**2. Chitosan: General considerations**

Chitosan is a natural co-polymers of chitin, composed by units of 2-amino-2-desoxi-Dglycopyranose and of 2-acetamide-2-desoxi-D-glycopyranose interconnected by glycosidic bonds β-1,4 in variable proportions. The first type of units is frequently present in chitosan. This polymer is naturally found in the cell wall of fungi, mainly in the Mucorales order [5, 13, 14]. Chitosan is formed by the chitin deacetylation, and the group N-acetyl can be suffer several degrees of deacetylation (Figure 1). Chitosan is characterized according to its deacetylation level and molar mass, once such features may influence the degradability and in the polysaccharide hydrolysis [15, 16]. According to the medium acetylation level (AL), chitosan may be obtained with physical-chemical properties differentiated regarding the solubility parameters, pKa and viscosity [17, 19]. It is difficult to obtain chitosan with high deacetylation level as due long process of isolation, and the degradation of the polymer also increases [5, 15].

**Figure 1.** Chemical structure of Chitin and Chitosan (Source: authors)

The synthesis of chitin from fungi and crustaceans differs significantly; however, steps of enzymatic machinery for biosynthesis and catalytic regulation are similar. In crustaceans is initiated by conversion of glucose 6-phosphate N-acetylglucosamine-1-phosphate, involving a series of stages that require several enzymatic reactions. N-acetylglucosamine-1-phosphate reacts with UTP forming the UDP N-acetylglucosamine, which finally transfers Nacetylglucosamine for the polymerization of chitin chains in a process mediated by the enzyme chitin synthase and Mg +2 ion as catalyst, resulting in the formation of a long chain of subunits monosaccharide's linked by β-1 → 4. Chitosan is thus obtained by deacetylation of chitin by chemical treatment with NaOH at high temperature [20].

230 Practical Applications in Biomedical Engineering

biological applications, mainly as antimicrobial agent [12].

**Figure 1.** Chemical structure of Chitin and Chitosan (Source: authors)

The synthesis of chitin from fungi and crustaceans differs significantly; however, steps of enzymatic machinery for biosynthesis and catalytic regulation are similar. In crustaceans is initiated by conversion of glucose 6-phosphate N-acetylglucosamine-1-phosphate, involving a series of stages that require several enzymatic reactions. N-acetylglucosamine-1-phosphate reacts with UTP forming the UDP N-acetylglucosamine, which finally transfers Nacetylglucosamine for the polymerization of chitin chains in a process mediated by the

Chitin Chitosan

**2. Chitosan: General considerations** 

increases [5, 15].

Recent developments in the areas of biomaterials devices have resulted in a number of advances in the searches of natural substances which may inhibit the dental plaque formation and/or the hydroxyapatite demineralization [11, 12]. In particular, this research is focused on novel macromolecules and biocompatible materials for use in clinical applications. Chitosan is a non-toxic and natural polysaccharide, which have many

Chitosan is a natural co-polymers of chitin, composed by units of 2-amino-2-desoxi-Dglycopyranose and of 2-acetamide-2-desoxi-D-glycopyranose interconnected by glycosidic bonds β-1,4 in variable proportions. The first type of units is frequently present in chitosan. This polymer is naturally found in the cell wall of fungi, mainly in the Mucorales order [5, 13, 14]. Chitosan is formed by the chitin deacetylation, and the group N-acetyl can be suffer several degrees of deacetylation (Figure 1). Chitosan is characterized according to its deacetylation level and molar mass, once such features may influence the degradability and in the polysaccharide hydrolysis [15, 16]. According to the medium acetylation level (AL), chitosan may be obtained with physical-chemical properties differentiated regarding the solubility parameters, pKa and viscosity [17, 19]. It is difficult to obtain chitosan with high deacetylation level as due long process of isolation, and the degradation of the polymer also Fungi the chitin and chitosan synthesis simultaneously were occurred. The synthesis of chitin is highly compartmentalized. The enzyme chitin synthase is the zymogen form and distributed in specific regions of the cell surface, vesicles in specialized, chitosome. The macromolecular assembly starts out of the cytoplasm, where the protease enzyme acts on the cell surface activating the zymogenes. In this way the UDP N-acetylglucosamine is produced from glucose, and chitin synthase catalyzes the transfer of N-acetylglucosamine for polymerization chain forming chitin. The chitosan present in cell walls of certain fungi (Mucorales) is formed from the deacetylation (chitin deacetylase) chain chitin source for the biosynthesis of chitin. The regulations of the synthesis of chitin and chitosan are determined by the spatial organization of the synthesis of chitin in the cell surface [20, 21].

Crustacean chitosan is inconsistent in its physical–-chemical properties due to the variability in raw materials, the harshness of the isolation and conversion processes, the caustic effects of the chemicals used in the isolation process, and variability in the levels of deacetylation and protein contamination [22-24].

The use of biomass from fungi have demonstrated a great advantages, such as: independence of seasonal factor, wide scale production, simultaneous extraction of chitin and chitosan, extraction process is simple and cheap resulting in reduction of the time and cost required for production, and also absence of proteins contamination, mainly the proteins that could cause allergy reactions in individuals with shellfish allergies. However, to optimize the production of chitin and chitosan from fungi, it's usually used complex or synthetics cultures media, which are expensive. It's becomes necessary to obtain economic culture media that promote the growth of fungi and stimulate the production of the polymers [25-32].

In order to obtain alternative sources of nutrients and low cost several research projects are being conducted. Table 1 shows the use of various synthetic media and low cost alternative media used for growth of fungi of the order Mucorales, and production of chitin and chitosan. The content of chitin and chitosan from fungal cell wall varies among different species and growth conditions. Many studies have been performed to verify the possibility of using the biomass of fungi, especially Mucorales, class Zygomycetes, as an alternative source of chitin and chitosan. Many of these studies test simple models to verify the production of chitin and chitosan, ie, the approach adopted experiments using only one variable at a time during the fermentation, eg after cultivation, agitation, pH, temperature and concentration of nutrients. However, the literature report in recent years the scientific interest to reduce the numbers of tests and increase the accuracy of the results has been increased. Therefore, multivariate approach, using factorial design, allows the observation of the synergistic effect between the independent variables, since all variables are considered simultaneously, resulting in the final optimize conditions [30-34].


Microbiological Chitosan: Potential Application as Anticariogenic Agent 233

Chitosan is a weak base insoluble in water but soluble in dilute aqueous solutions of various acids, the most widely used is acetic acid [43]. The acid solubility is explained by the protonation of the free amino group, characteristic in the chitosan *in natura*, which change to NH2 to NH3+, whereas in alkaline condition, the hydro solubility is due to the formation of carboxylate, from the introduced carboxylic group [19, 44]. The possibility to obtain a variety of polymer derivatives with differences solubility, thermal stability, reactivity with other substances and specificity regarding the binding site, providing several biological applications of the chitosan [15]. Some applications of the chitosan, it is highligh it's the use

Chitosan has a recognized antimicrobial activity, being this, one of the main properties of the polysaccharide. Several researchers demonstrated that this polysaccharide has antimicrobial action in a great variety of microorganisms, included gram-positive bacteria and various species of yeast [21, 45]. In the literature is described that chitosan acts in the cellular wall of the microorganism modifying the electric potential of the cellular membrane [46]. This polysaccharide also acts potentiating other inhibition drugs, as the chlorhexidine

In reference [39] the authors report that chitosan has demonstrated low toxicity and the resistance development have not occurred. The antimicrobial action of the chitosan and its derivatives suffers influence from factors, which depending on the performed role may be classified in four main categories: 1. Microbial factors as species, age of the cell); 2. Intrinsic factors of the chitosan as: positive charge density, molecular weight, hydrophobic and hydrophilic characteristics, chelation capacity; 3. Physical state factors (soluble and solid

The antimicrobial action mechanism of the chitosan is not yet fully elucidated, being several mechanisms are suggested by the literature. Some authors suggested the amino groups of the chitosan when in contact with physiological fluids are protonated and bind to anionic groups of the microorganisms, resulting in the agglutination of the microbial cells, and growth inhibition [49, 50]. On the other hand, reference [49] report that when interacting with the bacterial cell, the chitosan, promotes displacement of Ca++ of the anionic sites of the membrane, damaging them. Another postulate is the interaction between the positive load of the chitosan and the negative load of the microbial cell wall, because it causes the rupture and loss of important intracellular constituent of the microorganism life. Chitosan with low molecular weight penetrates in the cell and is linked to the microorganism DNA inhibiting the transcription and consequently the translation, whereas the chitosan of high molecular

Authors in reference [47] investigated the relation between antimicrobial activity of the chitosan and the characteristics of the cellular wall of bacteria. They verified that the chitosan is antibacterial agent more efficient to Gram-negative bacteria due the composition of phospholipids and carboxylic acids of the bacterial cellular wall. These results suggest that the effects of the chitosan are distinct in the two types of bacteria: in the case of the gram-positive, the hypothesis is that chitosan of high molecular mass may form films

in the pharmaceutical industry, more specifically related to dental clinic [43].

gel, once it increases the drug permanence time action place [47, 48].

state), and 4. Environmental factors (pH, ionic forces, temperature, and time).

weight acts as a chelate agent, binding to the cell membrane [16].


**Table 1.** Chitin and chitosan production by Mucorales strains compared with *Cunninghamella cirnelloides* using yam bean as substrate.
