**6. Chemical methods to prepare chitin**

Several procedures have been developed through the years to prepare chitin; they are at the basis of the chemical processes for industrial production of chitin and chitosan. Various methods are reported in Muzzarelli's book such as: Method of Rigby (1936 and 1937); Hackman (1954); Foster and Hackman (1957); Horowitz, Roseman and Blumenthal (1957); Whistler and Be Miller (1962); Takeda and Abe (1962); Takeda and Katsuura (1964); Broussignac (1968); Lovell, Lafleur and Hoskins (1968); Madhavan and Ramachandran (1974) [6]. There is also a review that summarizes methods of preparation of various chitin and its conversion to chitosan [17].

### **7. Enzymatic methods to prepare chitin**

A new process for deproteinization of chitin from shrimp head was studied [18]. Recovery of the protein fraction of the shrimp waste has been widely studied by enzymatic hydrolysis method [19,20].The enzymatic deproteinization process has limited value due to residual small peptides directly attached to chitin molecules ranging from 4.4% to 7.9% of total weight [21]. As these processes are costly because of the use of commercial enzymes, there is now a need to develop an efficient and economical method for extracting proteins from shellfish waste. One interesting new technology for extraction of chitin that offers an alternative to the more harsh chemical methods is fermentation by using microorganisms. Fermentation has been envisaged as one of the most ecofriendly, safe, technologically flexible, and economically viable alternative methods [22-28]. Fermentation of shrimp waste with lactic acid bacteria results in production of a solid portion of chitin and a liquor containing shrimp proteins, minerals, pigments, and nutrients [26,29]. Deproteinization of the biowaste occurs mainly by proteolytic enzyme produced by Lactobacillus [30]. Lactic acid produced by the process of breakdown of glucose, creating the low pH condition of ensilation; suppress the growth of microorganisms involved in spoilage of shrimp waste [31]. The lactic acid reacts with calcium carbonate component in the chitin fraction leading to the fermentation of calcium lactate, which gets precipitated and can be removed by washing. There is now a need to develop an efficient, simpler, eco- friendly, economical, and commercially viable method.

#### **8. Chitosan**

6 The Complex World of Polysaccharides

**5. Chitin from fungi** 

processing or in cultures of fungi [4].

corresponds to 24,000 ± 34,500 Daltons [4].

**6. Chemical methods to prepare chitin** 

has become commercially available [3].

**Figure 2.** Exosqueleton of crustacea, this is the source of commercial chitin

Another source of chitin that is more environmentally friendly, although much more limited in volume, is squid pen. This waste contains very little in the way of inorganic material and very little, if any CO2 would be released in the extraction and purification process. Another and perhaps more sustainable source in the long run is vegetable chitin from fungal sources such as waste mycelia. There is extensive literature on the topic, but it is only recently that it

Chitin is widely distributed in fungi, occurring in *Basidiomycetes, Ascomycetes,* and *Phycomycetes*, where it is a component of the cell walls and structural membranes of mycelia, stalks, and spores. The amounts vary between traces and up to 45% of the organic fraction, the rest being mostly proteins, glucans and mannans. However, not all fungi contain chitin, and the polymer may be absent in one species that is closely related to another. Variations in the amounts of chitin may depend on physiological parameters in natural environments as well as on the fermentation conditions in biotechnological

The chitin in fungi possesses principally the same structure as the chitin occurring in other organisms. However, a major difference results from the fact that fungal chitin is associated with other polysaccharides which do not occur in the exoskeleton of arthropods. The molecular mass of chitin in fungi is not known. However, it was estimated that bakers' yeast synthesizes rather uniform chains containing 120 ± 170 GlcNAc monomer units which

Several procedures have been developed through the years to prepare chitin; they are at the basis of the chemical processes for industrial production of chitin and chitosan. Various methods are reported in Muzzarelli's book such as: Method of Rigby (1936 and 1937); Hackman (1954); Foster and Hackman (1957); Horowitz, Roseman and Blumenthal (1957); Despite the wide spread occurrence of chitin, up to now the main commercial sources of chitin have been crab and shrimp shells. In industrial processing, chitin is extracted from crustaceans by acid treatment to dissolve calcium carbonate followed by alkaline extraction to solubilized proteins. In addition a decolorization step is often added to remove leftover pigments and obtain a colorless product. These treatments must be adapted to each chitin source but by partial deacetylation under alkaline conditions, one obtains "chitosan" [16]. Chitosan is the most important derivate of this naturally occurring polymer being one of the most abundant polysaccharides after cellulose. Chitosan is a copolymer composed of Nacetyl-D-glucosamine and D-glucosamine units. It is obtained in three different ways, thermochemical deacetylation of chitin in the presence of alkali, by enzymatic hydrolysis in the presence of a chitin deacetylase, or naturally found in certain fungi as part of their structure. In chitosan part of the amino groups remain acetylated. It is generally accepted that N-acetylglucosamine residues are randomly distributed along the whole polymer chain. In an acid medium, amino groups are protonated and thus determine the positive charge of

the chitosan molecule. Thus, chitosan behaves like a polycation in solution [32]. Properties of chitosan, such as the mean polymerization degree, the degree of N-deacetylation, the positive charge, and the nature of chemical modifications of its molecule, strongly influence its biological activity.

Chitin contains 6–7% nitrogen and in its deacetylated form, chitosan contains 7–9.5% nitrogen. In chitosan, between 60 to 80% of the acetyl groups available in chitin are removed [33]. The chain distribution is dependant on the processing method used to obtain biopolymer [34-36]. It is the N-deacetylated derivative of chitin, but the N-deacetylation is almost never complete [35]. Chitin and chitosan are names that do not strictly refer to a fixed stoichiometry. Chemically, chitin is known as poly-N-acetylglucosamine, and in accordance to this proposed name, the difference between chitin and chitosan is that the degree of deacetylation in chitin is very little, while deacetylation in chitosan occurs to an extent but still not enough to be called polyglucosamine [37].

**Figure 3.** Chitin and chitosan chemical structure

#### **9. Sources of chitosan**

Chitosan is commercially produced from deacetylated chitin found in shrimp and crab shell. However, supplies of raw materials are variable and seasonal and the process is laborious and costly [38]. Furthermore, the chitosan derived from such process is heterogeneous with respect to its physiochemical properties [38]. Recent advances in fermentation technology provide an alternative source of chitosan. Fungal cell walls and septa of *Ascomycetes, Zygomycetes, Basidiomycetes* and *Deuteromycetes* contain mainly chitin, which is responsible for maintaining their shape, strength and integrity of cell structure [38]. The production of chitosan from fungal mycelia has a lot of advantages over crustacean chitosans such as the degree of acetylation, molecular weight, viscosity and charge distribution of the fungal chitosan. They are more stable than crustacean chitosans. The production of chitosan by fungus in a bioreactor at a technical scale offers also additional opportunities to obtain identical material throughout the year. The fungal chitosan is free of heavy metal contents such as nickel, copper [39-41]. Moreover the production of chitosan from fungal mycelia gives medium-low molecular weight chitosans (1–12 × 104 Da), whereas the molecular weight of chitosans obtained from crustacean sources is high (about 1.5 ×106 Da) [41]. Chitosan with a medium-low molecular weight has been used as a powder in cholesterol absorption [42] and as thread or membrane in many medical-technical applications. For these reasons, there is an increasing interest in the production of fungal chitosan.

There are some examples of chitosan extracted from fungi. Chitosans isolated from *Mucorales* typically show Mw in the range 4 x 105 to 1.2 x 106 Daltons and FA values between 0.2 and 0.09. Amino acid analysis of chitosan prepared from *Aspergillus niger* reveals covalently bound arginine, serine, and proline. Nadarajah et. al., 2001, studied chitosan production from mycelia of *Rhizopus* sp KNO1 and KNO2, *Mucor sp* KNO3 and *Asperigullus niger* with the highest amount of extractable chitosan obtained at the late exponential phase. *Mucor* sp KNO3, produced the highest amount of 557mg per 2.26 g of dry cell weight /250 ml of culture. Kishore et. al.(1993), examined the production of chitosan from mycelia of *Absidia coerulea*, *Mucor rouxii*, *Gongronella butieri, Phvcomyces blakesleeanus* and *Absidia blakesleeana*. Chitosan yields of *A. coerulea*, *M. rouxii*, *G. butieri, P. blakesleeanus* and *A. blakesleeana* were 47–50, 29–32, 21–25, 6 and 7 mg/100 mL of medium, respectively. The degree of acetylation of chitosan ranged from 6 to 15%; the lowest was from strains of *A. coerulea*. Viscosity average molecular weights of fungal chitosans were equivalent, approximately 4.5 x 105 Daltons. Wei-Ping Wang et.al., (2008) evaluated the physical properties of fungal chitosan from *Absidia coerulea* (AF 93105*), Mucor rouxii* (Ag 92033), and *Rhizopus oryzae (*Ag 92033). Their glucosamine contents and degrees of deacetylation (DD) were over 80%, differences had been observed in their molecular weight (Mw), ranging from 6.6 to 560 kDa. Chitosan was isolated and purified from the mycelia of *Rhizomucor miehei* and *Mucor racemosus* with a degree of deacetylation of 97 y 98 respectively [43-45].

8 The Complex World of Polysaccharides

still not enough to be called polyglucosamine [37].

**Figure 3.** Chitin and chitosan chemical structure

**9. Sources of chitosan** 

its biological activity.

the chitosan molecule. Thus, chitosan behaves like a polycation in solution [32]. Properties of chitosan, such as the mean polymerization degree, the degree of N-deacetylation, the positive charge, and the nature of chemical modifications of its molecule, strongly influence

Chitin contains 6–7% nitrogen and in its deacetylated form, chitosan contains 7–9.5% nitrogen. In chitosan, between 60 to 80% of the acetyl groups available in chitin are removed [33]. The chain distribution is dependant on the processing method used to obtain biopolymer [34-36]. It is the N-deacetylated derivative of chitin, but the N-deacetylation is almost never complete [35]. Chitin and chitosan are names that do not strictly refer to a fixed stoichiometry. Chemically, chitin is known as poly-N-acetylglucosamine, and in accordance to this proposed name, the difference between chitin and chitosan is that the degree of deacetylation in chitin is very little, while deacetylation in chitosan occurs to an extent but

Chitosan is commercially produced from deacetylated chitin found in shrimp and crab shell. However, supplies of raw materials are variable and seasonal and the process is laborious and costly [38]. Furthermore, the chitosan derived from such process is heterogeneous with respect to its physiochemical properties [38]. Recent advances in fermentation technology provide an alternative source of chitosan. Fungal cell walls and septa of *Ascomycetes, Zygomycetes, Basidiomycetes* and *Deuteromycetes* contain mainly chitin, which is responsible for maintaining their shape, strength and integrity of cell structure [38]. The production of chitosan from fungal mycelia has a lot of advantages over crustacean chitosans such as the degree of acetylation, molecular weight, viscosity and charge distribution of the fungal chitosan. They are more stable than crustacean chitosans. The production of chitosan by fungus in a bioreactor at a technical scale offers also additional opportunities to obtain identical material throughout the year. The fungal chitosan is free of heavy metal contents such as nickel, copper [39-41]. Moreover the production of chitosan from fungal mycelia gives medium-low molecular weight chitosans (1–12 × 104 Da), whereas the molecular weight of chitosans obtained from crustacean sources is high (about 1.5 ×106 Da) [41]. Chitosan with a medium-low molecular weight has been used as a powder in cholesterol absorption [42] and as thread or membrane in many medical-technical applications. For

these reasons, there is an increasing interest in the production of fungal chitosan.

Considerable research has been carried out on using mycelium waste from fermentation processes as a source of fungal chitin and chitosan. It is argued that this would offer a stable non-seasonal source of raw material that would be more consistent in character than shellfish waste, but so far this route does not appear to have been taken up by chitosan producing companies. Currently there is only one commercial source of fungal chitosan and is produced by the company Kitozyme. However their raw material is not mycelium waste from a fermentation process, which is what is normally envisaged when fungal chitosan is referred to, but actually conventional edible mushrooms grown under contract in France and shipped to Belgium for processing. So mycelium waste still remains a vast and as yet untapped potential source of chitosan.

## **10. Genetic engineering approach to produce chitin**

It is difficult to obtain pure carbohydrates, especially chitin, through conventional techniques. Bacterial cells have been engineered in an effort to overcome this problem [46]. *E. coli* has been engineered to produce chitobiose. This method took advantage of NodC, which is a chito-oligosaccharide synthase, and genetically engineered chitinase to make a cell factory with the ability to produce chito-oligosaccharides [47]. Recombinant chitooligosaccharides have also been obtained using *E coli* cells which expressed nodC or nodBC genes [48]. By expressing different combinations of nod genes in *E. coli*, O-acetylated and sulfated chito-oligosaccharide have been produced [49].
