
constructive activity endures only about a month, it can give but two
or three days to the construction and provisioning of each of its ten
subterranean works. The provisions, according to Fabre, consist of a
large species of field-cricket, of which three or four individuals are
placed in each cell. Kohl states, however, that in Eastern Europe an
Insect that he considers to be the same species as Fabre's Sphex,
makes use of locusts as provisions, and he thinks that the habit may
vary according to the locality or to the species of Orthoptera that may
be available in the neighbourhood. However that may be, it is clear
from Fabre's account that this part of the Sphex's duties do not give
rise to much difficulty. The cricket, having been caught, is paralysed
so that it may not by its movements destroy the young larva for
whose benefit it is destined. The Sphex then carries it to the burrow
to store it in one of the cells; before entering the cell the Insect is in
the habit of depositing its prey on the ground, then of turning round,
entering the burrow backwards, seizing as it does so the cricket by
the antennae, and so dragging it into the cell, itself going backwards.
The habit of depositing its prey on the ground enabled Fabre to
observe the process of stinging; this he did by himself capturing a
cricket, and when the wasp had momentarily quitted its prey,
substituting the sound cricket for the paralysed one. The Sphex, on
finding this new and lively victim, proceeds at once to sting it, and
pounces on the cricket, which, after a brief struggle, is overcome by
the wasp; this holds it supine, and then administers three stings, one
in the neck, one in the joint between the pro- and meso-thorax, and a
third at the base of the abdomen, these three spots corresponding
with the situation of the three chief nervous centres governing the
movements of the body. The cricket is thus completely paralysed,
without, however, being killed. Fabre proved that an Insect so treated
would survive for several weeks, though deprived of all power of
movement. Three or four crickets are placed by the wasp in each
cell, 100 individuals or upwards being thus destroyed by a single
wasp. Although the sting has such an immediate and powerful effect
on the cricket, it occasions but a slight and evanescent pain to a
human being; the sting is not barbed, as it is in many bees and true
wasps, and appears to be rarely used by the Insect for any other
purpose than that of paralysing its victims. The egg is laid by the
Sphex on the ventral surface of the victim between the second and
third pairs of legs. In three or four days the young larva makes its
appearance in the form of a feeble little worm, as transparent as
crystal; this larva does not change its place, but there, where it was
hatched, pierces the skin of the cricket with its tiny head, and thus
begins the process of feeding; it does not leave the spot where it first
commenced to feed, but gradually enters by the orifice it has made,
into the interior of the cricket. This is completely emptied in the
course of six or seven days, nothing but its integument remaining;
the wasp-larva has by this time attained a length of about 12
millimetres, and makes its exit through the orifice it entered by,
changing its skin as it does so. Another cricket is then attacked and
rapidly consumed, the whole stock being devoured in ten or twelve
days from the commencement of the feeding operations; the
consumption of the later-eaten crickets is not performed in so
delicate a manner as is the eating of the first victim. When full-grown,
the process of forming a cocoon commences: this is a very elaborate
operation, for the encasement consists of three layers, in addition to
the rough silk that serves as a sort of scaffolding on the exterior: the
internal coat is polished and is of a dark colour, owing to its being
coloured with a matter from the alimentary canal: the other layers of
the cocoon are white or pale yellow. Fabre considers that the outer
layers of the cocoon are formed by matter from the silk-glands, while
the interior dark coat is furnished by the alimentary canal and applied
by the mouth of the larva: the object of this varnish is believed to be
the exclusion of moisture from the interior of the cocoon, the
subterranean tunnels being insufficient for keeping their contents dry
throughout the long months of winter. During the whole of the
process of devouring the four crickets, nothing is ejected from the
alimentary canal of the larva, but after the cocoon is formed the larva
ejects in it, once for all, the surplus contents of the intestine. Nine
months are passed by the Insect in the cocoon, the pupal state being
assumed only towards the close of this period. The pupa is at first
quite colourless, but gradually assumes the black and red colour
characteristic of the perfect wasp. Fabre exposed some specimens
of the pupa to the light in glass tubes, and found that they went
through the pupal metamorphosis in just the same manner as the
pupae that remained in the darkness natural to them during this
stage of their existence.
Sphex coeruleus is frequently stated to have the habit of provisioning

its nests with both Orthoptera and Spiders; but Kohl considers with
reason that this record is, as regards spiders, a mistake, arising
probably from a confusion with some other Insect of similar
appearance, such as Pelopaeus (Sceliphron) coeruleus. S.
coeruleus is no doubt the same as S. (Chlorion) lobatus, which
Rothney observed in East India, provisioning its nests with
Orthoptera. He discovered a nest in process of construction, and
during the absence of the mother-wasp abstracted from the burrow a
large field-cricket that she had placed in it; he then deposited the
Orthopteron near the cell; the parent Sphex on returning to work
entered the tunnel and found the provision placed therein had
disappeared; she came out in a state of excitement, looked for the
missing cricket, soon discovered it, submitted it to the process of
malaxation or kneading, and again placed it in the nest, after having
cleared it from some ants that had commenced to infest it. She then
disappeared, and Rothney repeated the experiment; in due course
the same series of operations was performed, and were repeated
many times, the Sphex evidently acting in each case as if either the
cricket had disappeared owing to its being incompletely stunned, or
to its having been stolen by ants. Finally, the observer placed the
cricket at a greater distance from the nest, when it recovered from
the ill-treatment it had received sufficiently to make its escape. The
points of interest in this account are the fact that the cricket was only
temporarily paralysed, and that the wasp was quite able to cope with
the two special difficulties that must frequently occur to the species
in its usual round of occupations.
The genus Ammophila is of wide distribution, and its species make

vertical tunnels in the ground. The habits of some of the species
found in France have been described by Fabre. The Insect does not
inhabit the burrow while it is in process of formation, but quits it; and
some of the species temporarily close the entry to the incomplete
nest with a stone. The tunnel is a simple shaft with a single cell at its
termination; this is stored with caterpillars, the different species of
Ammophila selecting different grubs for the purpose. A. hirsuta
hibernates in the perfect state, and carries on its work in the spring; it
chooses a single larva of considerable size belonging to one of the
nocturnal Lepidoptera, and this it paralyses by a series of about nine
stings, of which one is implanted in each segment from the first
thoracic ring backwards; it forms the burrow only after the food to be
placed therein has been obtained. The caterpillar used is
subterranean in habit, and the Ammophila detects the larva by some
sense, the nature of which appears at present quite uncertain. A.
holosericea chooses smaller larvae of the family Geometridae, and
uses only one or two stingings to paralyse each larva; several
caterpillars are used to provision a single cell, and they are often
selected of different colours.
Marchal has also published an important account of the proceedings

of A. affinis; he confirms Fabre's observations, and even adds to
their interest by suggesting that the Ammophila administers special
stings for the purpose of paralysing the mandibles of the caterpillar
and depriving it of any power of afterwards injuring the larva that will
feed on it. He thinks the mother-Ammophila herself profits by
appropriating an exudation from the victim.
Some species of Sphegides have the curious habit of choosing the

interiors of human habitations as the spots most suitable for the
formation of their own domestic establishments. Fabre has given a
charming account of the habits of Pelopaeus (Sceliphron) spirifex, a
species that inhabits the South of Europe, and that forms its nests in
the cottages of the peasants. The spot usually selected is a nook in
the broad, open fireplace, out of reach of the flames, though not of
the smoke; here the Pelopaeus forms a nest of earth, consisting of
ten to fifty cells, the material being mud or clay brought in little balls
by the aid of the Insect's mandibles; about twenty visits are required
in order to complete one cell, so that for the construction of a large
nest of fifty cells, about one thousand visits must be made by the
Insect. It flies in and out of the house apparently not at all
incommoded by the human habitants, or by the fact that the
peasant's potage may be simmering on the fire quite close to where
the fearless little creature is carrying on its architectural operations.
The cells are stored with spiders, of which the wasp has to bring a
plentiful supply, so that its operations extend over a considerable
period. The prey is captured by the Pelopaeus whilst on the wing,
and carried off at once, being probably stung by the wasp during the
process of transit; apparently it is killed by the operation, not merely
paralysed. Only small spiders are taken by this species, and the
larva of the Pelopaeus consumes them in a short time, one by one,
before the process of decomposition sets in; the egg, too, is laid on
the first spider introduced, and this is of course at the bottom of the
cell, so that the spiders are eaten by the wasp's larva in the order in
which they were brought to the cell. The cell is sealed up when full,
the number of spiders placed in it being on the average about eight.
The larva completes its task of consuming the store in about ten
days, and then forms a cocoon for its metamorphosis. Two or three
generations are produced in a single year, the autumnal one passing
eight or nine months in the clay cells, which are lodged in a nook of
the peasant's hearth, and exposed to the smoke of his fire during all
the months of winter. Pelopaeus (Sceliphron) is a genus including
many species;[51] several of them are known to be specially attached
to the habitations of human beings. Roth has given an account of the
habits of P. (Sceliphron) laetus in Australia; he says that in some
parts it is very difficult to keep these wasps out of the houses; the
nest is formed of mud, and constructed on the furniture or in any part
of a room that suits the fancy of the Insect. This it must be admitted
is, according to human ideas, liable to the charge of being very
capricious. Roth timed a wasp building its nest, and found that it
brought a fresh load of mud every two or three minutes. If the wasp
be allowed to complete the nest undisturbed, she does so by adding
to the exterior diagonal streaks of mud, so giving to the nest the look
of a small piece of the bark of a common acacia. The construction
consists of from ten to twenty cells, and when completed is
provisioned with spiders for the use of the young. This wasp is much
pestered by parasites, some of which prevent the development of
the larvae by consuming the spiders intended by the mother-wasp
for its young. A fly, of the Order Diptera, is said to follow the wasp
when carrying a spider, and to deposit also an egg on the food; as
the Dipterous larvae have more rapid powers of assimilation, the
Pelopaeus larvae are starved to death; and their mildewed remains
may be found in the cell, after their enemies have become fully
developed and have flown away. Another parasite is said to eat the
wasp-larva, and attains this end by introducing an egg through the
mud wall and the cocoon of the wasp—a habit that seems to indicate
a Leucospid parasite. Tachytes australis, a wasp of the sub-family
Larrides also dispossesses this Pelopaeus in a manner we shall
subsequently describe. This fragment of natural history from
Australia has a special interest, for we find repeated there similar
complex biological relations to those existing in the case of the
European congeners.
P. (Sceliphron) madraspatanus is common in the north-west

provinces of Hindostan, and is called the "mud-dauber" by the
European residents. According to Horne it constructs its cells in the
oddest places, but chiefly about the inhabited apartments in houses.
It is perfectly fearless when engaged in building: the cells are four to
six in number, and are usually provisioned with spiders to the
number of about twenty. On one occasion it was observed that green
caterpillars were stored instead of spiders. The species is said to be
protected by a peculiar odour as well as by its sting; it is also stated
that it disguises its edifice when completed by making it look like a
dab of mud, and on one occasion "rays of mud were observed round
the nest, even more exactly imitating a lump of mud thrown with
some force." P. (Sceliphron) bilineatus, formerly thought to be a
variety of P. madraspatanus, builds its nests in hedges and trees.
Sub-Fam. 2. Ampulicides.—Prothorax long and narrow,

forming a neck in front; clypeus beak-like; four submarginal cells,
the outer one being complete; metathorax elongate, the
posterior part of the metasternum deeply divided to allow a
perfect inflection of the abdomen.
Fig. 44—Ampulex compressa. Male. East India.
This is one of the smallest of the divisions of the Sphegidae, but has
a very wide distribution, being represented in both the Eastern and
Western Hemispheres. It is allied to the Sphegides, but differs by the
prolongation of the neck and of the head, and by the articulation
between the petiole and thorax being placed on the under surface of
the body; the wing-nervures are said to be of inferior importance
owing to their frequently differing in individuals of the same species.
These Insects appear to be rare in individuals, as well as few in
species, and but little has been recorded as to their habits; but it is
known that they live on cockroaches. Perkins has given a brief
sketch of the habits of Ampulex sibirica that is of great interest, but
requires confirmation. He says that this Insect, in West Africa, enters
apartments where cockroaches abound, and attacking one, that may
probably be four times its own size, succeeds, after a struggle, in
stinging it; the cockroach instantly becomes quiet and submissive,
and suffers itself to be led away and placed in confinement in some
spot such as a keyhole, and in one case was apparently prevented
from afterwards escaping, by the wasp carrying some heavy nails
into the keyhole. The larva of the Ampulex may be presumed to live
on the Blattid, as it is added that dead bodies of the cockroaches are
frequently found with the empty cocoon protruding from them. This
account, if correct, points to some features in the habits of this Insect
that are unique. A remark made by Rothney in reference to the
habits of A. (Rhinopsis) ruficornis seems to indicate some similar
instinct on the part of that species; he says, "I also saw two or three
of these wasps collar a peculiar cockroach by the antennae and lead
it off into a crack in the bark, but as the cockroach reappeared
smiling each time, I don't know what was up." The same observer
records that this species associates with Sima rufonigra, an ant it
greatly resembles in appearance, as well as with a spider that is also
of similar appearance (Fig. 72). Schurr has given a brief account of
the proceedings of Ampulex compressa, and his statements also
tend to confirm the correctness of Perkins' report. The habits of a
species of Ampulex were partially known to Réaumur, who described
them on the authority of M. Cossigni. The species is believed to be
A. compressa, which occurs not only in East India, but also in the
island of Bourbon, the locality where M. Cossigni made his
observation: his account is, like the others, a mere sketch of certain
points observed, the most important of which is that when Ampulex
cannot introduce the cockroach into a hole that it has selected as
suitable, it bites off some portions of the body in order to reduce the
poor Insect to the necessary extent.
From these fragmentary observations it would appear that the sting

of the Ampulex has not so powerful a paralysing effect as that of
most other Fossores; and that the Ampulex does not form any nest,
but takes advantage of suitable holes and crevices to store the victim
in; also that it displays considerable ingenuity in the selection of
materials with which to block up the cavity in which it has placed the
partially incapacitated creature.
The genus Dolichurus is by some entomologists considered the type

of a sub-family allied to the Ampulicides; it long consisted of a small
and rare European Insect, but some exotic species have recently
been added to it. It will probably prove not sufficiently distinct from
Ampulicides, although the pronotum is much shorter, but Handlirsch
has recently observed that the European species attacks Blattidae
as do the normal Ampulicides; and Ferton has recorded that D.
haemorrhous lives at the expense of Loboptera decipiens, the wasp
depositing its egg on the left intermediate femur of the prey. This is
placed in a solitary cell, and is entirely consumed by the larva, life
being preserved till within a few hours of the end of the repast, which
occupies altogether eight days.

Chitin and Chitosan Properties and Applications Lambertus A M Van Den Broek Full Chapter

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Chitin and Chitosan: Properties and

Applications Lambertus A. M. Van Den

Titles in the Series:

Renewables‐Based Technology: Sustainability Assessment

Handbook of Natural Colorants

Surfactants from Renewable Resources

Industrial Applications of Natural Fibres: Structure, Properties and Technical Applications

Thermochemical Processing of Biomass: Conversion into Fuels, Chemicals and Power

Biorefinery Co‐Products: Phytochemicals, Primary Metabolites and Value‐Added Biomass

Bio‐Based Plastics: Materials and Applications

Introduction to Wood and Natural Fiber Composites

Cellulosic Energy Cropping Systems

Introduction to Chemicals from Biomass, 2nd Edition

Cellulose Nanocrystals: Properties, Production and Applications

Nanoporous Catalysts for Biomass Conversion

Thermochemical Processing of Biomass: Conversion into Fuels, Chemicals and Power,

Waste Valorization: Waste Streams in a Circular Economy

Process Systems Engineering for Biofuels Development

Biobased Packaging: Material, Environmental and Economic Aspects

LAMBERTUS A.M. VAN DEN BROEK

Limit of Liability/Disclaimer of Warranty

Library of Congress Cataloging‐in‐Publication data applied for

Cover Design: Wiley

Set in 10/12pt Times by SPi Global, Pondicherry, India

List of Contributors xvii

1 Sources of Chitin and Chitosan and their Isolation 1

2 Methods of Isolating Chitin from Sponges (Porifera) 35

2.3 The Modern Approach to Chitin Isolation from Sponges 40

3 Physicochemical Properties of Chitosan and its Degradation Products 61

4 New Developments in the Analysis of Partially Acetylated Chitosan

6 Beneficial Health Effects of Chitin and Chitosan 145

6.5 Outlook 164

7 Antimicrobial Properties of Chitin and Chitosan 169

8 Enzymes for Modification of Chitin and Chitosan 189

9 Chitin and Chitosan as Sources of Bio‐Based Building Blocks

9.2.1 Chitosan Production 232

10 Chemical and Enzymatic Modification of Chitosan to Produce New

11 Chitosan‐Based Drug Delivery Systems 259

11.5 Outlook 276

13 Food Applications of Chitosan and its Derivatives 315

14 Potential of Chitosans in the Development of Edible Food Packaging 349

14.2.1 Definitions and Interests 353

15 The Use of Chitosan‐Based Nanoformulations for Controlling Fungi

16 Chitosan Application in Textile Processing and Fabric Coating 395

16.4.2 Chitosan Spinning Processes 402

17 Chitin and Chitosan for Water Purification 429

18 Chitosan for Sensors and Electrochemical Applications 461

19 Marketing and Regulations of Chitin and Chitosan from Insects 477

Artur Bartkowiak Center of Bioimmobilisation and Innovative Packaging Materials,

Bogdan Ionel Tamba A&B Pharm Corporation, Roman, Neamt ̦, Romania

Lambertus A.M. van den Broek and Carmen G. Boeriu

Chitin and Chitosan: Properties and Applications, First Edition.

1.1 Chitin and Chitosan

1.1.2 Different Crystalline Forms of Chitin

1.2 Sources of Chitin and Chitosan

% (w/w) chitin in biomass

0004461239.INDD 6 10/26/2019 1:16:41 PM

0004461239.INDD 7 10/26/2019 1:16:41 PM

% (w/w) chitin in biomass

Cuttlefish (Cephalopoda) Sepia spp. Shells 91.25 1.35 7.4 [26]

0004461239.INDD 8 10/26/2019 1:16:41 PM

1.2.1.1 Crustaceans (Part of Arthropoda)

1.2.1.2 Insects (Part of Arthropoda)

List of Contributors xvii

1 Sources of Chitin and Chitosan and their Isolation 1

2 Methods of Isolating Chitin from Sponges (Porifera) 35

2.3 The Modern Approach to Chitin Isolation from Sponges 40

3 Physicochemical Properties of Chitosan and its Degradation Products 61

6 Beneficial Health Effects of Chitin and Chitosan 145

6.5 Outlook 164

7 Antimicrobial Properties of Chitin and Chitosan 169

8 Enzymes for Modification of Chitin and Chitosan 189

9.2.1 Chitosan Production 232

11 Chitosan‐Based Drug Delivery Systems 259

11.5 Outlook 276

13 Food Applications of Chitosan and its Derivatives 315

14 Potential of Chitosans in the Development of Edible Food Packaging 349

14.2.1 Definitions and Interests 353

16 Chitosan Application in Textile Processing and Fabric Coating 395

16.4.2 Chitosan Spinning Processes 402

17 Chitin and Chitosan for Water Purification 429

18 Chitosan for Sensors and Electrochemical Applications 461

19 Marketing and Regulations of Chitin and Chitosan from Insects 477