Firstly, Thanks to Allah S. W. T Because Giving Me Success for My Final Year Project

Acknowledgement

Firstly, thanks to Allah s. w. t because giving me success for my final year project.

I wish to express my gratitude to individuals who have helped me with creating this. This would have never come to light without their massive efforts and help.

I'm deeply grateful to my supervisor,"Dr- Ayman Salah El-deen Al-Husini "who has advice me and always helping to complete of my final year project .I consider myself very fortunate for being able to work with a very considerate and encouraging lecture like him .

Also I'd like to thank"Dr-Ibrahim Mohey"head chemistry department Portsaid university for his contributions.I would like to express my deepest thanks to all doctors and demonstrators who taught me during the four years. I would have never forget the contributions,encouraging and supporting of family , fiancé and friends.Their contributions were very essential to me ….

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List of figures ………………………………………………………. 5
Abbreviations …………………………………………………….... 6
Introduction ………………………………………………………… 7:8
chemistry of chitosan …………………………………………. 9:11
Production of chitosan ……………………………………….... 11:22
History of chitosan ……………………………………………… 22:23
Properties of Chitosan ………………………………………… 23:24
Degradation ……………………………………………………….. 24:25
Molecular Weight……………………………………………….. 25:26
Solvent Properties……………………………………………… 26:27
Degree of Deacetylation ……………………………………… 27:29
Solubility of chitosan …………………………………………. 29:33
It’s application …………………………………………………. 33:34
Water Treatment Applications ……………………………. 34:36
Medical & pharmacutical Applications ………………… 36:38
Orthopedics ………………………………………………………… 38
Tissue Engineering ……………………………………………… 38:40
Wound Healing …………………………………………………… 40
Drug Delivery……………………………………………………… 40:41
Surgical Adhesion………………………………………………. 41:42
Hemostatic Agent ……………………………………………….. 42:43
other bio-medical applications …………………………….. 43
Biotechnological Applications ……………………………. 44
Cell-Stimulater …………………………………………………… 44
Fat-Net………………………………………………………………. 44:45
several potential clinical applications …………………. 46:47
Administering Chitosan ………...…………………………… 47
Potential industrial use ………………………………………. 48
Agricultural & Horticultural use ……………………….. 48:49
Applied as seed coating agents ……………………………. 49:50
Applied as foliar treatment agents ……………………… 50:52
Applied as soil amendment …………………………………. 52
Applications of chitosan derivatives ……………………. 52
Application of glycol chitosan for entrapment of protein molecules……………………………………………………………. 52:53
Amphiphilic derivatives of glycol chitosan ………….. 53
Glycol chitosan-coated MRI(Magnetic-Resonance-Imaging) agent safer effective in detecting breast cancer….….. 53-54
Cosmetics ………………………………………………………….... 54:57
References………………………………………………………..... 58:64

** List of figures** : *Page.No*

Fig (1) : chemical structure of chitosan………………………. 9

Fig (2): Deactylation of chitin to chitosan …………………….. 9

Fig(3):chitin & chitosan manufacturing process ………….. 11

Fig(4) : preparation of chitin & chitosan ………………………. 15

Fig(5) : Fungus Gongronella butleri USDB 0201 was grown on sweet potato pieces in a tray-type solid substrate fermentor…………………………………………………………………. 17

Fig(6) : Extraction of chitosan from mycelia of fungus G.butleri grown in solid substrate ………………………………………………………………………………….. 18

Fig(7) : Extraction of chitosan and glucan from the AIM suspended in 0.35M acetic acid by treatment with Termamyl Type LS (Nwe & Stevens, 2002 and Nweetal.,2008) ……………………………….. 21

Fig (8): types of methods of extraction of chitosan

from fungi …………………………………………………………………… 22

Fig (9) :Chemical structure of CS-g-CMC biomaterials ……. 36

Fig(10) : cell scaffold interaction …………………………………… 39

Abbreviations :-

(SSF) : Solid Substrate Fermentation.
(SMF) : Submerged Fermentation .
(M.W) : Molecular Weight.
(HPLC): High Performance Liqud Chromatography.
(D.D) : Degree of Deacetylation.
(CS-g-CMC):Chitosan-g Carboxy Methyl Cellulose .
(CD) : Cyclodextrin .
(HDL) : High-Density Lipoprotein.
(LDL) : Low Density Lipoprotein.
(ABA) : Abscisic Acid .
(GC) : Glycol chitosan .
(BSA) : Bovine Serum Albumin.

1-Introduction :-

A polymer is a chemical compoundor mixture of compounds consisting of repeating structural units created through a process of polymerization.([1])The term derives from the ancient Greek word πολύς (polus, meaning "many, much") and μέρος (meros, meaning "parts"), and refers to a molecule whose structure is composed of multiple repeating units, from which originates a characteristic of high relative molecular massand attendant properties.([2])

Hence, the terms polymer and polymeric material encompass very large, broad classes of compounds, both natural and synthetic, with a wide variety of properties. Because of the extraordinary range of properties of polymeric materials([3]).they play an essential and ubiquitous roles in everyday life. ([4])

Over the last years, many attempts have been made toreplace petrochemical products by renewable, biosourcedcomponents. Abundant naturally occurring polymers – asstarch, collagen, gelatin, alginate, cellulose and chitin– represent attractive candidates as they could reducethe actual dependence on fossil fuels, and consequentlyhave a positive environmental impact.

In this respect, chitosanis a quite unique bio-based polymer: its intrinsicproperties are so singular and valuable that chitosan possessesnoactualpetrochemical equivalent.Consequently,the inherent characteristics of chitosan make it exploitabledirectly for itself.Chitosan, a natural and linear biopolyaminosaccharide, has receivedmuch attention as a functional biopolymer with applications inpharmaceuticals, food, cosmetics and medicines.([5])

Chitosan is soluble in aqueous acids because of the protonation of amino groups, but it is insoluble in water and most of organic solvent so as to restrict the applications.

Nevertheless, chitosanapplication is limited by its solubility in aqueous solution An elegant way to improve or toimpart new properties to chitosan is the chemical modification of the chain,generally by grafting of functional groups, without modification of the initialskeleton in order to conserve the original properties. The functionalization iscarried out on the primary amine group, generally by quaternization, or on thehydroxyl group.

Chitosan is a safe and friendly substance for the human organism; therefore, it has become of great interest not only as an underutilized resource, but also as a new functional material of high potential in various fields.

Some unique properties make chitosan an excellent material for the development of new industrial applications and recent progress in chitosan material is quite noteworthy. In this review, we mainly take a closer look at various chitosanApplications.

2-Chemistry of Chitosan:-

Fig (1) : chemical structure of chitosan

Itis a heteropolymer consists of β(1-4) 2-acetamido-2-deoxy-β-Dglucopyranose(N-acetylglucosamine) and 2-amino-2-deoxy-β-D-glucopyranose(D-glucosamine) units, randomly or block distributed throughout the biopolymer.The chain distribution is dependent on the processing method used to derivethe biopolymer (Dodane and Vilivalam, 1998; Kumar, 2000; Khor and Lim,2003). It is the N-deacetylated derivative of chitin, but the N-deacetylation isalmost never complete (Kumar, 2000; Santos et al., 2005).

Fig (2): Deactylation of chitin to chitosan

Chitin and chitosanare names that do not strictly refer to a fixed stiochiometry. Chemically, chitin isknown as poly-N-acetylglucosamine,homopolymer of b-(1fi4)-linked-acetyl-D-glucosamine (Muzzarelli, 1997).([6]) and in accordance to this proposed name,the difference between chitin and chitosan is that the degree of deacetylation inchitin is very little, while deacetylation in chitosan occurred to an extent but stillnot enough to be called polyglucosamine (Muzzarelli, 1973).Chitosan has one primary amine and two free hydroxyl groups foreach monomer with a unit formula of C6H11O4N.This natural biopolymer is aglucosaminoglycan and is composed of two common sugars, glucosamine andN-acetylglucosamine, both of which are constituents of mammalian tissues(Khan, 2001; Snyman et al., 2002).Chitosan is the second abundant polysaccharide next to cellulose (Duarte et al.,2002; Sinha et al., 2004), but it is the most abundant natural aminopolysaccharide and is estimated to be produced annually almost as much ascellulose (Kumar, 2000).

Chitosan can be chemically considered as analoguesof cellulose, in which the hydroxyl at carbon-2 has been replaced by acetamidoor amino groups (Krajewska, 2004). As a point of difference from otherabundant polysaccharides, chitin and chitosan contain nitrogen in addition tocarbon, hydrogen and oxygen.Chitin and chitosan are of commercial interestdue to their high percentage of nitrogen (6.89%) compared to syntheticallysubstituted cellulose (1.25%) (Muzzarelli and Muzzarelli, 1998; Kumar, 2000).

As most of the present-day polymers are synthetic materials, theirbiocompatibility and biodegradability are much more limited than those ofnatural polymers such as cellulose, chitin, chitosan and their derivatives.However, these naturally abundant materials also exhibit a limitation in theirreactivity and processability.Chitosan is recommended as suitable functionalmaterial, because this natural polymer has excellent properties such asbiocompatibility, biodegradability, non-toxicity and adsorption properties.Recently, much attention has been given to chitosan as a potentialpolysaccharide source (Kumar, 2000).

Chitosan can be degraded by soilmicroorganisms and water microorganisms. This makes chitosan environmentalfriendly.This was acknowledged by the US Environmental Protection Agencywhen it exempted chitosan from tolerance level testing (Hennen, 1996).Chitin and chitosan are obtained from the shells of crustaceans such as crabs,prawns, lobsters and shrimps, the exoskeletons of insects, and the cell walls offungi such as aspergillus and mucor where it provides strength and stability(Dodane and Vilivalam, 1998; Kumar, 2000; Khor and Lim, 2003; Krajewska,2004; Sinha et al., 2004; Qin et al., 2006).([7])

3 -Production of Chitosan:-

Fig(3):chitin & chitosan manufacturing process

Chitin and chitosan are found as supporting materials in manyaquatic organisms (shells of shrimps and crabs and bone plates of squids and cuttlefishes)([8]),in many insects (mosquitoes, cockroaches, honey bees, silkworms, Drosophila melanogaster,Extatosoma tiaratum and Sipyloidea sipylus), in terrestrial crustaceans (Armadillidium vulgare,Porcellio scaber), in nematode, in mushrooms (Agaricus bisporus, Auricularia auriculajudae,Lentinula edodes, Trametes versicolor, Armillaria mellea, Pleurotus ostreatus, Pleurotus sajo-cajuand Pleurotus eryngii) and in some of microorganisms (yeast, fungus, and algae) (Carlberg,1982; Nemtsev et al., 2004; Veronico et al., 2001; Paulino et al., 2006; Moussian et al., 2005;Tauber, 2005; Hild et al., 2008; Anantaraman & Ravindranath, 1976; Pochanavanich &Suntornsuk, 2002; Mario et al., 2008; Yen & Mau, 2007 cited in Nwe et al., 2010).([9])

Crab and shrimp shell wastes arecurrently utilized as the major industrial source of biomass for the large-scaleproduction of chitin and chitosan. Processing wastes from marine food factorieshelp to recycle the wastes and make the derivatives or by-products for use inother fields. These crustacean shell wastes are composed of protein, inorganicsalts, chitin and lipids as main structural components. Therefore, extraction ofchitin and chitosan was mainly employed by stepwise chemical methods (Kimand Rajapakse, 2005).

The production of chitosan from fungal mycelia has a lot of advantages over crustaceanchitosans such as the degree of acetylation, molecular weight, viscosity and chargedistribution of the fungal chitosan are more stable than that of crustacean chitosans; theproduction of chitosan by fungus in a bioreactor at a technical scale offers also additionalopportunities to obtain identical material throughout the year and to obtain chitosans with aradioactive label or with specific changes in its polymeric composition; and the fungalchitosan is free of heavy metal contents such as nickel, copper (Tan et al., 1996, Arcidiacono& Kaplan, 1992([10]), Nwe & Stevens, 2002a). Moreover the production of chitosan from fungalmycelia give medium-low molecular weight chitosans (1–12 × 104 Da), whereas themolecular weight of chitosans obtained from crustacean sources is high (about 1.5 ×106Da)(Nwe & stevens, 2002). Chitosan with a medium-low molecular weight has been used as apowder in cholesterol absorption (Ikeda et al., 1993) and as thread or membrane in manymedical-technical applications.

For these reasons, there is an increasing interest in theproduction of fungal chitosan. However, so far, the extraction of high yield pure chitosanproduction from fungal cell wall material has not been accomplished upto 2001 (Stevens,2001).

*Steps:

In the first stage, chitin production was associated withfood industries such as shrimp canning. In the second stage, the production ofchitosan was associated with fermentation processes, similar to those for theproduction of citric acid from Aspergillus niger, Mucor Rouxii, andStreptomyces, which involved alkali treatment yielding chitosan. Briefly, shellswere ground to smaller sizes and minerals, mainly calcium carbonate, wereremoved by extraction (demineralization, decalcification) with dilute hydrochloricacid followed by stirring at ambient temperature.

The protein was extracted(deproteinisation) from the residual material by treatment with dilute aqueoussodium hydroxide and thereby prevents contamination of chitin products fromproteins. The resulting chitin was deacetylated in 40 - 45% sodium hydroxide at120ºC for 1- 3 hours with exclusion of oxygen, and followed by purificationprocedures to form chitosan with a cationic nature. The alkali removed theprotein and the deacetylated chitin simultaneously. Depending on the alkaliconcentration, some soluble glycans would be removed.In the deacetylation process, some of the acetyl groups were removed from themolecular chain of chitin.

This shortened the chain lengths of the chitinmolecule, eventually leaving behind a polymer with a complete amino groupcalled chitosan. This treatment produces 70% of deacetylated chitosan (Kumar,2000; Khan, 2001; Krajewska, 2004; Kim and Rajapakse, 2005). Methodsbased on alkaline treatments were employed to achieve N-deacetylation, as Nacetylgroups cannot be removed by acidic reagents as effectively as withalkaline treatment. However, partial deacetylation could occur under this harshtreatment (Muzzarelli, 1973).

The extent of deacetylation mainly depends uponalkali concentration, time and temperature employed throughout the process.For example, increasing temperature or strength of sodium hydroxide solutioncan remove acetyl groups, resulting in a range of chitosan molecules withdifferent physicochemical properties and applications (Khan, 2001).

According to Kumar (2000), to produce 1 kg of 70% deacetylated chitosan fromshrimp shells, 6.3 kg of HCl and 1.8 kg of NaOH are required in addition tonitrogen, water (1.4 tons). Commercially, chitosan is available in the form of dry flakes, solution and fine powder (Duarte et al., 2002; Sinha et al, 2004). The hydrolysis of chitin with concentrated acids under drastic conditions produces relatively pure D-glucosamine (Kumar, 2000).

In India, the Central Institute ofFisheries Technology, Kerala, initiated research on chitin and chitosan.From their investigation, they found that dry prawn wastecontained 23% and dry squilla contained 15% chitin. Chitin and chitosan are now producedcommercially in India, Japan, Poland, Norway and Australia (Kumar, 2000). It is likely that future sources of chitin and chitosan will come from biotechnologyinnovation, especially when medical applications are the focus (Khor and Lim,2003). Thus, production and utilization of chitosan constitutes an economicallyattractive means of crustacean shell wastes disposals, which is soughtworldwide.

Fig(4) : preparation of chitin & chitosan

Investigation of a method to produce high quality and quantity of fungal chitosan :-

**Growth of fungus and extraction of chitosan by traditional method:

Chitosan is a substantial component of cell wall of certain fungi, particularly thosebelonging to the class Zygomycetes (Bartniki-Garcia, 1968).([11]) Tan et al., 1996 evaluated theyield of chitosan from several Zygomycetes fungi including Absidia, Gongronella, Mucor andRhizopus and concluded that G.butleri gave the highest yield of chitosan.

At the same time,Crestini et al., 1996 reported that the yield of chitosan produced from Lentinus edodes grownin solid state fermentation, 6.18 g/kg was higher than that in submerged fermentation, 0.12g/l. In 1998, fungus Gongronella butleri was selected to produce chitosan in our research.

Firstly, a comparison was made between the yield of chitosan from fungal mycelia grown insolid substrate fermentation (SSF) and in submerged fermentation (SMF) using variousnitrogen sources. The Termamyl assayed extraction method was not discovered yet at thattime. The chitosan was extracted using vacuum filtration and β-glucanase treatmentmethod. It was observed that the yield of chitosan obtained from fungal mycelia grown inSSF (3.7 g chitosan/kg of solid substrate) was higher than that in SMF (0.6 g chitosan/L offermentation medium) due to the low amount of mycelia produced in SMF (Nwe et al.,2002). Based on the results obtained from our research and Crestini et al., 1996, solidsubstrate fermentation was selected as the best fermentation method to produce chitosan byfungus Gongronella butleri.([12])

Fig. 5. Fungus Gongronella butleri USDB 0201 was grown on sweet potato pieces in a tray-type solid substrate fermentor.

Sweet potato pieces were used as solid support and ascarbon source. The dried fungal mycelia were used to extract chitosan.The history of the development of chitosan extraction procedure by enzymatic methodstarted with the work of Mr. Su Ching Tan from the National University of Singapore,Singapore. In his method, mycelia were treated with 1 M NaOH and the resultant alkalineinsoluble material (AIM) was treated with 0.35 M acetic acid at 25oC for 2 h (Tan et al., 1996).

The yield ofchitosan extracted from fungal mycelia grown in solid substrate fermentation was 2-3 g/100g of mycelia. An effective chitosan extraction procedure is essential for an economicalproduction of fungal chitosan.). Most methods used

1 M NaOH to remove protein and other cell wall materialsand then the chitosan was extracted with 2 % acetic acid. The yield of chitosan producedfrom the fungal mycelia treated in this way is very low. The extraction procedure for highyield production of pure chitosan from the fungal cellwall material has not yet beenaccomplished up to 2001 (Stevens, 2001).([13])

Fig. 6. Extraction of chitosan from mycelia of fungus G.butleri grown in solid substrate

chitin/chitosan occurs in two forms, as free aminoglucoside and covalently bonded toβ-glucan (Bartnicki-Garcia, 1968; Gooday, 1995; Robson, 1999; Wessels et al., 1990). In 1990,Wessels et al. proposed that initially chitin and β-glucan chains accumulate individually inthe fungal cell wall and thereafter form the interpolymer linkage. The formation of thechitin/chitosan–glucan complex chains results in a rigid cross-linked network in the cellwall (Gooday, 1995; Robson, 1999) and causes a considerable problem for the extraction ofintact chitosan and glucan. It does not break down easily under mild extraction condition(Muzzarelli et al., 1980).

Under the above mentioned conditions only free chitosan, that ischitosan unbounded to other cell wall components is extracted (Nwe & Stevens, 2002).([14])Chitosan bounded to insoluble cell wall components will not be extracted. To extract the high quality and quantity of chitosan and glucan from cell wall of fungi, thebond between chitosan and glucan in cell wall of fungi must be investigated.

Most of theresearchers are trying to find the linkage between the chitosan and glucan in the fungal cellwall by digestion with glucanase, chitinase and amylase. In 1979, Sietsma and Wesselsreported that 90%of β-glucan obtained from the chitin-glucan complex by digestion with (1-3)-β-glucanase and N-acetyl-glucosamine, lysine and/or citrulline were identified asproducts after digestion with chitinase. Therefore they proposed that the bridge linking theglucan chain with the chitin contains lysine, citrulline, glucose and N-acetyl-glucosamine.Similar evidence was obtained by Gopal et al., 1984 for the degradation of chitin-glucancomplex by (1,3)-β- and (1,6)-β-glucanase, and subsequently by chitinase.

Carbohydrateexpressed as glucose and N-acetyl-glucosamine monomers was detectable in equivalentamounts in the hydrolysate. The residue after chitinase treatment was further treated with α-amylase but additional release of glucose could not be detected colorimetrically. Surarit et al.,1988 suggested a glycosidic linkage between position 6 of N-acetyl-glucosamine in chitin andposition 1 of glucose in β-(1-6)-glucan in the cell wall of Candida albicans.

In 1990, Wessels et al.,proposed that a direct link between free amino groups in the glucosaminoglycan and thereducing end of the glucan chains forming the inter-polymer linkages in the chitin-glucancomplex. Up to 1992, no cleare evidence of the identity of the chemical link between thechitosan and glucan polymer chains had been uncovered (Roberts, 1992).

After 1995, Kollar etal., 1995 and Fontaine et al. 2000 digested the cell wall of Saccharomyces cerevisae and Aspergillusfumigatus with 1%SDS and 1 M NaOH respectively and the insoluble fractions were digestedwith (1,3)-β-endoglucanase and chitinase.

Firstly, Thanks to Allah S. W. T Because Giving Me Success for My Final Year Project

Firstly, thanks to Allah s. w. t because giving me success for my final year project.

I wish to express my gratitude to individuals who have helped me with creating this. This would have never come to light without their massive efforts and help.

I'm deeply grateful to my supervisor,"Dr- Ayman Salah El-deen Al-Husini "who has advice me and always helping to complete of my final year project .I consider myself very fortunate for being able to work with a very considerate and encouraging lecture like him .

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