Chitosan: Derivatives, Composites and Applications

This book delves deeply in to the preparation, characterization and multiple applications of chitin and chitosan. The 17 chapters written by leading experts is an excellent reference source and state-of-the-art review for researchers and scientists using chitosan or biopolymers in their respective areas.
This book is divided into following sections:

• Production and derivatives of chitosan
• Chitosan in the textile and food industries
• Chitosan in biomedical applications
• Chitosan in agriculture and water treatment

The book is practical as readers will be able to see descriptions of chitosan production methods as well as techniques that can be used to estimate and modify their physical and chemical properties. It provides a full description not only of the traditional and recent developments in the applications of chitosan in the fields of biotechnology, environmental studies, food, medicine, water treatments, drug delivery, but it includes all of the  therapeutically usages as well.

• Language: English
• Publisher: Wiley
• Release date: Aug 4, 2017
• ISBN: 9781119364818

Book preview

Section I

PRODUCTION AND DERIVATIVES OF CHITOSAN

Chapter 1

Chitin and Chitosan: History, Composition and Properties

Annu1*, Shakeel Ahmed1,2 and Saiqa Ikram1*

1Bio/Polymers Research Laboratory, Department of Chemistry, Jamia Millia Islamia (A Central University), New Delhi, India

2Department of Textile Technology, Indian Institute of Technology Delhi, New Delhi, India

*Corresponding authors: [e-mail]; [e-mail]

Abstract

Chitin and chitosan are most abundant naturally occurring polymers, ranked second after cellulose. Chitin is generally extracted from natural sources viz. terrestrial organisms, marine organisms, microorganisms like fungi and enzymatically from crustaceans shell waste materials. On the other hand, chitosan is obtained from the deacetylation of the former, chitin.

Nowadays, chitin and chitosan are commercially manufactured from biowastes obtained from aquatic organisms. But due to the seasonal and capricious availability of raw materials, terrestrial crustaceans and mushrooms are the alternative source for their production. Structurally, chitin and chitosan are N-acetyl-D-glucosamine units and D-glucosamine units, respectively, with only difference in hydroxyl group of cellulose. Both the biopolymers are biodegradable and possess many beneficial properties such as biocompatibility, antimicrobian, hemostatic, anti-inflammatory, antioxidant, mucoadhesion, analgesic, non-toxicity, adsorption enhancing, antihypertensive, anticholesterolemic, anticancer, and antidiabetic. Due to having such respectable properties chitin, chitosan, and their corresponding derivatives are greatly exploiting by the scientists and also getting tremendously better results in medical and engineering fields.

Keywords: Chitin, chitosan, history, structure, properties, solubility, viscosity, biomedical, anticancer

1.1 Chitin

1.1.1 History

French scientist Prof. Henri Braconnot for the very first time discovered chitin in 1811 in mushroom. After that Odier found the same compound in the cuticles of insects in 1823 and called it Chitin from the Greek word meaning tunic, covering or envelope [1].

This was how it begins the research in a new direction of polymers present in our nature. Gradually as the time passed away in 1859, Prof. C. Rouget coined another noval biopolymer, actually derived from previous chitin, and it was known as Chitosan. In 1878, Ledderhose revealed that the chitin consists of glucosamine and acetic acid. Thereafter, in 1930s and 1940s, both former and latter attract considerable attention as evidenced by about 50 patents. Chitin is the most abundant naturally occurring polymer, ranked second after cellulose and also most abundant naturally occurring polysaccharide possesses amino acid and sugars. Chemically, chitin is composed of N-acetyl-D-glucosamine units with β-(1-4) glycosidic linkage [2].

1.1.2 Sources of Chitin

Chitin is generally produced from natural sources viz. terrestrial organisms, marine organisms, microorganisms like fungi and enzymatically from crustaceans shell waste materials. On the other hand, chitosan is obtained from the deacetylation of the former, chitin. Nowadays, chitin and chitosan are commercially manufactured from biowastes obtained from aquatic organisms. But due to the seasonal and capricious availability of raw materials terrestrial crustaceans and mushrooms are the alternative source for their production [2]. The industrial manufacturing of synthetic polymers was restricted by the time because of the inadequate facilities as well as the cutthroat competition in synthetic polymers. Therefore, again the shellfish shells, crustaceans and shrimps revitalized the interest in late 1970s (Global industry analysis since 2004). Various sources for extraction and production of chitin can be categorized as follows:

Terrestrial organisms

Marine or Aquatic organisms

Microorganisms (e.g., Fungi)

1.1.2.1 Terrestrial Organisms

From commercial point of view, these organisms are mainly used for the extraction of chitin, due to their easy availability and processability. Terrestrial species generally includes crustaceans such as Porcellio scaber, Armadillidium vulgare; arthropods, nematodes, insects, silkworms, mosquitoes, honeybee, Sipyloidea sipylus, Drosophila melanogaster, Extatosoma tiaratum, and many more [3]. As the composition of these organisms is quite different, there is a variation in the contribution to the percentage of chitin produced as depicted in Table 1.1.

Table 1.1 Percentage of chitin produced from different sources [3].

1.1.2.2 Marine or Aquatic Organisms

Chitin produced from aquatic species includes diatoms, algae, crabs, shrimps, lobster, squids, and krill. The hazardous waste materials generated from head, thorax, shells, and claws of shellfish are utilized as raw materials for processing, containing 15–40% chitin, 20–40% proteins, and 20–50% CaCO3. The waste streams of molluscs and crustaceans are the main source of chitin. Also they constitute a rich source of proteins, flavor compounds, and various pigments and hence are of great attention for different research fields and industries as well. Actually, if they are disposed of in the open environment of the water bodies, such as sea or river, then they are problematic due to the higher biochemical oxygen demand and eutrophication [4].

1.1.2.3 Microorganisms (e.g., Fungi or Mushroom)

Chitin can be obtained from microorganisms either by fermentative methods or biotechnological methods. Utilization of various microbes makes it easier for industries to produce chitin widely and hence microbes are considered as the economic source of extracting chitin as well as chitosan. Microbial world mainly includes fungi (cell wall, mycelia, septa) molds, chrysophyte algae, yeasts, prosthecate bacteria, spores of streptomycete and ciliates. Except Oomycetes, remaining Ascomycetes, Basidiomycetes, Deuteromycetes, and Zygomycetes consists of 2–65% chitin/chitosan. Especially, Mucoralean strains viz. Syncephalastrum racemosum and Cunninghamella echinulata cell wall exhibited maximum chitin/chitosan yield of 7% per mycelia dry weight under optimum conditions [5–7]. Some of the examples of extraction of chitin from terrestrial, marine and microorganisms are listed in Table 1.2.

Table 1.2 Examples of sources of extraction of chitin [5].

1.1.3 Extraction of Chitin

Chitin can be extracted from insect cuticle, tracheae and peritrophic matrix [25], shellfish waste such as shrimps, crabs, krill, lobster, fishes and microorganisms such as fungi or mushroom mycelia and some bacteria as discussed above. Generally, extraction of chitin involves the following steps:

Demineralization

Deproteination

Decolorization

There may be difference in the sequence of these steps [3, 8]. For instance, Kumari et al. described the extraction of chitin from fish scales of Labeo rohita but after demineralization they performed decolorization followed by deproteination [9].

1.1.3.1 Demineralization

It can be performed by using strong acids such as HCl, H2SO4, HNO3 and weak acids such as CH3COOH and HCOOH. But generally HCl seems to be best one. Previous researchers showed that the concentration of HCl was about 1N or 2N for 0.3–96 h from 25–100 °C (for insect cuticles). But from last decade, it has been found that only 0.25M to 2M concentration of HCl for 1–36 or 48 h is sufficient at temperature 0–100 °C for just 15 min [8]. This step particularly performed to remove the minerals, especially CaCO3.

1.1.3.2 Deproteination

It is generally performed under alkaline medium of 0.75N–2.5N or 1M NaOH aqueous solution for 2–42 or 1–72 h at temperature 40 or 65–100 °C in crustaceans and marine shrimp shells, respectively. NaOH, KOH, NaHCO3, Na2CO3, K2CO3, Ca(OH)2, NaHSO3, and Na2SO3 are the reagent that can be used for deproteination of the crustaceans. Due to the adverse effect of these perilous chemicals on environment, nowadays, proteolytic enzymes such as trypsin, pepsin, or papain have been used to raise the efficiency of deproteination [10, 11].

1.1.3.2.1 Enzymatic Deproteination

Proteolytic enzymes can be extracted from various sources such as Bacillus mojavensis A21, B. subtilis A26, B. licheniformis NH1, B. licheniformis MP1, Vibrio metschnikovii J1, alkaline protease from Sardinelle (Sardinella aurita), Goby (Zosterisessor ophiocephalus), Aspergillus clavatus ES1, and Grey triggerfish (Balistes capriscus). One unit of protease activity can be defined as the amount of enzyme required to liberate 1 μg of tyrosine per minute [12]. Degree of Deproteination can be calculated as follows:

(1.1)

Graphic

where Pi and Pf are the concentrations of protein at initial and final hydrolysis; while S and R are the mass of original sample and hydrolysed residue, respectively [12].

1.1.3.3 Decolorization

In order to remove pigmentation and melanin, the mixture has been treated either with hydrogen peroxide or potassium permanganate solution [3].

According to Mohammed et al. approximately 35% of chitin (dry weight) were obtained from prawn shell after deproteination, decolorization, and demineralization [13]. Chitin can be extracted either via using biotechnological method or via chemical method as depicted in Figures 1.1 and 1.2.

Figure 1.1 Extraction of chitin.

Figure 1.2 Extraction of chitin by alkaline solution.

1.1.4 Structure and Composition

Chitin is a linear copolymer of β-(1-4)-linked 2-amino-2-deoxy-D-glucan and 2-acetamido-2-deoxy-D-glucan. Structurally, chitin is composed of β-(1-4)-linked D-glucosamine units. Unlike cellulose, the hydroxyl group of cellulose is replaced by N-acetyl group (–NHCOCH3) as shown in Figure 1.3. Chitin has been existed mainly in three solid state polymorphic forms viz. α, β, and γ. Out of these allomorphs, α-chitin is the most abundant one followed by β and then rarest one is γ-chitin. The major source of α-form of chitin is generally shrimps, insect cuticle, crab, krill, lobster, cell wall of yeast and Zygomycetes. The abundancy of α-chitin favors the significant quality of chitin as high crystallinity and purity due to the absence of calcium carbonate, proteins, and pigments. Instead β-chitin is found in connotation with proteins in squid pens while γ-chitin is found in cuttlefish stomach lining. X-Ray diffraction revealed that the inner ring present in α-form of chitin is unaffected from hydration while the inner ring of β-chitin is sensitive to hydration. Moreover crystallographically, α-chitin exhibits two antiparallel molecules per unit cell, whereas β-chitin exhibits one parallel arrangement as depicted in Figure 1.4. As far as similarity is concerned, both the allomorphs have same moiety of N-acetylglycosamine [14–17].

Figure 1.3 Structural comparison of chitin and cellulose.

Figure 1.4 Antiparallel and parallel arrangements of different allomorphs of chitin.

1.1.5 Properties of Chitin

1.1.5.1 Solubility, Reactivity, and Viscosity

Like cellulose, chitin as biopolymer is highly crystalline materials having specific solvent behavior. As chitin is β-(1,4)-linked N-acetyl-D-glucosamine therefore, its solubility and reactivity are highly influenced by –NH2 and –OH functional groups. Being a typical natural polymer obtained from different sources, chitin possesses excessive hydrogen bonding which in turn degrade it before melting and thus making it essential to be dissolve in suitable solvent. Chitin is generally hydrophobic in nature and hence insoluble in water and organic solvents at room temperature but soluble in hexafluoroisopropanol, chloroalcohols, and hexafluoroacetone in association with mineral acid aqueous solution and dimethylacetamide having 5% lithium chloride [18]. In other words, water is a thermodynamically poor solvent for uncharged chitin unit due to strong interactions. Thus, charge on the polymeric chain can make the chitin soluble by releasing the attractive forces. It has also been reported that small amount of chitin can be dissolved in 1% acidic solution at 121 °C for 20 min and on reacting it with HCl and NaOH, decrement in crystallinity index [19, 20]. The hydrolysis of chitin with concentrated acids under drastic conditions produces relatively pure D-glucosamine. Additionally, the intrinsic viscosity of chitin can be considered as a function of demineralization time. Temperature is also an important criterion for purity of chitin which if carried out at high temperature produces better results as compared to the low temperature and obtains stable viscosities with time as well. Nitrogen content available in chitin depends on the extent of degree of deacetylation and is found to be 5–8%. However, the nitrogen content available in chitosan is present in the form of primary aliphatic amino groups and hence undergo important amine N-acylation and Schiff reactions [18].

1.1.5.2 Miscellaneous Properties

Chitin has also been known for its various properties such as water-binding and fat-binding capacity. Knorr revealed from his studies that 0.5–2.0% of microcrystalline chitin on treating with wheat flour bread or with potato protein white bread, the water binding, fat binding capacity, and emulsifying ability were enhanced from 170–315% (w/w) which is better than microcrystalline cellulose. Generally, chitin cannot be able to yield emulsions but the microcrystalline chitin showed improved emulsifying properties which increases on increasing its concentration (0.12–0.8 g/100 ml water) and 65% water addition is suitable for manufacturing the loaf of bread of chitin [21]. Chitin exhibits almost similar properties as chitosan including biodegradability, biocompatibility, nontoxicity, antimicrobial, anti-inflammatory, anticancer, antioxidant, etc. [22].

1.2 Chitosan

1.2.1 History

In 1859, C. Rouget published his findings that modified chitin could be prepared by treating chitin with boiling, concentration solutions of potassium hydroxide in water and he observed that chitin can be manipulated via chemical- and temperature-dependent treatment. Chitin was renamed as Chitosan by Hoppe-Seiler in 1894, pronounced as kite-O-san. Till 1920s, chitosan had been studied by the researchers as a subject and it seeks its importance in different fields and different ways of its extraction. In 1930s chitosan derived from chitin source such as shrimp shells, crabs, lobster, krill, and mushroom had been confirmed by Rammelberg. Till 1950s, some advance diffraction techniques had already been developed such as X-Ray diffraction, which were the most reliable one and it was therefore being proved that the cell wall of fungi, mycelia, and septa consists of chitin and chitosan. In 1960s, chitosan was studied as a hemostatic agent for its ability to bind with red blood cells, for water treatments and detoxification. Recently, it is considered as fat magnet because it acts as a fat inhibitor and hence beneficial dietary supplement for weight [23, 24].

1.2.2 Sources and Extraction

Chitosan is found in similar sources as of chitin. Similarly, terrestrial organisms include silkworm, insects, honeybee, arthropods, and nematodes. The exoskeleton of crustaceans, shrimp shells, crab, krill, lobster, etc. has been successfully utilized in the extraction of chitosan. On the other hand, mushrooms; cell wall of fungi, mycelia, and septa; spores of Zygomycetes and Ascomycetes are good source of chitosan besides Basidiomycetes. Generally, chitosan is extracted from the deacetylation of the chitin. As already explained above, chitin can be extracted from different sources, mainly crustaceans and shellfish waste via demineralization, deproteination, and decolorization. After the decolorization of the shellfish (or any other source), deacetylation can be performed in order to obtain the desired product, chitosan. The deacetylation is a chemical process and can be achieved by different methods. The methods involved either chemical or enzymatic method. The degree of deacetylation of chitosan obtained from insect were found to be 70–95% [3, 25]. The flow chart representation of extraction of chitosan has been depicted in Figure 1.5.

Figure 1.5 Extraction of chitosan.

1.2.2.1 Deacetylation of Chitin by Chemical Method

Previously, chitosan had been extracted chemically by means of fungi cell wall using alkali and acid treatments. In that process, cell wall constituents mainly proteins, lipids, and chitosan were first treated with 2–4% NaOH for 15–120 min at 100 °C and then the chitosan containing material (alkali insoluble) of cell wall with 2–10% CH3COOH solution for 1–24 h at 25–95 °C. The components of cell wall which neither dissolve in alkali nor in acid medium are called alkali-acid insoluble material and the acetic acid soluble material is referred to fungal chitosan [26, 27].

Chemically, deacetylation can be achieved by treating the washed and purified chitin with 25% and 50% NaOH by 1:5 (w/v) at 80 and 100 °C for 5 and 10 h, respectively. Thereafter, deacetylation the product is washed with deionized water till neutral pH and placed under vacuum oven at 60 °C for getting dried weight chitosan product [13]. Zamani et al. extracted chitosan from Zygomycetes fungus Rhizomucor pusillus cell wall by using sulphuric acid as a medium to dissolve it. He revealed from his studies that 45.3% of chitosan can be extracted from the fungi cell wall when treated with 1% H2SO4 for 20 min. at 121 °C followed by NaOH alkali solution. Contrary, he concluded that the acetic acid soluble material did not contain chitosan while the alkali-acid insoluble material contained chitosan and phosphate was the major source of impurity of acetic acid soluble material. Moreover, chitosan present in 8% of biomass and 45.3% in alkali insoluble material [28].

1.2.2.2 Deacetylation of Chitin by Enzymatic Method

In order to achieve chitosan, the decolorized and purified chitins attained after demineralization and deproteination can be reacted with NaOH. The concentration of NaOH could be 12.5M at 140 °C for 4 h. The ratio of the mixture of chitin to NaOH can be around 1:10 (w/v). The chitosan thus obtained is completely water soluble in acidic conditions. The residues are washed by distilled water and after 12 h drying in incubator at 50 °C, dry weight chitosan is obtained.

1.2.2.3 Deacetylation of Chitin by Microwave Assisted Method

Recently, it has been found that the three extraction steps of chitin can also be achieved by microwave assisted mechanism as a time saving as well as eco-friendly method. Knidri et al. successfully produced chitosan after deacetylation with the help of microwave irradiation, with degree of deacetylation of 82.73% within just 24 min. which is quite good as compared to the conventional method where it took 81.5% degree of deacetylation in much longer time of 5–10 h [29].

1.2.3 Structure and Composition

Chitosan has similar structure like cellulose and chitin, the only difference is the functional group at C-2 position. Cellulose and chitin possess hydroxyl group (–OH) and N-acetylamine group (–NHCOCH3), respectively, on the other hand, chitosan consists of amino group (–NH2) at C-2 position. Chitosan is a linear-chain polysaccharide which consists of N-acetyl-2-amino-2-deoxy-D-glucopyranose (acetylated unit) and 2-amino 2-deoxy-D-glucopyranose (deacetylated unit), where the repeating units are linked by β-(1 → 4)-glycosidic bonds as shown in Figure 1.6 [30]. Formation of chitosan from deacetylation of chitin can be confirmed by the Fourier transform infrared spectroscopy (FT-IR) due to the two amide bands I and II at 1655 cm–1 and 1583 cm–1, respectively. Higher intensity of band I and lower intensity of band II indicate efficient deacetylation and formation of –NH2 group [31]. On the basis of the molecular weight or density of the polymeric chain, chitosan can be divided into two types viz. low density chitosan (LDC) and high density chitosan (HDC).

Figure 1.6 Structure of chitosan.

1.2.4 Properties

Chitosan obtained from chitin after various procuring steps can have different conditions such as temperature, concentration, time, and deacetylation which can affect the physical, chemical, or biological properties of the product.

1.2.4.1 Physical Properties

1.2.4.1.1 Viscosity

Generally, viscosity of the chitosan solution is affected by many factors such as temperature, pH, concentration, molecular weight, degree of deacetylation, and method of extraction. Starting with temperature, if the temperature of the chitosan solution increases, its viscosity will decrease. Reverse is observed in case of concentration where there is an increase in viscosity as the concentration increases. In acidic medium, chitosan acts as an excellent viscosity enhancer. Type of acid used as a solvent to dissolve the chitosan is the criteria of pH of the solution which in turn affects the viscosity of the solution. The intrinsic viscosity of chitosan plays a vital role in its storage and stability. Basically, intrinsic viscosity deals with the ability of the solution to become viscous with suitable solvent and temperature and is directly proportional to the polymer average molecular weight. The intrinsic viscosity can be evaluated by using Mark-Houwink equation:

(1.2)

Graphic

where, η is intrinsic viscosity of the chitosan solution, k and α are polymer conformation constants, and Mw is the viscosity average molecular weight. Compact spherical structure of chitosan is defined by α = 0, random coil by α = 0.5–0.8, and rigid coil by α = 1.8. Moreover, viscosity of chitosan also influences biomedical properties such as wound dressing and biodegradation as well [17, 32, 33].

1.2.4.1.2 Molecular Weight

Molecular weight of chitosan greatly influences the physicochemical properties of the biopolymer. Generally molecular weight of chitosan can deal with the average of all the molecules present in the sample and can be evaluated with the help of advanced techniques such as light scattering, osmometry, NMR, viscometry, gel permeation chromatography, and size exclusion chromatography. These techniques applied on chitosan give varied results and hence comparison of finally achieved polymeric material from different manufacturers is tedious and complicated. Low and high molecular weight chitosan shows different effect on their physicochemical properties such as viscosity, hydrophilicity, moisture content, thermal properties, and stability. As chitosan polymeric chains of shorter length are of low molecular weight therefore indicates less interaction with hot sulfuric acid and hence lower degree of hydrolysis in comparison with longer ones. Additionally, the low molecular weight chitosan has ability to penetrate inside the bacterial cell thereby inhibits the RNA transcription leading to the death of the cell [34, 35]. Furthermore, the degree of deacetylation decreases the molecular weight of the chitosan [36]. Polydispersity, ratio of molecular weight and average molecular weight, indicates the uniformity and functionality of the polymer which is considered as respectable if occur in between 0.85 and 1.15 because of the good polymer homogeneity at this value. Also, the high molecular weight chitosan is considered as more stable. Various factors viz. thermal stability, temperature, pH, and mechanical shearing of chitosan affect the molecular weight of chitosan and are responsible for the variation in polydispersity index [32].

1.2.4.1.3 Degree of Deacetylation

Degree of deacetylation is an extremely important property of chitosan as it influences almost all the other properties to a significant extent. Ample of studies and different methods of production of chitosan revealed that most of the properties including physical properties such as viscosity, thermal, swelling, stability and chemical properties such as solubility, pH of the solution and reactivity are greatly influenced by the degree of deacetylation. It has been generally found that the temperature and time can modify the characteristic pattern of the deacetylation of chitosan which leads to alterations in its physicochemical as well as biological behavior. The high positive charge density on the polymeric chain leads to a high degree of deacetylation of around 97.5% which in turn responsible for good antimicrobial activity as compared to mild or moderate degree of deacetylation of about 83.7% [16]. Actually, the degree of deacetylation is the ratio of glucosamine to the N-acetylated glucosamine units. Commercially, the appropriate degree of deacetylation of chitosan should be 75–98% for biomedical purpose, as manufactured by pharmaceutical industries. Since, higher the degree of deacetylation, higher is its purity hence is of great interest in studying their degradation behavior. This is due to the fact that chitosan with high degree of deacetylation does not induce inflammation because of its lower affinity toward enzyme and therefore, exhibit slower rate of enzymatic degradation as compared to the lower (chitosan with lower degree of deacetylation) one [32]. Additionally, increase in concentration of the alkaline solution can increase the degree of deacetylation of the polymeric chain with increasing temperature and reaction time [36, 37].

1.2.4.1.4 Stability or Polymer Degradation

Stability is also an important property of chitosan biopolymer. The degradation of the polymeric chain of chitosan defines its stability under various conditions. Generally, during acid hydrolysis at low pH, the polymeric chain of chitosan cleaves and thus degrades the polymer. The cleavage mechanism involves depolymerisation followed by deacetylation by the splitting of β-1,4-glycosidic bonds followed by N-acetylglucosamine linkage, respectively. This depolymerisation leads to the generation of free radical species in the solution which in turn induces oxidation reactions. On the other hand, increased deacetylation results in decrease in molecular weight. After splitting, there has been observed strong intermolecular attractions among them as interchain cross-linking which can change its structure and structure-based properties. The degradation of chitosan mainly depends on degree of deacetylation, molecular weight, temperature, moisture content, polydispersity, and purity level. Besides this, in vivo degradation of chitosan can be performed by several enzymes such as lysozyme, a protease found in mammalian cells, generating oligosaccharides which are nontoxic and can be introduced into glycoproteins and glycosaminoglycans. Amid them, in vitro degradation of chitosan can be carried out under controlled conditions by various chemical reactions such as oxidation and enzymatic hydrolysis in order to obtain low molecular weight chitosan [32].

1.2.4.1.5 Thermal Properties

Elevated temperatures can alter the physicochemical properties of the chitosan solution such as solubility, viscosity, structural changes, etc. Generally, heating of polymeric solution causes the degradation and loss of stability. This degradation due to excessive heating at different rate is referred to thermal degradation and can be measured by thermogravimetric analysis (TGA) of the solution. This degradation can be performed in three steps:

At temperature range 30–110 °C, evaporation takes place in order to remove moisture from the polymeric solution.

At temperature range 180–340 °C, decomposition takes place.

At temperature 470 °C, subsequent weight loss of chitosan has been observed.

However, it has been found that the physicochemical properties of chitosan are unaffected below glass transition temperature. Also, elevated glass transition temperature leads to an increase in molecular weight of chitosan. As far as biomedical application is concerned, the temperature of chitosan should not exceed above 100 °C because excessive and uncontrolled heating may cause discoloration as well as depolymerisation which in turn alter its rheological properties [32].

1.2.4.1.6 Hygroscopicity and Swelling Ability

Chitosan is a hygroscopic pseudo-plastic natural polymer. It can form hydrogen bond with the functional group present in the polymeric chain, i.e., –NH2 and –OH with the O-atom and H-atom of water molecule, respectively. The rate of water take up ability can be determined by inherent moisture already present in the sample and the conditions in which it has been stored. The water content present in dry chitosan is found to be increased on decreasing the degree of deacetylation. The swelling property of chitosan decreases with an increase in the concentration of cross-linking agent [17]. Apart from this, viscosity, compressibility, and flow properties have also been affected by the absorbed water content and cause a little decrease in tensile strength. However, 6% (w/w) water content can enhance the binding ability due to weak hydrogen bonding interactions. It has also been reported that the storage of chitosan for long duration can increase the moisture content of chitosan but it reduces its ability to further binding to water as well as increase its degradation time [38]. This leads to the pronounced disintegration of chitosan by hydrolysis reaction. In order to measure the water content present in chitosan, a modest and fast technique, known as loss on drying technique can be used in which chitosan sample is weighed, heated, and then again weighed after cooling. Besides, for long duration storage of chitosan, swelling index test has also been utilized. The important point to be kept in mind is that the selection of type of medium erstwhile to the water studies. This is because of the varying effect of ionic strength, solubility, swelling behavior, and viscosity on chitosan. The swelling ratio can be evaluated by using the following formula after getting constant value of weighed sample at a predefined duration, usually at 37 °C temperature. The formula is:

(1.3)

Graphic

where, SR is the swelling ratio, Wi represents the initial weight, and Wf is the final weight of dosage form before and after the swelling, respectively [32].

1.2.4.2 Chemical Properties

1.2.4.2.1 Solubility

Chitosan is generally soluble in some organic as well as inorganic acids having pH < 6.0 of the solution and form a non-Newtonian, thin shearing fluid. Usually, the organic acids display solubility includes methanoic acid (HCOOH), acetic acid (CH3COOH), hydrochloric acid (1% HCl), dilute nitric acid (HNO3), lactic acid, etc. Out of these 0.2–100% aqueous methanoic acid considered as the best solvent to dissolve chitosan. But the most frequently used solvent is 1% CH3COOH at pH 4.0. It should be note here that at high temperature concentrated CH3COOH can cause depolymerisation of chitosan. Chitosan usually insoluble in phosphoric and sulphuric acid but at elevated temperatures around 95–121 °C both low as well as medium molecular weight chitosan can be dissolved. Chitosan can form the water soluble salts such as pyruvate, malate, lactate, malonate, ascorbate, acetate, tartarate, glyoxylate, and glycolate. The formation of water soluble salts takes place by the neutralization of acids viz. HCl, HCOOH, CH3COOH, and lactic acid. On the other hand, chitosan is insoluble in water and aqueous bases. This is due to the fact that the –NH2 group of chitosan cannot be protonated in basic or neutral medium but at low pH due to the electrostatic repulsion the free –NH2 group become protonated and hence soluble and allowing polymer solvation. Additionally, chitosan is also insoluble in organic nonpolar solvents such as dimethylformamide and dimethylsulphoxide but substantively soluble in acidified polyol. Solubility of chitosan solution mainly depends on the degree of deacetylation, method of extraction, time, temperature, concentrations, and molecular weight [17, 28, 32].

1.2.4.2.2 pH of Chitosan Solution

The dilute acidic chitosan solutions are found to have pH less than 6.0. Since chitosan itself known to be a strong base due to the presence of free –NH2 groups throughout the chain having pKa value of 6.3, hence pH of the solution can alter the properties and charge on chitosan. At low pH, the free –NH2 groups get protonated and acquire positive charge which lead chitosan a cationic polyelectrolyte and hence water soluble. Besides, it can form quaternary salts with nitrogen at low pH values. Amid them, at high pH, i.e, <6.0, again the amino groups become deprotonated thus made chitosan insoluble. The pKa value of chitosan also greatly depends on degree of deacetylation and method of extraction. Furthermore, chitosan has gelation ability to form gels with anionic hydrocolloids because of its acidic pH and hence can be utilized in slow release drug delivery [39].

1.2.4.2.3 Reactivity

Chitosan is a linear chain biopolymer having –NH2 and –OH functional group as a reactive site of its backbone. The extent of these functional groups, especially, amino group are protonated, that gives rise to more reactive the chitosan is. More the protonated –NH2 group available in the polymeric chain more is its reactivity and hence ability to bind with toxic metal ions and also form chelates with transition metal ions. This chelating ability of chitosan exemplifies its importance in water treatment and air purification. The nitrogen content available in chitosan is present in the form of primary aliphatic amino groups and hence undergo important amine N-acylation and Schiff reactions. Besides, chitosan can react with aldehydes and ketones to yield aldimines and ketimines, respectively, at room temperature. Additionally, proteic and nonproteic amino groups containing glucans can also be obtained by reacting chitosan with ketoacids followed by reaction with sodium borohydride, e.g., N-Carboxybenzyl chitosans (nonproteic glucan) obtained from o- and p-phthalaldehydic acids. Furthermore, on treating chitosan with simple aldehydes, hydrogenation takes place producing N-alkyl chitosan. Since, presence of hindered bulky group tends to decrease the strength of hydrogen bond of chitosan, this leads to enhance its swelling ability instead of being hydrophobic and hence can be utilized in film formation [18]. Amid them, the cationic nature of chitosan is being high positive charge density, greatly affect the reactivity, solubility, adsorption, and biodegradability of chitosan [17].

1.2.4.3 Miscellaneous Properties

Besides above physical and chemical properties, chitosan possesses many biological and miscellaneous properties. These includes [17, 33, 40]:

Non-toxicity

Gelation ability

Biodegradability

Biocompatibility

Chelating ability

Antimicrobial activity

Haemostaticity

Fungistaticity

Spermicidal activity

Anticancer activity

Anti-inflammatory

Mucoadhesion ability

Wound healing ability

Bone regeneration ability

Drug releasing ability

Immunoadjuvant ability

Being a natural polysaccharide, chitosan has been illustrated with many beneficial properties. The high positive charge density on the polymeric chain leads to a high degree of deacetylation of around 97.5% which in turn responsible for good antimicrobial activity as compared to mild or moderate degree of deacetylation of about 83.7%. Actually, the polycationic nature of chitosan tends to interact it with the negative charge density or anionic components of the bacterial cell and thus alters the function as well as permeability of the cell thereby causing cell death because of the rupture and leakage of intercellular components, sometimes by inhibiting RNA transcription. That’s why chitosan exhibits remarkable antibacterial or antimicrobial activity. This is the most accepted mechanism of its antibacterial activity as put forth by many researchers but the actual mechanism is still unknown. Chitosan also acquired excellent mucoadhesion property as it possesses free –OH and –NH2 groups which permit the polysaccharide chain to interact with mucin via electrostatically as well as via hydrogen bonding [32].

1.3 Conclusion

Chitin and chitosan are not unknown to the researchers and scientists of polymeric fields. And nowadays, these are not only confined to polymeric fields, instead being applied in electronics and pharmaceutical to biomedical fields also. Both chitin and chitosan are the naturally occurring polysaccharide with N-acetyl-D-glucosamine long chain polymeric unit having β-(1,4) linkage and glycosidic linkage. Chitin can be extracted from terrestrial (silkworm or honeybee), marine (crab, krill, lobster, squids), microorganisms (fungi), and enzymes (protease or lipase). In the extraction of chitin from these sources, there are three major steps termed as: demineralization, deproteination, and decolorisation. Chitosan is extracted from the same sources as of chitin and can be obtained after deacetylation of chitin. Various properties of chitin and chitosan such as purity, solubility, pH, viscosity, hygroscopicity, thermal, swelling, and reactivity have been greatly influenced by each other, especially the molecular weight and degree of deacetylation. Degree of deacetylation plays an important role in altering almost all the properties of chitosan and hence the results too. Viewing biomedical aspects of chitin as well as chitosan, it has been observed that marine originated more deacetylated products gives better results in terms of its properties, as compared to less deacetylated products.

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Chapter 2

Nitrogenated Polysaccharides – Chitin and Chitosan, Characterization and Application

Michael Ioelovich

Designer Energy Ltd, Rehovot, Israel

Corresponding author: [e-mail]

Abstract

In this chapter preparation, characterization and applications of chitin and chitosan have been described and discussed. These nitrogenated polysaccharides occupy second place prevalence in the nature after cellulose. Diverse physical, physicochemical, and chemical methods are applied to characterize structure and properties of these biopolymers. Being nanostructured, chitin and chitosan can be isolated from natural sources in a form of nanoscale particles, fibrils, and filaments having unique features such as small dimensions, variety shapes, enhanced specific surface, high sorption and absorption ability, and other specific characteristic in combination with biocompatibility, biodegradability, and complex of unique therapeutic properties. In addition, these biopolymers serve as a basis for the production of some therapeutically active substances. Therefore, chitin and chitosan find a great commercial interest in biomedicine, pharmaceutics, cosmetics, personal care, and some other areas. Small particles and fibrils made of the biopolymers can be used as fillers for biocomposites and thickeners imparting to liquid systems an increased viscosity and gel consistence. The nanocarriers of nitrogenated polysaccharides can bind various therapeutic active substances, which expand application areas of these biopolymers. Antibacterial nonwoven materials made of nanofilaments have an increased absorption ability and accelerate healing process. Some other application areas of the nitrogenated polysaccharides are also described.

Keywords: Chitin, chitosan, features, nanomaterials, preparation, characterization, applications

2.1 Introduction

As is known, chitin is one from the most widespread biopolymers that occupies second place prevalence in the nature after cellulose. Resources of this biopolymer are estimated in 100 billion tons [1]. In the nature, chitin is found in shells of mollusks, shrimps and lobsters, pens and beaks of cephalopods, exoskeletons of arthropods, crustaceans and insects, cell walls and membranes of some fungi and microorganisms, etc. Chitin implements a skeleton function in lower eukaryotes similar to that of cellulose in plants. Commercially, chitin can be isolated from shells of crabs, shrimps and lobsters by acidic treatment to remove of calcium carbonate followed by alkaline extraction of proteins and bleaching [2]. Annual production volume of chitin in the world is estimated at about 80–100 thousand metric tons.

Macromolecules of this nitrogenated polysaccharide consist of 1,4-β-N-acetylglucosamine (more precise 1,4-β-N-acetyl-2-aminodeoxyglucose) units [2, 3]. Chitin can be regarded as a nitrogenated derivative of cellulose, where the hydroxyl group at C2 atoms in each repeat units is replaced with an acetylamino group. Inter and intramolecular hydrogen bonds impart to chitin chains an increased stiffness. Molecular weight (MW) of chitin samples varies in the range of 100–1000 kDa. Similar to cellulose, chitin is a linear semicrystalline biopolymer [4, 5]. The linear macromolecules joined by hydrogen bonds form a supramolecular structure of the polymer that consists of nanoscale fibrils [6, 7]. Each fibril is built of ordered crystallites and low-ordered noncrystalline (amorphous) domains statistically alternating along the fibril.

Structural studies showed that crystallites of chitin can be in three allomorph forms: α, β, and γ [8-11]. The most abundant α-form is present in chitin samples isolated from sea animals such as crabs, shrimps, lobsters, krill, etc., insects, fungi, and some microorganisms. The rare β-allomorph is found in the chitin of squid pens, tube-worms and some others sources. The γ-form of chitin can be isolated from some beetles [12]. The β- and γ-forms of chitin are instable and can undergo an intra-crystalline swelling; moreover, after some treatments these forms of chitin are transformed irreversibly into more stable α-polymorph [10, 11].

Chitin is also feedstock for production of another known nitrogenated polysaccharide – chitosan, by means of alkaline deacetylation process. Global market of chitosan in 2015 was around 20–25 thousand metric tons. The Asia-Pacific region has the leading chitosan market with a share of 55%, while the US represents the second biggest market for chitosan, with an estimated share of 25%.

Native chitosan occurs in cell walls of some fungi, for example, Mucoraceae [3]. Chitosan is a linear semicrystalline nitrogenated cellulose derivative composed of 1,4-β-2-glucosamine units. Degree of deacetylation (DD) of chitosan samples isolated from chitin ranges from 60% to 100%. MW of chitosan samples can be in the range 5–500 kDa. Unlike cellulose and chitin, chitosan dissolves in an aqueous medium having a weak acidic pH value. After neutralization, acidic solutions of chitosan turn into hydrogels [13].

Both chitin and chitosan can be isolated from natural sources in a form of nanoscale particles, fibrils and filaments having unique features, such as small dimensions, variety shapes, enhanced specific surface, high sorption and absorption ability, and other specific characteristic in combination with biocompatibility, biodegradability, and complex of unique therapeutic properties – antibacterial, analgesic, fungistatic, haemostatic, etc. In addition, these biopolymers serve as a basis for the production of diverse therapeutically active substances, for example, glucosamine, N-acetylglucosamine, nitrogenated oligosaccharides, etc. Therefore, chitin and chitosan can be applicable in biomedicine, pharmaceutics, cosmetics, personal care, and some other areas.

Small particles and fibrils made of the biopolymers can be used as fillers for biocomposites and thickeners imparting to liquid systems an increased viscosity and gel consistence. The solid and gel-like nanocarriers of the nitrogenated polysaccharides can bind various therapeutic active substances, which expand application areas of these biopolymers. Antibacterial nonwoven materials made of nanofilaments have an increased absorption ability and accelerate healing process. Multilayer dressings containing the nitrogenated polysaccharides are used for active treatment of injuries, wounds, and burns. Moreover, both biopolymers can find application in some other areas such as production of membranes, adhesives, coatings, sorbents, cleansing additives, fining agents, etc.

The purpose of this papers was to describe preparation methods, structure, properties and main applications of chitin and chitosan, and their based materials, including nanoscale particles, fibrils, and filaments.

2.2 Extraction of Nitrogenated Polysaccharides from Natural Sources

Chitin can be extracted from broad variety of natural sources such as aquatic animals (crustaceans, arthropods, cephalopods, mollusks, shrimps, lobsters, etc.), insects (scorpions, spiders, beetles, ants, etc.), microorganisms (yeast, microalgae, etc.), fungi and some other sources [14]. The chitin sources can also contain proteins, lipids, minerals, mainly calcium carbonate, and some other admixtures [15]. For example, nanofibrils of chitin in sea animals are strongly bound with proteins, and this organic complex is surrounded by a mineral layer [16, 17]. The different sources of chitin have different structure and chemical composition. Content of chitin in shells of various crustaceans can be from 14% to 70%, in shells of insects from 18% to 65% and in cell walls of microorganisms from 20% to 45% [2, 17, 18]. The average chitin content in shrimp shells was 18–20% of the dry weight of the shells. Furthermore, shell of crustaceans contains 10–50% of proteins and 20–60% of minerals.

Since natural sources contain proteins, minerals, lipids, and pigments, these admixtures should be removed to isolate pure chitin. Extraction procedure of chitin usually involves steps of demineralization, deproteinization, and decoloration [19, 20]. The chosen raw-material is preliminary purified by washing with water and organic solvents,