# Conversion of Aquaculture Waste into Biomedical Wealth: Chitin and Chitosan Journey

Authors: Jerrine Joseph; Kaari Manigundan; Mary Shamya Arokia Rajan; Manikkam Radhakrishnan; Venugopal Gopikrishnan; Subramanian Kumaran; Rajasekar Thirunavukkarasu; Wilson Aruni; Velmurugan Shanmugam
Journal: Advances in Materials Science and Engineering
 (2022)

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## Abstract

Biowaste originating from aquaculture sector represents a potential feedstock to produce value-added substances and materials like chitin and chitosan. They are the long chain polymers of N-acetylglucosamine polymers with huge industrial and biomedical significance. Chitin has long been recognized as a useful biomaterial for drug delivery and neurological therapy. Similarly, chitosan oligosaccharide, a short chain polymer derived from chitin/chitosan, has been identified for its potential biomedical applications, including antibacterial, anti-inflammatory, and anti-Alzheimer action. Chitosan nanoparticles are also used extensively in biomedical applications. Here we have critically summarized various methods for the extraction of chitin, chitosan, and chitooligossacharides and chitosan nanoparticle preparation and their diverse biomedical applications.


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## Body

## 1. Introduction

Aquaculture is the world’s most prominent, high-growth agribusiness enterprise. It caters to the elite needs and consumer satisfaction. Aquatic organisms make up more than 150 billion USD per year in the market. Similarly, in last 20 years, the shrimp production went up to 3 million tons in every year. This scenario is made feasible by increasing shrimp production through high stocking density and the use of high-quality protein feeds [1]. Aquaculture derived byproducts are mixed with items produced in industrial processing that are commercially important. In particular the chitin and chitosan source are epitomized by the exoskeleton [2]. Aquaculture waste materials are the abundant source of chitin [3]. The freshwater aquaculture contains fish pens, fish cages, and ponds on a smaller scale in rice paddies. The brackish water is commonly carried out in coastal fish ponds. The rafts, stakes, and ropes are used in marine culture to hold a fish cage of mollusks and seaweeds. The disposal of large quantities of environmentally harmful effluents uneaten feed and feces such as fish waste, natural pigments (usually incorporated into feeds), disinfectants and chemotherapeutants, collagen, and chitosan has resulted in an increase in negative environmental impacts [4].Chitin and its derivatives, which are a significant aquaculture waste, are increasingly being recognized as imperative biopolymers [5, 6]. They are used by aquatic inhabitants as nourishing material. The biological uses of chitin and its derivatives in the realm of therapeutics are vast, notably those related to anti-inflammatory, antioxidant, antibacterial, immunity-enhancing, and anticancer effects and drug transport [6, 7]. As a result of its biological importance, this review focuses on the biomedically valuable products of chitin, chitosan, and its derivatives.


## 2. Chitin

Chitin (Figure1(a)) is a primary polysaccharide found in crab shells and their byproducts. In nature, they are cationic polysaccharides linked to glycosaminoglycans. (1,4)-N-acetyl-D-glucose-2-amine is the molecular name for it, and it appears in three polymorphic forms: d-chitins. This is due to extraordinarily strong hydrogen interactions between adjacent chains’ amide groups and carbonyl groups. Deacetylated chitosan is a linear polysaccharide composed of randomly dispersed -(1–4)-linked D-glucosamine and N-acetyl-D-glucosamine with varying degrees of deacetylation (DD; 40–99%) and molecular weight (MW) [8, 9]. Their degree of deacetylation is one of the most important factors influencing both its chemical and biological properties [9–11]. It has the ability to chelate different metal ions because of their neutral or negatively charged hydroxyl groups of D-glucosamine, GlcN residues, and amino groups [12, 13].Figure 1
Structure of chitin (a), chitosan (b), and COS (c).
(a)(b)(c)Furthermore, alkali concentration, polymer chemical form, chitin type and polymorphism type, chitin particle size, heterogeneous or homogeneous N-deacetylation, single or multiple procedures, atmosphere, and incubation duration have all been demonstrated to alter chitin and its deacetylated chitosan characteristics. All these traits, as well as its excellent mechanical properties, biocompatibility, and biodegradability, open the door to a wide range of applications [14, 15].


### 2.1. Extraction of Chitin

Chitin is found in crustaceans and shellfish as part of a complex protein network onto which calcium carbonate deposits to form the hard shell. Chitin separation from shellfish necessitates deproteinization and demineralization of two primary constituents: proteins and inorganic calcium carbonate. Over the years, many techniques for preparing pure chitin have been developed and employed; however, no standard approach has been accepted universally. Chemical or enzymatic treatments might be used for both deproteinization and demineralization.


#### 2.1.1. Chemical Method

(1) Chemical Deproteinization. This is done in a heterogeneous manner with chemicals that depolymerize the biopolymer as well. Chemical approaches were used to begin the deproteinization process. A variety of chemicals have been attempted as deproteinization reagents, including NaOH, Na2CO3, NaHCO3, KOH, K2CO3, Ca(OH)2, Na2SO3, NaHSO3, CaHSO3, Na3PO4, and Na2S. NaOH is the chosen reagent, which can be employed in concentrations ranging from 0.125 to 5.0 M, at various temperatures (up to 160°C) and for various treatment periods (from few minutes up to few days). In addition to deproteinization, NaOH causes partial deacetylation of chitin as well as hydrolysis of the biopolymer, lowering the molecular weight of the biopolymer [16].(2) Chemical Demineralization. Acid treatment with HCl, HNO3, H2SO4, and CH3COOH is the most common method for removing the minerals and calcium carbonate from chitin. Demineralization is simple to accomplish since it entails the breakdown of calcium carbonate into water-soluble calcium salts while also releasing carbon dioxide. The degree of mineralization of each shell, extraction duration, temperature, particle size, acid content, and solute/solvent ratio are all factors that influence these treatments. Calcium chloride is dependent on the acid content since it takes two molecules of HCl to convert one molecule of calcium carbonate into calcium chloride [17, 18].Acid intake should be equal to, or even more than, the stoichiometric quantity of minerals in order to achieve a full reaction. Because the heterogeneity of the material makes it difficult to extract all minerals, a greater volume or more concentrated acid solution is required. Acidimetric titration may be used to evaluate it. The consumption of acid is correlated with the advancement of pH toward neutrality, but the persistence of acidity in the medium indicates that the reaction is complete. Demineralization has historically been achieved at room temperature with dilute HCl at various concentrations (up to 10% w/v), 90 percent formic acid, and 22% HCl, respectively [19].


#### 2.1.2. Biological Method

Chitin extraction necessitates the use of proteases, which can be found in plants, bacteria, and animals. Proteases such as alcalase, pepsin, papain, pancreatin, devolvase, and trypsin remove proteins from crustacean shells while reducing deacetylation and depolymerization during chitin separation. This treatment can be used after or before the solid material’s demineralization phase, which changes the accessibility of the reactant. Both pure and coarsely extracted proteases are used in the deproteinization process. Crude proteases, on the other hand, are not only economically cheaper but also more efficient due to the presence of coexisting proteases [20].(1) Biological Deproteinization. Bacterial proteases have been found to be useful in the deproteinization process in numerous investigations. One example is the enzymatic deproteinization of previously demineralized shrimp waste to produce chitin and a nutrient-dense protein hydrolysate. Alcalase, a serine endopeptidase derived from Bacillus licheniformis, was used for deproteinization because of its selectivity for terminal hydrophobic amino acids, which results in nonbitter hydrolysate and allows for simple regulation of the degree of hydrolysis. Alcalase was employed for chitin, protein hydrolysate, and astaxanthin recovery [21]. The residual protein concentration of chitin separated by enzymatic deproteinization was considerably greater than that of chitin isolated by alkali treatment [22]. Shrimp shell deproteinization has recently used alkaline crude proteases from a variety of fish and marine invertebrates. Younes et al., 2014, investigated the use of crustacean feces as both a substrate and a source of proteases. For shrimp waste deproteinization, alkaline proteases are utilized from the red scorpion fish Scorpaena scrofa [23].(2) Nonenzymatic Process. Deproteinization via fermentation, which can be done by endogenous microorganisms (also known as autofermentation) or by introducing chosen strains of microorganisms, can reduce the expense of employing enzymes. This method can use single-stage fermentation, two-stage fermentation, cofermentation, or sequential fermentation. For the fermentation of crustacean shells, a variety of microbial species have been proposed. Lactic acid fermentation and non-lactic-acid fermentation are the two types of fermentation that can be used [24].(1)
Lactic Acid Fermentation. To ferment crustacean shells, inoculum generated from lactic acid and protease producing Lactobacillus sp. can be utilized. Lactic acid is formed when glucose is converted to alcohol, resulting in a lower pH in the silage, which inhibits the growth of spoilage bacteria. Calcium lactate precipitates when lactic acid reacts with calcium carbonate. The number of species and inoculum, carbon supply and concentration, starting pH and pH variation throughout fermentation, temperature, and fermentation length are all factors that influence the success of lactic acid fermentation [25–27].(2)
Non-Lactic-Acid Fermentation For crustacean shell fermentation without lactic acid, bacteria (Bacillus sp., Pseudomonas sp.) and fungus (Aspergillus sp.) were employed. Ghorbel-Bellaaj et al. [28] tested 6 proteolytic Bacillus sp. (B. pumilus A1, B. mojavensis A21, B. licheniformis RP1, B. cereus SV1, B. amyloliquefaciens An6, and B. subtilis A26) on fermentation of shrimp waste. The highest degree of deproteinization was achieved with B. cereus SV1. B. subtilis was employed to ferment shrimp shells in jaggery broth, eliminating 84 percent of the proteins and 72 percent of the minerals [29]. The fermentation process and hence deproteinization and demineralization efficiency are influenced by a variety of variables. Ghorbel-Bellaaj et al. [30] used a Plackett-Burman factorial design to investigate the important parameters influencing fermentation efficacy using P. aeruginosa A2. Shrimp shell concentration, glucose concentration, inoculum size, and incubation length were found to alter deproteinization and demineralization degrees [30].


## 2.1. Extraction of Chitin

Chitin is found in crustaceans and shellfish as part of a complex protein network onto which calcium carbonate deposits to form the hard shell. Chitin separation from shellfish necessitates deproteinization and demineralization of two primary constituents: proteins and inorganic calcium carbonate. Over the years, many techniques for preparing pure chitin have been developed and employed; however, no standard approach has been accepted universally. Chemical or enzymatic treatments might be used for both deproteinization and demineralization.


### 2.1.1. Chemical Method

(1) Chemical Deproteinization. This is done in a heterogeneous manner with chemicals that depolymerize the biopolymer as well. Chemical approaches were used to begin the deproteinization process. A variety of chemicals have been attempted as deproteinization reagents, including NaOH, Na2CO3, NaHCO3, KOH, K2CO3, Ca(OH)2, Na2SO3, NaHSO3, CaHSO3, Na3PO4, and Na2S. NaOH is the chosen reagent, which can be employed in concentrations ranging from 0.125 to 5.0 M, at various temperatures (up to 160°C) and for various treatment periods (from few minutes up to few days). In addition to deproteinization, NaOH causes partial deacetylation of chitin as well as hydrolysis of the biopolymer, lowering the molecular weight of the biopolymer [16].(2) Chemical Demineralization. Acid treatment with HCl, HNO3, H2SO4, and CH3COOH is the most common method for removing the minerals and calcium carbonate from chitin. Demineralization is simple to accomplish since it entails the breakdown of calcium carbonate into water-soluble calcium salts while also releasing carbon dioxide. The degree of mineralization of each shell, extraction duration, temperature, particle size, acid content, and solute/solvent ratio are all factors that influence these treatments. Calcium chloride is dependent on the acid content since it takes two molecules of HCl to convert one molecule of calcium carbonate into calcium chloride [17, 18].Acid intake should be equal to, or even more than, the stoichiometric quantity of minerals in order to achieve a full reaction. Because the heterogeneity of the material makes it difficult to extract all minerals, a greater volume or more concentrated acid solution is required. Acidimetric titration may be used to evaluate it. The consumption of acid is correlated with the advancement of pH toward neutrality, but the persistence of acidity in the medium indicates that the reaction is complete. Demineralization has historically been achieved at room temperature with dilute HCl at various concentrations (up to 10% w/v), 90 percent formic acid, and 22% HCl, respectively [19].


### 2.1.2. Biological Method

Chitin extraction necessitates the use of proteases, which can be found in plants, bacteria, and animals. Proteases such as alcalase, pepsin, papain, pancreatin, devolvase, and trypsin remove proteins from crustacean shells while reducing deacetylation and depolymerization during chitin separation. This treatment can be used after or before the solid material’s demineralization phase, which changes the accessibility of the reactant. Both pure and coarsely extracted proteases are used in the deproteinization process. Crude proteases, on the other hand, are not only economically cheaper but also more efficient due to the presence of coexisting proteases [20].(1) Biological Deproteinization. Bacterial proteases have been found to be useful in the deproteinization process in numerous investigations. One example is the enzymatic deproteinization of previously demineralized shrimp waste to produce chitin and a nutrient-dense protein hydrolysate. Alcalase, a serine endopeptidase derived from Bacillus licheniformis, was used for deproteinization because of its selectivity for terminal hydrophobic amino acids, which results in nonbitter hydrolysate and allows for simple regulation of the degree of hydrolysis. Alcalase was employed for chitin, protein hydrolysate, and astaxanthin recovery [21]. The residual protein concentration of chitin separated by enzymatic deproteinization was considerably greater than that of chitin isolated by alkali treatment [22]. Shrimp shell deproteinization has recently used alkaline crude proteases from a variety of fish and marine invertebrates. Younes et al., 2014, investigated the use of crustacean feces as both a substrate and a source of proteases. For shrimp waste deproteinization, alkaline proteases are utilized from the red scorpion fish Scorpaena scrofa [23].(2) Nonenzymatic Process. Deproteinization via fermentation, which can be done by endogenous microorganisms (also known as autofermentation) or by introducing chosen strains of microorganisms, can reduce the expense of employing enzymes. This method can use single-stage fermentation, two-stage fermentation, cofermentation, or sequential fermentation. For the fermentation of crustacean shells, a variety of microbial species have been proposed. Lactic acid fermentation and non-lactic-acid fermentation are the two types of fermentation that can be used [24].(1)
Lactic Acid Fermentation. To ferment crustacean shells, inoculum generated from lactic acid and protease producing Lactobacillus sp. can be utilized. Lactic acid is formed when glucose is converted to alcohol, resulting in a lower pH in the silage, which inhibits the growth of spoilage bacteria. Calcium lactate precipitates when lactic acid reacts with calcium carbonate. The number of species and inoculum, carbon supply and concentration, starting pH and pH variation throughout fermentation, temperature, and fermentation length are all factors that influence the success of lactic acid fermentation [25–27].(2)
Non-Lactic-Acid Fermentation For crustacean shell fermentation without lactic acid, bacteria (Bacillus sp., Pseudomonas sp.) and fungus (Aspergillus sp.) were employed. Ghorbel-Bellaaj et al. [28] tested 6 proteolytic Bacillus sp. (B. pumilus A1, B. mojavensis A21, B. licheniformis RP1, B. cereus SV1, B. amyloliquefaciens An6, and B. subtilis A26) on fermentation of shrimp waste. The highest degree of deproteinization was achieved with B. cereus SV1. B. subtilis was employed to ferment shrimp shells in jaggery broth, eliminating 84 percent of the proteins and 72 percent of the minerals [29]. The fermentation process and hence deproteinization and demineralization efficiency are influenced by a variety of variables. Ghorbel-Bellaaj et al. [30] used a Plackett-Burman factorial design to investigate the important parameters influencing fermentation efficacy using P. aeruginosa A2. Shrimp shell concentration, glucose concentration, inoculum size, and incubation length were found to alter deproteinization and demineralization degrees [30].


## 2.1.1. Chemical Method

(1) Chemical Deproteinization. This is done in a heterogeneous manner with chemicals that depolymerize the biopolymer as well. Chemical approaches were used to begin the deproteinization process. A variety of chemicals have been attempted as deproteinization reagents, including NaOH, Na2CO3, NaHCO3, KOH, K2CO3, Ca(OH)2, Na2SO3, NaHSO3, CaHSO3, Na3PO4, and Na2S. NaOH is the chosen reagent, which can be employed in concentrations ranging from 0.125 to 5.0 M, at various temperatures (up to 160°C) and for various treatment periods (from few minutes up to few days). In addition to deproteinization, NaOH causes partial deacetylation of chitin as well as hydrolysis of the biopolymer, lowering the molecular weight of the biopolymer [16].(2) Chemical Demineralization. Acid treatment with HCl, HNO3, H2SO4, and CH3COOH is the most common method for removing the minerals and calcium carbonate from chitin. Demineralization is simple to accomplish since it entails the breakdown of calcium carbonate into water-soluble calcium salts while also releasing carbon dioxide. The degree of mineralization of each shell, extraction duration, temperature, particle size, acid content, and solute/solvent ratio are all factors that influence these treatments. Calcium chloride is dependent on the acid content since it takes two molecules of HCl to convert one molecule of calcium carbonate into calcium chloride [17, 18].Acid intake should be equal to, or even more than, the stoichiometric quantity of minerals in order to achieve a full reaction. Because the heterogeneity of the material makes it difficult to extract all minerals, a greater volume or more concentrated acid solution is required. Acidimetric titration may be used to evaluate it. The consumption of acid is correlated with the advancement of pH toward neutrality, but the persistence of acidity in the medium indicates that the reaction is complete. Demineralization has historically been achieved at room temperature with dilute HCl at various concentrations (up to 10% w/v), 90 percent formic acid, and 22% HCl, respectively [19].


## 2.1.2. Biological Method

Chitin extraction necessitates the use of proteases, which can be found in plants, bacteria, and animals. Proteases such as alcalase, pepsin, papain, pancreatin, devolvase, and trypsin remove proteins from crustacean shells while reducing deacetylation and depolymerization during chitin separation. This treatment can be used after or before the solid material’s demineralization phase, which changes the accessibility of the reactant. Both pure and coarsely extracted proteases are used in the deproteinization process. Crude proteases, on the other hand, are not only economically cheaper but also more efficient due to the presence of coexisting proteases [20].(1) Biological Deproteinization. Bacterial proteases have been found to be useful in the deproteinization process in numerous investigations. One example is the enzymatic deproteinization of previously demineralized shrimp waste to produce chitin and a nutrient-dense protein hydrolysate. Alcalase, a serine endopeptidase derived from Bacillus licheniformis, was used for deproteinization because of its selectivity for terminal hydrophobic amino acids, which results in nonbitter hydrolysate and allows for simple regulation of the degree of hydrolysis. Alcalase was employed for chitin, protein hydrolysate, and astaxanthin recovery [21]. The residual protein concentration of chitin separated by enzymatic deproteinization was considerably greater than that of chitin isolated by alkali treatment [22]. Shrimp shell deproteinization has recently used alkaline crude proteases from a variety of fish and marine invertebrates. Younes et al., 2014, investigated the use of crustacean feces as both a substrate and a source of proteases. For shrimp waste deproteinization, alkaline proteases are utilized from the red scorpion fish Scorpaena scrofa [23].(2) Nonenzymatic Process. Deproteinization via fermentation, which can be done by endogenous microorganisms (also known as autofermentation) or by introducing chosen strains of microorganisms, can reduce the expense of employing enzymes. This method can use single-stage fermentation, two-stage fermentation, cofermentation, or sequential fermentation. For the fermentation of crustacean shells, a variety of microbial species have been proposed. Lactic acid fermentation and non-lactic-acid fermentation are the two types of fermentation that can be used [24].(1)
Lactic Acid Fermentation. To ferment crustacean shells, inoculum generated from lactic acid and protease producing Lactobacillus sp. can be utilized. Lactic acid is formed when glucose is converted to alcohol, resulting in a lower pH in the silage, which inhibits the growth of spoilage bacteria. Calcium lactate precipitates when lactic acid reacts with calcium carbonate. The number of species and inoculum, carbon supply and concentration, starting pH and pH variation throughout fermentation, temperature, and fermentation length are all factors that influence the success of lactic acid fermentation [25–27].(2)
Non-Lactic-Acid Fermentation For crustacean shell fermentation without lactic acid, bacteria (Bacillus sp., Pseudomonas sp.) and fungus (Aspergillus sp.) were employed. Ghorbel-Bellaaj et al. [28] tested 6 proteolytic Bacillus sp. (B. pumilus A1, B. mojavensis A21, B. licheniformis RP1, B. cereus SV1, B. amyloliquefaciens An6, and B. subtilis A26) on fermentation of shrimp waste. The highest degree of deproteinization was achieved with B. cereus SV1. B. subtilis was employed to ferment shrimp shells in jaggery broth, eliminating 84 percent of the proteins and 72 percent of the minerals [29]. The fermentation process and hence deproteinization and demineralization efficiency are influenced by a variety of variables. Ghorbel-Bellaaj et al. [30] used a Plackett-Burman factorial design to investigate the important parameters influencing fermentation efficacy using P. aeruginosa A2. Shrimp shell concentration, glucose concentration, inoculum size, and incubation length were found to alter deproteinization and demineralization degrees [30].


## 3. Chitosan

Chitosan is a common term for a group of polymers formed by different degrees of chitin deacetylation. Figure1(b) depicts the structure of chitosan. In reality, the degree of acetylation distinguishes chitin and chitosan, indicating the balance between the two types of residues (Figure 2). To convert chitin to chitosan, enzyme preparations or a chemical method are used [31, 32]. Chemical techniques are widely utilized in the commercial manufacturing of chitosan due to its low cost and adaptability for large scale production [33]. Because of its positive charges (NH3+) at pH (pH 6.5), chitosan is insoluble in neutral and acidic environment, which is one of its benefits.Figure 2
Flowchart of chemical and biological processes for chitin, chitosan, and COS.Alkali deacetylation is used more frequently because glycosidic connections are particularly susceptible to acid [33]. N-Deacetylation of chitin can be accomplished in two ways: heterogeneously or homogeneously [34, 35]. In the heterogeneous method, chitin is treated for a few hours with a hot concentrated NaOH solution, and chitosan is produced as an insoluble residue that is deacetylated up to 85% to 99%. The homogeneous approach, which involves dispersion of chitin in concentrated NaOH (30 g NaOH/45 g H2O/3 g Chitin) at 25°C for 3 hours or longer and then dissolving it in crushed ice at 0°C, is used to make alkali chitin.The solubility of chitosan can be measured not only by the number of 2-acetamido-2-deoxy-D-glucose units in the molecule, but also by the distribution of N-acetyl groups [36]. According to Aiba [37], deacetylation reactions in heterogeneous conditions result in an uneven distribution of N-acetyl-D-glucosamine and D-glucosamine residues, as well as some blockwise acetyl group distribution along polymeric chains. As a result, solubility and degree of chitosan aggregation in aqueous solutions may differ, leading to variances in average characteristics. Changes in DA, acetyl group distribution throughout chains, MW, and viscosity in solution may also be caused by differences in chitosan manufacturing [38]. In actuality, a number of factors in the deacetylation process might affect the characteristics of the finished chitosan [39]. Temperature and processing time have a significant impact on the DA and MW of chitosan, according to Rege and Block [40].


### 3.1. Extraction of Chitosan

#### 3.1.1. Chemical Deacetylation

In industrial applications, the chemical conversion of chitin to chitosan, which is the most difficult and costly step, has been done with 50 percent NaOH at high temperatures. Chitin deacetylase performs this step-in nature at lower temperatures and a neutral pH [41]. In N-acetyl-D-glucosamine residues of chitin, the enzyme hydrolyzes the linking between the acetyl group and the amine group. Chitin oligosaccharides are bioconverted into chitosan oligosaccharides and vice versa. DY-9 chitin deacetylase from Absidia corymbifera was found to be active against water-soluble chitin (WSCT-50), glycol chitin, chitosan (DD 71–88%), and chitin oligosaccharides [42, 43]. Chitin oligosaccharides were a suitable substrate because of their solubility, which improved the CDA’s availability and accessibility. Size affects the rate of deacetylation of (GlcNAc) 2–6, as longer lengths result in higher rate activity. These data indicate that chitin solubilization is a limiting factor in CDA-mediated enzymatic chitin to chitosan bioconversion.


#### 3.1.2. Enzymatic Method

Chitin deacetylases convert chitin to chitosan in a manner that is controlled and nondegradable and results in chitosan that is fresh and well defined [44]. Chitosan oligomers, in particular, are made using this method. Chitin deacetylase catalyzes the hydrolysis of N-acetamido links in chitin to produce chitosan. Glycoproteins called chitin deacetylases are released into the periplasmic or culture media. Furthermore, all enzymes have a strong selectivity for -(1,4)-linked N-acetyl-D-glucosamine polymers and are thermally stable at their optimal temperature (50°C). It is worth mentioning that acetate (a deacetylation product) has no effect on the chitin deacetylases produced by C. lindemuthianum and A. nidulans, making them suitable for biotechnological applications [45–47].The impact of chitin deacetylase, isolated from the fungusM. rouxii, on the chitosan synthesis was examined using chitin as a substrate (in both its crystalline and amorphous form) [48]. The enzyme is inefficient on insoluble chitins because the degree of deacetylation stays low. As a result, it appears that pretreatment of chitin substrates prior to enzyme addition is necessary to increase the accessibility of the acetyl groups to the enzyme and therefore the deacetylation yield. Experiments with partially deacetylated water-soluble chitosan’s and M. rouxii chitin deacetylase were carried out under controlled conditions to achieve this goal. The enzyme may deacetylate chitosan up to 97% under certain conditions (deacetylation from an initial chitosan with DA = 0.32 and a number-average degree of polymerization of 30) [49].


### 3.2. Chitosan Nanoparticles

The addition of a polyanion, such as tripolyphosphate, to a chitosan solution under continuous stirring can produce nanoparticles. Ionotropic gelation, microemulsion, emulsification solvent diffusion, and emulsion-based solvent evaporation are the most common ways for creating chitosan-based nanoparticles. Drugs are trapped inside the polymeric matrix through electrostatic contact, hydrogen bonding, and hydrophobic interactions, among other mechanisms. With or without a stabilizing agent like poloxamer, a positively charged chitosan solution is dissolved in acetic acid or any other negatively charged polyanionic solution. Nanoparticles are easily formed as a result of the complexation of positive and negative charged species during mechanical stirring at room temperature, resulting in the separation of chitosan into spherical particles of varied sizes and surface charges [50].


## 3.1. Extraction of Chitosan

### 3.1.1. Chemical Deacetylation

In industrial applications, the chemical conversion of chitin to chitosan, which is the most difficult and costly step, has been done with 50 percent NaOH at high temperatures. Chitin deacetylase performs this step-in nature at lower temperatures and a neutral pH [41]. In N-acetyl-D-glucosamine residues of chitin, the enzyme hydrolyzes the linking between the acetyl group and the amine group. Chitin oligosaccharides are bioconverted into chitosan oligosaccharides and vice versa. DY-9 chitin deacetylase from Absidia corymbifera was found to be active against water-soluble chitin (WSCT-50), glycol chitin, chitosan (DD 71–88%), and chitin oligosaccharides [42, 43]. Chitin oligosaccharides were a suitable substrate because of their solubility, which improved the CDA’s availability and accessibility. Size affects the rate of deacetylation of (GlcNAc) 2–6, as longer lengths result in higher rate activity. These data indicate that chitin solubilization is a limiting factor in CDA-mediated enzymatic chitin to chitosan bioconversion.


### 3.1.2. Enzymatic Method

Chitin deacetylases convert chitin to chitosan in a manner that is controlled and nondegradable and results in chitosan that is fresh and well defined [44]. Chitosan oligomers, in particular, are made using this method. Chitin deacetylase catalyzes the hydrolysis of N-acetamido links in chitin to produce chitosan. Glycoproteins called chitin deacetylases are released into the periplasmic or culture media. Furthermore, all enzymes have a strong selectivity for -(1,4)-linked N-acetyl-D-glucosamine polymers and are thermally stable at their optimal temperature (50°C). It is worth mentioning that acetate (a deacetylation product) has no effect on the chitin deacetylases produced by C. lindemuthianum and A. nidulans, making them suitable for biotechnological applications [45–47].The impact of chitin deacetylase, isolated from the fungusM. rouxii, on the chitosan synthesis was examined using chitin as a substrate (in both its crystalline and amorphous form) [48]. The enzyme is inefficient on insoluble chitins because the degree of deacetylation stays low. As a result, it appears that pretreatment of chitin substrates prior to enzyme addition is necessary to increase the accessibility of the acetyl groups to the enzyme and therefore the deacetylation yield. Experiments with partially deacetylated water-soluble chitosan’s and M. rouxii chitin deacetylase were carried out under controlled conditions to achieve this goal. The enzyme may deacetylate chitosan up to 97% under certain conditions (deacetylation from an initial chitosan with DA = 0.32 and a number-average degree of polymerization of 30) [49].


## 3.1.1. Chemical Deacetylation

In industrial applications, the chemical conversion of chitin to chitosan, which is the most difficult and costly step, has been done with 50 percent NaOH at high temperatures. Chitin deacetylase performs this step-in nature at lower temperatures and a neutral pH [41]. In N-acetyl-D-glucosamine residues of chitin, the enzyme hydrolyzes the linking between the acetyl group and the amine group. Chitin oligosaccharides are bioconverted into chitosan oligosaccharides and vice versa. DY-9 chitin deacetylase from Absidia corymbifera was found to be active against water-soluble chitin (WSCT-50), glycol chitin, chitosan (DD 71–88%), and chitin oligosaccharides [42, 43]. Chitin oligosaccharides were a suitable substrate because of their solubility, which improved the CDA’s availability and accessibility. Size affects the rate of deacetylation of (GlcNAc) 2–6, as longer lengths result in higher rate activity. These data indicate that chitin solubilization is a limiting factor in CDA-mediated enzymatic chitin to chitosan bioconversion.


## 3.1.2. Enzymatic Method

Chitin deacetylases convert chitin to chitosan in a manner that is controlled and nondegradable and results in chitosan that is fresh and well defined [44]. Chitosan oligomers, in particular, are made using this method. Chitin deacetylase catalyzes the hydrolysis of N-acetamido links in chitin to produce chitosan. Glycoproteins called chitin deacetylases are released into the periplasmic or culture media. Furthermore, all enzymes have a strong selectivity for -(1,4)-linked N-acetyl-D-glucosamine polymers and are thermally stable at their optimal temperature (50°C). It is worth mentioning that acetate (a deacetylation product) has no effect on the chitin deacetylases produced by C. lindemuthianum and A. nidulans, making them suitable for biotechnological applications [45–47].The impact of chitin deacetylase, isolated from the fungusM. rouxii, on the chitosan synthesis was examined using chitin as a substrate (in both its crystalline and amorphous form) [48]. The enzyme is inefficient on insoluble chitins because the degree of deacetylation stays low. As a result, it appears that pretreatment of chitin substrates prior to enzyme addition is necessary to increase the accessibility of the acetyl groups to the enzyme and therefore the deacetylation yield. Experiments with partially deacetylated water-soluble chitosan’s and M. rouxii chitin deacetylase were carried out under controlled conditions to achieve this goal. The enzyme may deacetylate chitosan up to 97% under certain conditions (deacetylation from an initial chitosan with DA = 0.32 and a number-average degree of polymerization of 30) [49].


## 3.2. Chitosan Nanoparticles

The addition of a polyanion, such as tripolyphosphate, to a chitosan solution under continuous stirring can produce nanoparticles. Ionotropic gelation, microemulsion, emulsification solvent diffusion, and emulsion-based solvent evaporation are the most common ways for creating chitosan-based nanoparticles. Drugs are trapped inside the polymeric matrix through electrostatic contact, hydrogen bonding, and hydrophobic interactions, among other mechanisms. With or without a stabilizing agent like poloxamer, a positively charged chitosan solution is dissolved in acetic acid or any other negatively charged polyanionic solution. Nanoparticles are easily formed as a result of the complexation of positive and negative charged species during mechanical stirring at room temperature, resulting in the separation of chitosan into spherical particles of varied sizes and surface charges [50].


## 4. Chitin Oligosaccharides

Chitooligosaccharides (COS), also called chitosan oligomers, contain oligomers ofβ-1, 4-linked 2-amino-2-deoxy-d-glucopyranose and 2-acetamido-2-deoxy-d-glucopyranose in Figure 1(c). It is produced by partial hydrolysis of chitosan and has a wide range of uses in the food and pharmaceutical industries due to its nontoxic nature. Anticoagulation, antioxidation, inflammatory resistance, immunological stimulation, antibacterial, antitumor, and other biological activities are among its numerous biological uses [51–53].


### 4.1. Extraction of Chitin Oligosaccharides

Chitin oligosaccharides can be made through enzyme hydrolysis, physical procedures, or chemical processes. Chitosanases and nonspecific enzymes such as cellulase, lysozyme, lipase, protease, amylase, and others were used for enzymatic hydrolysis [54]. Acid hydrolysis and hydrogen peroxide oxidation were straightforward chemical procedures to use, but their detrimental environmental impact limited their use. Physical techniques, such as ultrasonic treatment, ultraviolet radiation, and microwave therapy, have a lower cost and produce easier-to-purify goods than the other two methods. The transglycosylation process of glycolytic enzymes such as chitinase, chitosanase, and other glycosidases produced chitin oligosaccharides. Using a transglycosylation reaction, it was discovered that chitinase isolated from Trichoderma reesei KDR-11 transforms (GlcNAc) 4 to (GlcNAc) 2 (55.7%) and (GlcNAc) (39.6%) [55]. COS with DP 4–12 were successfully generated using a lysozyme-catalyzed transglycosylation reaction with N,N′,N″-tri (monochloro) acetylchitotriose and N,N′,N″-triacetylchitotriose as substrates, followed by a base-catalyzed removal technique to remove the N-monochloroacetyl groups [56, 57].


## 4.1. Extraction of Chitin Oligosaccharides

Chitin oligosaccharides can be made through enzyme hydrolysis, physical procedures, or chemical processes. Chitosanases and nonspecific enzymes such as cellulase, lysozyme, lipase, protease, amylase, and others were used for enzymatic hydrolysis [54]. Acid hydrolysis and hydrogen peroxide oxidation were straightforward chemical procedures to use, but their detrimental environmental impact limited their use. Physical techniques, such as ultrasonic treatment, ultraviolet radiation, and microwave therapy, have a lower cost and produce easier-to-purify goods than the other two methods. The transglycosylation process of glycolytic enzymes such as chitinase, chitosanase, and other glycosidases produced chitin oligosaccharides. Using a transglycosylation reaction, it was discovered that chitinase isolated from Trichoderma reesei KDR-11 transforms (GlcNAc) 4 to (GlcNAc) 2 (55.7%) and (GlcNAc) (39.6%) [55]. COS with DP 4–12 were successfully generated using a lysozyme-catalyzed transglycosylation reaction with N,N′,N″-tri (monochloro) acetylchitotriose and N,N′,N″-triacetylchitotriose as substrates, followed by a base-catalyzed removal technique to remove the N-monochloroacetyl groups [56, 57].


## 5. Biological or Biomedical Applications of Chitin and Its Derivatives

The chitin and chitosan or chitosan derivatives have recently been reported to several biological applications such as antimicrobial, anti-inflammatory activity, anticancer, wound-dressing and healing activity, drug delivery, vaccine adjuvant, tissue engineering, etc. Figure3 shows a review of the potential applications and side effects of chitin, chitosan, and COS.Figure 3
Biomedical applications of chitin, chitosan, and COS.


### 5.1. Antimicrobial Activity

Chitin has no antibacterial properties in general [58], despite the fact that chitosan and chitosan-derived compounds from various sources can potentially inhibit a wide range of pathogens. Furthermore, numerous studies have shown that chitosan oligosaccharide has been shown to have antibacterial action against Staphylococcus aureus [59], Cryptosporidium parvum [60], Candida auris [61], and Ceratocystis fimbriata in several investigations [62]. Similarly, chitosan oligosaccharide silver and gold nanoparticles exhibited potential antibacterial properties [63–65] (Table 1).Table 1
Antimicrobial activity of chitosan or chitosan-based commercial products.
ProductAntimicrobial activityMICReferencesPentafluoropropionyl-chitosanS. aureus, S. sanguinis, S. epidermidis, B. subtilis, E. coli, P. aeruginosa, and S. enterica256, 128, 128, 128, >512, >512 and >512μg/ml[66]2,3,4,5,6-Pentafluorobenzayl-chitosanS. aureus, S. sanguinis, S. epidermidis, B. subtilis, E. coli, P. aeruginosa, and S. enterica512, 128, 128, 128, >512, >512 and >512μg/ml[66]Pentadecafluorooctanoyl-chitosanS. aureus, S. sanguinis, S. epidermidis, B. subtilis, E. coli, P. aeruginosa, and S. enterica512, 256,256, 256, >512, >512 and >512μg/ml[66]Pentafluoropropionyl-HTCCS. aureus, B. subtilis, S. epidermidis, S. sanguinis, E. coli, P. aeruginosa, and S. enterica256, 128, 256, 128, 512, 128, 512[66]2,3,4,5,6-Pentafluorobenzyl-HTCCS. aureus, B. subtilis, S. epidermidis, S. sanguinis, E. coli, P. aeruginosa, and S. enterica128, 64, 512, 128, 512, 128 and 256μg/ml[66]Pentadecafluorooctanoyl-HTCCS. aureus, B. subtilis, S. epidermidis, S. sanguinis, E. coli, P. aeruginosa, and S. enterica256, 128, 512, 128, 512, 128 and 256μg/ml[66]Carboxymethyl chitosanE. coli and S. aureus0.05 and 0.1μg/ml[67]Quaternized chitosanE. coli and S. aureus0.0125 and 0.025μg/ml[67]Quaternized carboxymethyl chitosanE. coli and S. aureus0.00625 and 0.0125μg/ml[67]ChitosanE. coli, F. oxysporum, B. cinerea, and D. sorokiniana20, 100, 10, and 10 ppm[68]ChitosanX. campestris500 ppm[69]ChitosanS. enterica2000 ppm[70]ChitosanP. aeruginosa, A. hydrophila, S. dysenteriae, A. fumigates, and C. albicans>200, 1000, >200, >2000, and 500 ppm[71]ChitosanE. aerogenes250 ppm[72]ChitosanB. cereus and L. brevis1000 and 1000 ppm[73]ChitosanM. canis and T. mentagrophytes1100, 2200 ppm[74]Chitosan oligosaccharide (10 kDa)Propionibacterium acnes32–64μg/mL[75]


### 5.2. Anti-Inflammatory Activity

Inflammation is a critical human body defensive mechanism against infections, injuries, and poisons. The ability of chitosan, chitin, and chitosan oligosaccharide to inhibit and induce inflammation makes them useful anti-inflammatory agents. It has been shown that chitin, for example, promotes inflammation in obese people’s peripheral blood mononuclear cells [76]. In an experimental atopic dermatitis mouse model, chitin nanofibril was used to suppress NF-B to reduce skin inflammation [77]. In DSS-induced colitis, chitosan improved intestinal barrier function by boosting the expression of tight junction proteins such claudin-1, occludin, and ZO-1 [78]. Furthermore, chitosan oligosaccharide inhibited NF- B downstream targets like COX-2 and upstream targets like TLR-4 in lipopolysaccharide-induced inflammation in intestinal epithelial cells [79]. Furthermore, through downregulating IL-4, IL-5, and IL-13 and upregulating IFN, chitosan oligosaccharide protects sensitized mice from shrimp tropomyosin-induced food allergy [80]. Chitosan oligosaccharide also lowered liver IL-1 levels, reducing inflammatory reactions caused by heat stress [81].


### 5.3. Anticancer

Chitin, chitosan, and their derivatives have anticancer efficacy in a variety of cancer cells. They serve as a vehicle for the delivery of anticancer drugs as well. Previous studies stated that chitin suppressed cancer cells by inhibiting the mediators chitinase-3 like protein-1 (CHI3L1) and vascular endothelial growth factor C (VEGF-C) [82, 83]. Silver and poly-L-lactic acid encapsulated chitin nanoparticles also exhibited potential anticancer action in MCF7 breast cancer cell line [84]. Similarly, chitosan was also found to inhibit human melanoma cell lines such as SKMEL38, RPMI7951, and A375 significantly [85]. It also demonstrated anticancer potential in human cancers such as human ovarian cancer, breast cancer, and cervical carcinoma [86]. When chitosan was combined with additional compounds like carboxymethyl chitosan and chitosan selenate, it had a huge cytotoxic impact on cancer cell lines [87].


### 5.4. Wound-Dressing and Healing Activity

Chitin has been utilized as a tissue adhesive and as a key component of biomaterials for wound healing and tissue repair [88]. Chitin and chitin-based compounds, such as diacetyl chitin, chitin hydrogel, and chitin-amphiphilic ion, improved wound healing effectiveness [89–91]. Similarly, the wound healing ability of chitin nanoparticles/nanofibers was studied in diabetic induced rats [92, 93]. Furthermore, chitin nanofibrils are used as biomaterial for bleeding purposes which are significantly better than commercially available products [94]. Chitosan and chitosan oligosaccharide/chitooligosaccharide, on the other hand, are used for wound healing. Infectious animal models were treated with chitosan/chitosan oligosaccharide nanoparticles to see whether they could heal [95–97]. The wound healing ability of chitosan-based materials such as chitosan-polyurethane hydrogel, chitosan-curcumin complex, quaternary ammonium chitosan nanoparticles, and carboxymethyl chitosan with alginate was investigated using a diabetic rat model [98, 99].


### 5.5. Drug Delivery

In recent years, chitosan has shown to be an excellent carrier for variety of active substances [100]. Because of its biocompatibility, biodegradability, bioabsorption, and pH sensitivity, low-molecular-weight chitosan is utilized as a drug carrier for anticancer chemotherapy and other treatments [101]. Furthermore, the transport of proteins/peptides, growth hormones, antibiotics, anticancer, and anti-inflammatory medicines is considerably more effective using chitosan/chitosan-based materials or nanoparticles. Other research has been directed to treat diseases such as depression, schizophrenia, migraine, brain tumors, general anxiety disorder, epilepsy, pain, viral and bacterial infections, and so on [102].Doxorubicin, an anticancer medication, can be encapsulated with chitosan nanoparticles to decrease adverse effects and enhance absorption and survival time [103]. Chitosan nanoparticles, for example, are utilized as a drug carrier for anticancer medicines that inhibit the activity of human colorectal adenocarcinoma, lung cancer, and prostate-3 cancer cell lines. Antimicrobial and antioxidant activities of chitosan nanoparticles are encapsulated with the antibiotic gentamicin. With the release of chitosan nanoparticles against pneumonia, antibiotics are more effective while reducing their toxicity [104].


### 5.6. Tissue Engineering

The tissue engineering is developed as a key field of regenerative medicine, with the potential to overcome some of the drawbacks of traditional replacement treatments that rely on pure synthetic materials. The development of hybrid materials resulting from the integration of cells into 3D porous scaffolds or hydrogels is one of the most prevalent ideas connected with tissue engineering research. In three dimensions, the scaffold material plays an important role in cell anchoring, proliferation, and tissue development. The material should be able to construct porous scaffold structures, with porosity properties tailored to the application. A recent study highlighted chitosan and its derivatives as potential scaffold materials since they are predicted to dissolve when new tissue forms, reducing inflammation responses and hazardous breakdown products [105–107].A CMC/HA composite was used for bone regeneration since chitin and its derivatives have been demonstrated to speed up the release of cell growth factor during wound healing. The effects of a CMC/HA composite on bone healing in cell lines and animals were investigated [108]. The nanofibers made from a water-soluble carboxymethyl chitin CMC/PVA scaffold may promote cell adhesion/attachment and proliferation, implying that these scaffolds could be beneficial in tissue engineering [109]. Another form of nanofibers produced from N-CECS/PVA acted as a biocomposite nanofiber and showed substantial cellular adhesion and proliferation, making it useful as a wound dressing for skin regeneration [110].In another study, Liu et al. [111] employed chitosan hydrogel as a scaffold for chondrocytes to repair articular cartilage abnormalities. They created the hydrogels by combining chitosan with sodium glycerophosphate (GP) and hydroxyethyl cellulose (HEC) and discovered that both in vivo and in vitro methods produced good outcomes. They came to the conclusion that this hydrogel may promote matrix buildup and could be used to repair articular cartilage abnormalities in the future [111]. Zhang et al. [112] developed and studied a calcium phosphate chitosan composite as a scaffold for bone tissue engineering, and the mechanical property of the ceramic phase was shown to be improved by matrix reinforcement without altering the osteoblast phenotype.A 3D porous scaffold made of gelatin chitosan nanobioglass was created for bone tissue engineering. The scaffold produced in this example had around 80% porosity, greater compressive strength, proliferation, adhesion, and the capacity to transform into artificial bone grafts quickly [113, 114]. The nano-TiO2-chitosan artificial skin with collagen proved to be a promising artificial skin substitute, demonstrating nano-unique TiO2’s bactericidal action and chitosan’s immune-enhancing impact [115]. Chitosan derived from sponges and composed with sodium alginate has been shown to be effective in skin tissue engineering [116]. A lyophilized glutaraldehyde crosslinked chitosan extracted from a sponge used for blood hemostasis performed similarly. Chitosan served as a mechanical barrier to blood, forcing it to rapidly coagulate [117].


### 5.7. Gene Therapy

Gene therapy is a type of medication delivery in which the cellular machinery is manipulated to achieve a therapeutic effect [118]. Mao et al. [119] looked at the variables that influence siRNA and DNA delivery and transfection effectiveness. PEGylation has been found to improve biocompatibility and stability of siRNA loaded complexes, and chitosan derivatives are also utilized for therapeutic gene delivery to the brain [120].Despite the flexibility of chitosan, nanoparticles have become the preferred treatment for a range of neurological illnesses: glioblastoma, medulloblastoma, Parkinson’s disease, and Alzheimer’s disease [121]. This method of treatment has been utilized to treat viral infections like HIV-infected brain. The search for less invasive procedures has recently driven the development of new formulations for nose-to-brain gene transfer. The search for less invasive procedures has recently driven the development of new formulations for nose-to-brain gene transfer. Rassu et al. [122], for example, created chitosan-coated solid lipid nanoparticles containing BACE1 siRNA for intranasal use in the treatment of Alzheimer’s disease. This opens the door to further research into developing an effective combination of NAG-loaded CSNPs as a fish feed to boost growth and immune response in commercial aquaculture production [123].


## 5.1. Antimicrobial Activity

Chitin has no antibacterial properties in general [58], despite the fact that chitosan and chitosan-derived compounds from various sources can potentially inhibit a wide range of pathogens. Furthermore, numerous studies have shown that chitosan oligosaccharide has been shown to have antibacterial action against Staphylococcus aureus [59], Cryptosporidium parvum [60], Candida auris [61], and Ceratocystis fimbriata in several investigations [62]. Similarly, chitosan oligosaccharide silver and gold nanoparticles exhibited potential antibacterial properties [63–65] (Table 1).Table 1
Antimicrobial activity of chitosan or chitosan-based commercial products.
ProductAntimicrobial activityMICReferencesPentafluoropropionyl-chitosanS. aureus, S. sanguinis, S. epidermidis, B. subtilis, E. coli, P. aeruginosa, and S. enterica256, 128, 128, 128, >512, >512 and >512μg/ml[66]2,3,4,5,6-Pentafluorobenzayl-chitosanS. aureus, S. sanguinis, S. epidermidis, B. subtilis, E. coli, P. aeruginosa, and S. enterica512, 128, 128, 128, >512, >512 and >512μg/ml[66]Pentadecafluorooctanoyl-chitosanS. aureus, S. sanguinis, S. epidermidis, B. subtilis, E. coli, P. aeruginosa, and S. enterica512, 256,256, 256, >512, >512 and >512μg/ml[66]Pentafluoropropionyl-HTCCS. aureus, B. subtilis, S. epidermidis, S. sanguinis, E. coli, P. aeruginosa, and S. enterica256, 128, 256, 128, 512, 128, 512[66]2,3,4,5,6-Pentafluorobenzyl-HTCCS. aureus, B. subtilis, S. epidermidis, S. sanguinis, E. coli, P. aeruginosa, and S. enterica128, 64, 512, 128, 512, 128 and 256μg/ml[66]Pentadecafluorooctanoyl-HTCCS. aureus, B. subtilis, S. epidermidis, S. sanguinis, E. coli, P. aeruginosa, and S. enterica256, 128, 512, 128, 512, 128 and 256μg/ml[66]Carboxymethyl chitosanE. coli and S. aureus0.05 and 0.1μg/ml[67]Quaternized chitosanE. coli and S. aureus0.0125 and 0.025μg/ml[67]Quaternized carboxymethyl chitosanE. coli and S. aureus0.00625 and 0.0125μg/ml[67]ChitosanE. coli, F. oxysporum, B. cinerea, and D. sorokiniana20, 100, 10, and 10 ppm[68]ChitosanX. campestris500 ppm[69]ChitosanS. enterica2000 ppm[70]ChitosanP. aeruginosa, A. hydrophila, S. dysenteriae, A. fumigates, and C. albicans>200, 1000, >200, >2000, and 500 ppm[71]ChitosanE. aerogenes250 ppm[72]ChitosanB. cereus and L. brevis1000 and 1000 ppm[73]ChitosanM. canis and T. mentagrophytes1100, 2200 ppm[74]Chitosan oligosaccharide (10 kDa)Propionibacterium acnes32–64μg/mL[75]


## 5.2. Anti-Inflammatory Activity

Inflammation is a critical human body defensive mechanism against infections, injuries, and poisons. The ability of chitosan, chitin, and chitosan oligosaccharide to inhibit and induce inflammation makes them useful anti-inflammatory agents. It has been shown that chitin, for example, promotes inflammation in obese people’s peripheral blood mononuclear cells [76]. In an experimental atopic dermatitis mouse model, chitin nanofibril was used to suppress NF-B to reduce skin inflammation [77]. In DSS-induced colitis, chitosan improved intestinal barrier function by boosting the expression of tight junction proteins such claudin-1, occludin, and ZO-1 [78]. Furthermore, chitosan oligosaccharide inhibited NF- B downstream targets like COX-2 and upstream targets like TLR-4 in lipopolysaccharide-induced inflammation in intestinal epithelial cells [79]. Furthermore, through downregulating IL-4, IL-5, and IL-13 and upregulating IFN, chitosan oligosaccharide protects sensitized mice from shrimp tropomyosin-induced food allergy [80]. Chitosan oligosaccharide also lowered liver IL-1 levels, reducing inflammatory reactions caused by heat stress [81].


## 5.3. Anticancer

Chitin, chitosan, and their derivatives have anticancer efficacy in a variety of cancer cells. They serve as a vehicle for the delivery of anticancer drugs as well. Previous studies stated that chitin suppressed cancer cells by inhibiting the mediators chitinase-3 like protein-1 (CHI3L1) and vascular endothelial growth factor C (VEGF-C) [82, 83]. Silver and poly-L-lactic acid encapsulated chitin nanoparticles also exhibited potential anticancer action in MCF7 breast cancer cell line [84]. Similarly, chitosan was also found to inhibit human melanoma cell lines such as SKMEL38, RPMI7951, and A375 significantly [85]. It also demonstrated anticancer potential in human cancers such as human ovarian cancer, breast cancer, and cervical carcinoma [86]. When chitosan was combined with additional compounds like carboxymethyl chitosan and chitosan selenate, it had a huge cytotoxic impact on cancer cell lines [87].


## 5.4. Wound-Dressing and Healing Activity

Chitin has been utilized as a tissue adhesive and as a key component of biomaterials for wound healing and tissue repair [88]. Chitin and chitin-based compounds, such as diacetyl chitin, chitin hydrogel, and chitin-amphiphilic ion, improved wound healing effectiveness [89–91]. Similarly, the wound healing ability of chitin nanoparticles/nanofibers was studied in diabetic induced rats [92, 93]. Furthermore, chitin nanofibrils are used as biomaterial for bleeding purposes which are significantly better than commercially available products [94]. Chitosan and chitosan oligosaccharide/chitooligosaccharide, on the other hand, are used for wound healing. Infectious animal models were treated with chitosan/chitosan oligosaccharide nanoparticles to see whether they could heal [95–97]. The wound healing ability of chitosan-based materials such as chitosan-polyurethane hydrogel, chitosan-curcumin complex, quaternary ammonium chitosan nanoparticles, and carboxymethyl chitosan with alginate was investigated using a diabetic rat model [98, 99].


## 5.5. Drug Delivery

In recent years, chitosan has shown to be an excellent carrier for variety of active substances [100]. Because of its biocompatibility, biodegradability, bioabsorption, and pH sensitivity, low-molecular-weight chitosan is utilized as a drug carrier for anticancer chemotherapy and other treatments [101]. Furthermore, the transport of proteins/peptides, growth hormones, antibiotics, anticancer, and anti-inflammatory medicines is considerably more effective using chitosan/chitosan-based materials or nanoparticles. Other research has been directed to treat diseases such as depression, schizophrenia, migraine, brain tumors, general anxiety disorder, epilepsy, pain, viral and bacterial infections, and so on [102].Doxorubicin, an anticancer medication, can be encapsulated with chitosan nanoparticles to decrease adverse effects and enhance absorption and survival time [103]. Chitosan nanoparticles, for example, are utilized as a drug carrier for anticancer medicines that inhibit the activity of human colorectal adenocarcinoma, lung cancer, and prostate-3 cancer cell lines. Antimicrobial and antioxidant activities of chitosan nanoparticles are encapsulated with the antibiotic gentamicin. With the release of chitosan nanoparticles against pneumonia, antibiotics are more effective while reducing their toxicity [104].


## 5.6. Tissue Engineering

The tissue engineering is developed as a key field of regenerative medicine, with the potential to overcome some of the drawbacks of traditional replacement treatments that rely on pure synthetic materials. The development of hybrid materials resulting from the integration of cells into 3D porous scaffolds or hydrogels is one of the most prevalent ideas connected with tissue engineering research. In three dimensions, the scaffold material plays an important role in cell anchoring, proliferation, and tissue development. The material should be able to construct porous scaffold structures, with porosity properties tailored to the application. A recent study highlighted chitosan and its derivatives as potential scaffold materials since they are predicted to dissolve when new tissue forms, reducing inflammation responses and hazardous breakdown products [105–107].A CMC/HA composite was used for bone regeneration since chitin and its derivatives have been demonstrated to speed up the release of cell growth factor during wound healing. The effects of a CMC/HA composite on bone healing in cell lines and animals were investigated [108]. The nanofibers made from a water-soluble carboxymethyl chitin CMC/PVA scaffold may promote cell adhesion/attachment and proliferation, implying that these scaffolds could be beneficial in tissue engineering [109]. Another form of nanofibers produced from N-CECS/PVA acted as a biocomposite nanofiber and showed substantial cellular adhesion and proliferation, making it useful as a wound dressing for skin regeneration [110].In another study, Liu et al. [111] employed chitosan hydrogel as a scaffold for chondrocytes to repair articular cartilage abnormalities. They created the hydrogels by combining chitosan with sodium glycerophosphate (GP) and hydroxyethyl cellulose (HEC) and discovered that both in vivo and in vitro methods produced good outcomes. They came to the conclusion that this hydrogel may promote matrix buildup and could be used to repair articular cartilage abnormalities in the future [111]. Zhang et al. [112] developed and studied a calcium phosphate chitosan composite as a scaffold for bone tissue engineering, and the mechanical property of the ceramic phase was shown to be improved by matrix reinforcement without altering the osteoblast phenotype.A 3D porous scaffold made of gelatin chitosan nanobioglass was created for bone tissue engineering. The scaffold produced in this example had around 80% porosity, greater compressive strength, proliferation, adhesion, and the capacity to transform into artificial bone grafts quickly [113, 114]. The nano-TiO2-chitosan artificial skin with collagen proved to be a promising artificial skin substitute, demonstrating nano-unique TiO2’s bactericidal action and chitosan’s immune-enhancing impact [115]. Chitosan derived from sponges and composed with sodium alginate has been shown to be effective in skin tissue engineering [116]. A lyophilized glutaraldehyde crosslinked chitosan extracted from a sponge used for blood hemostasis performed similarly. Chitosan served as a mechanical barrier to blood, forcing it to rapidly coagulate [117].


## 5.7. Gene Therapy

Gene therapy is a type of medication delivery in which the cellular machinery is manipulated to achieve a therapeutic effect [118]. Mao et al. [119] looked at the variables that influence siRNA and DNA delivery and transfection effectiveness. PEGylation has been found to improve biocompatibility and stability of siRNA loaded complexes, and chitosan derivatives are also utilized for therapeutic gene delivery to the brain [120].Despite the flexibility of chitosan, nanoparticles have become the preferred treatment for a range of neurological illnesses: glioblastoma, medulloblastoma, Parkinson’s disease, and Alzheimer’s disease [121]. This method of treatment has been utilized to treat viral infections like HIV-infected brain. The search for less invasive procedures has recently driven the development of new formulations for nose-to-brain gene transfer. The search for less invasive procedures has recently driven the development of new formulations for nose-to-brain gene transfer. Rassu et al. [122], for example, created chitosan-coated solid lipid nanoparticles containing BACE1 siRNA for intranasal use in the treatment of Alzheimer’s disease. This opens the door to further research into developing an effective combination of NAG-loaded CSNPs as a fish feed to boost growth and immune response in commercial aquaculture production [123].


## 6. Conclusion and Future Perspectives

Chitin, chitosan, and COS may be utilized as nanodelivery system carrier materials and have a wide range of biomedical applications, including drug delivery, antimicrobial agents, and wound healing. Researchers should also investigate novel applications for chitin, chitosan, and COS as well as animal trials to learn more about their human-related consequences. However, more measures must be taken to increase the safety and effectiveness of chitin in biological applications. These materials, which are made from aquaculture waste, are gaining popularity and have a wide range of applications. However, while developing chitin/chitosan-based goods for applications in many sectors for human benefit, we must consider environmental protection and green manufacturing.COS has been used to treat a variety of human illnesses and pathological situations, as opposed to chitin or chitosan. Low-molecular-weight chitosan oligosaccharide with >90% deacetylation suppresses inflammatory responses by boosting anti-inflammatory pathways. Chitosan oligosaccharide also possesses antioxidative and anticancer properties. Interestingly, chitosan oligosaccharide, as a prebiotic for gut microbiota, modifies gut microbiota, reducing systemic illnesses, including atherosclerosis. The degree of deacetylation and polymerization determines the biological effects of these compounds, which remains a problem in the development of chitosan oligosaccharide as a useful dietary supplement. More research is needed to uncover the exact molecular/cellular processes of these polymers’ biological impacts, particularly the function of their prebiotic effect and direct influence on disease-specific cells or therapeutic targets.



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*Source: 2897179-2022-04-22.xml*