Chapter 1 - The Relevance of Chitin

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C H A P T E R 1: T H E R E L E V A N C E O F C H IT I N

1.1 C H I T I N : F R O M T H E N T O N O W

Biopolymer i s a t e rm commonly used to re fe r to po lymers b io log ica l ly syn thes ized by

na ture. Po lysacchar ides a re one such class o f b iopo lymers , compr i s ing o f s imple

monosacchar ide ( sugar ) molecu les connec ted by e ther type l inkages to g ive h igh

mo lecu la r weigh t po lymers . Com pared to the i r r enowned cous ins , the nuc le ic ac ids

and proteins , the polysaccharides have t radi t ional ly been considered of less

s ignif icance and regarded pr imari ly as s t ructural mater ia ls and metabol ic energy

sources . On ly in the la t ter ha lf of the 2 0 th Century has interest in the chemistry of the

po lysacchar ides wi tnessed a rena i ssance b rought abou t by the deve lopment o f new

methods o f i so la t ion , ex t rac t ion , separa t ion , chemica l and enzymat ic modi f ica t ion

coupled wi th the adven t o f sens i t ive and power fu l ins t rumenta l ana lys i s t echn iques .

This has led to the "re-discovery", ident i f icat ion and funct ional e lucidat ion oft rad i t iona l and newly d i scovered po lysacchar ides , i l lumina t ing the i r inheren t and

essent ia l roles in biological funct ion. I

Among the po lysacchar ides , ce l lu lose and ch i t in a re the two mos t abundan t

biopo lym ers in the biosphere. Desp i te being scient i fical ly discovered ear l ier than

cel lulose, chi t in ini tia l ly received l im ited at tent ion. By contrast , extensive research and

development has centered on cel lulose s ince the la te 19 h Century. 2

The f i rs t expl ic i t account of chi t in was in 1811, a t t r ibuted to the Frenchman,

Braconnot , P rofessor o f Natura l His to ry , Di rec to r o f the Botan ica l Garden and m em ber

of the A cadem y in Sc iences o f Nancy , France . 3 Braconnot descr ibed and na med the

alkal i - resis tant fract ion from isolates of som e higher fungi "Fung ine". In 1823, Od ier

found a mater ia l with the same general propert ies as fungine in the cut ic le of beet les

and de signated i t "chi t in" af ter the Greek w ord "chi ton" that denotes "coa t of mail" in

reference to the cutic le . Subseq uent ly , the chem ical character of chi t in began to be

elucidated. The presence o f ni t rogen in chi t in is a t t r ibuted to Payen in 1 84 3 wh ile

Ledderho se iden t if i ed g lucosam ine and ace t ic ac id as the hydro lys i s p roduc ts o f ch it in

in 1876. T iem ann proposed tha t the amino sugar was based on e i the r g lucose o r

man nose and F ischer and Leuchs conf i rmed the a t t achment o f the amino group a t the

C-2 pos i t ion o f the monosacchar ide un i t . Kar re r and co-workers and Zechm eis te r and

co-workers separa te ly per fo rmed exper iments to de te rmine tha t N-ace ty lg lucosaminewas the pr im ary con st i tuent o f chi t in . Last , the (1-4) l inkage and i ts 13-D confo rma tion

was conf i rmed by X-ray d i f f rac t ion , enzymat ic and deamina t ion reac t ions by Meyer

and others in the f i rs t half of the 2 0 th Century .

The discovery of chi tosan is ascr ibed to Rouget in 1859 when he found that boi l ing

chi t in in potassiu m hy droxide rendered chi t in soluble in organic acids . In 1894 Ho ppe-

Seyler nam ed this mater ia l chitosan. Only in 1950 was the s t ructure of chi tosan f inal ly

resolved.

I t has taken more than a hundred years to arr ive at the chemical ident i ty of chi t in and

the revelat ion o f its polym eric propert ies. This is not surpr is ing consid er ing that the

term poly me r was not even acceptable unt il the 1930 's onwards! Nev ertheless , the

necessary foundat ion was la id for the eventual emergence of chi t in as an important

b iomedica l mate r ia l.



1 . 2 D E F I N I T I O N S

Before p roceed ing fu r ther , i t i s neces sary to def ine what ch i t in and i t s p r inc ipa l

der iva t ive , c h i tosan , i s . The ear l i e r l i te r a tu re i s l i tt e r ed wi th exam ples o f the casua l

i n t e r c h a n g e b e t w e e n t h e s e t w o t e r m s a n d t h e r e h a v e b e e n i n s t a n c e s w h e n c h i t i n i s

r e fe r r ed , w here i t i s c l ear f rom a r ead ing o f the con ten t , tha t ch i tosan wa s be ing

di scus sed . On ly in the pas t t en year s , has there been m ore cons i s t en cy in the usage of

the t e rm s ch i tin and ch i tosan . The def in i t ion and descr ip t ion tha t fo l lows incorp ora tes

w h a t i s w i d e l y a c c e p t e d b y t h e c h i t i n / c h i t o s a n c o m m u n i t y t o d a y .

The idea l s t ruc ture o f ch i t in is a l inear po lysacch ar ide o f [3 - (1-4) -2-ace tamido-2-de oxy-

D - g l u c o p y r a n o s e w h e r e a l l r e s id u e s a r e c o m p r i s e d e n t i r e ly o f N - a c e t y l - g l u c o s a m i n e

res idues o r in ch i t in j a rgo n , fu l ly ace ty la ted . L ike wise , the idea l s t ruc ture o f ch i tosan ,the p r inc ipa l der iva t ive o f ch i tin i s a l inear po ly m er o f 13- ( l -4 ) -2-amino-2-de oxy- D -

g l u c o p y r a n o s e w h e r e a ll re s i d u e s a r e c o m p r i s e d e n t i re l y o f N - g l u c o s a m i n e r e s i d u e s o r

fu l ly deac e ty la ted . The shor t - fo rm n o ta t ions fo r idea l ch i t in and ch i tosan a re dep ic ted

in F igure 1 .1 and employed th roughout th i s book .

C H 2 O H

N H A c

\

C H 2 O H

O

O H O

N H 2 n

C h i t in C h i t o s a n

A c - -

OII

- - C - - C H 3

Figure 1 .1 : Idea l i zed r epresen ta t ion o f ch i t in and ch i tosan

T r a d i t i o n a l s o u r c e s o f t h e b i o p o l y m e r , h o w e v e r , d o n o t r e s u l t in 1 0 0 % a c e t y l a t e d c h i ti no r 1 0 0 % d e a c e t y l a t e d c h i t o s a n a n d i n r e a l i t y , t h e b i o p o l y m e r e x i s t s a s a c o - p o l y m e r a s

represen ted in F igure 1 .2 , where the numbers in square b racke t s on the ex t r eme le f t

r ing as s igns the s ix carbon s in the g lucopy rano se r ing f rom C-1 to C-6 . Spec i f i ca l ly ,

t h e s u b s t i tu t i o n a t t h e C - 2 c a r b o n o f t h e g l u c o p y r a n o s e r in g c a n b e e i t h e r w i t h t h e

a c e t a m i d o g r o u p o r a n a m i n o g r o u p . T h e d i f f e r e n ti a t io n b e t w e e n c h i t i n a n d c h i t o s a n i s



to consider their respective acetyl content . W hen the num ber of acetam ido group s is

more than 50% (m ore comm only 70-90% ) the b iopolym er i s te rmed chi tin . In chi tin

terminology, the num ber of ace tamido groups i s te rmed the degree of ace ty la t ion (DA).On the o ther hand, when the degree of deacety la t ion (DD) or the amino group i s

predom inant , the b iopolym er i s te rmed chi tosan .

[6]C H 2 O H

[4 ] / ~ O [ ' , , , , ] F C H 2 O H q

I I I ~ 4, ~ , V n / l ~ _ ) - - - 0 O H

_ IR

OR = - - C / /

" C H 3

R = - - H

a n d x > 5 0 % - - - > c h i t in

a n d y > 5 0 % - - > c h i to s a n

Figure 1.2: Chem ical structural representat ion of chit in and chitosan

depic t ing the co -polym er charac ter o f the b iopolym ers

Instant differentiat ion between chit in and chitosan can be at tained from their solubil i ty

and n itrogen content . 4 Chit in is soluble in 5% Lithium chloride/N , N-

Dimethylacetamide solvent [LiCI /DMAc] and insoluble in aqueous ace t ic ac id whi le

the conv erse is t rue of chitosan. The nitrogen content in purif ied samp les is less than

7% for chit in and mo re than 7% for chitosan .

In this book, the term chit in wil l be used general ly to refer to chit in and i ts derivatives,

including chitosan. W hen direct references are ma de to chitosan or any chit in

deriv ative, the spe cific nam es will be used. Ch itin also has three crysta lline form s, c~, 13

and T. Unless oth erwise specif ied, chit in denotes a-chit in.



1 . 3 O B S C U R I T Y

Despi te i t s ear ly discovery, and being the second most abundant polysacchar ide af ter

cellulose with an est im ated annual production of at least 10 l~ tons per yea r in the

biosphere, chit in has remained an almost unused biomass resource late into the 20 th

Century. 5 W hy did ch it in lang uish in obscurity for so long?

First, i f we contrast chit in to cellulose, one explana tion cou ld be the source o f the raw

material . Cellulos e is readily available from trees and cotton plants; two tradit ional

sources that date back centuries, almost to the beginning o f civil ization. Coup led with

the centuries of experienc e in exploit ing and using this resource, i t is readily

unders tandable that ce l lu lose has been wel l ent renched as a b iopolymer of s igni f icant

uti li ty. Con versely, sources o f chit in comprising o f shells of crustaceans, insects andfungi, are more difficult to exploit and therefore, perceived to be of insignificant

usefulness except as food and decorative ornaments . Second, looking back a t the br ief

historical account of chit in and chitosan described above, the biopolym er belonged

prima rily in the realm o f the naturalists , who se preocc upation lay in identifying and

charac terizing chit in and chitosan in plants and animals. These included isolating and

determining the composit ion, the structure, macrostructure and physical propert ies and

establishing the ir biochemica l and biological roles. 6 A mo dicum of chit in che mistry

only began to appea r in the late 1930 's. Very l i t tle else was reported in the ensuin g

years from its discovery unti l the 1950's. A third factor is the difference in the physical

proper t ies of chi t in in compar ison to cel lu lose where the s t rong hydrogen-bondingnetw ork in the former led to a mo me ntary dead-end to i ts man ipulation and use. The

search for an effective solution to this si tuation was the beginning of a series of on-

going e fforts to defy the chemical and structural inertness o f chit in.

The turnaround from anonymity can probably be at tr ibuted to two unrelated yet vital

events. This was the advent o f the canning industry that came into being in the 1800's.

Canning enabled the preservat ion of food for extended per iods of time. Obviously , the

seafood process ing industry o f which crabmeat and other shel lf i sh p roducts were a par t

of embraced canning and grew. Coupled wi th an increas ing consumpt ion by a growing

world population and the globalization of food taste, the shellf ish waste that was

generated gradually increased, eventually leading to the need to resolve thisaccu mu lating industrial waste prob lem in the 20 h Century.

1.4 F R O M W A S T E T O N IC H E M A T E R I A L S

Commencing in the 1950's, a more concerted effort began to appear in the scientif ic

l i terature. A natural course in f inding a solution to the abundan t shellf ish waste from

seafood process ing i s the in teraction between indust ry and Academ ia. One exam ple i s

Tottori , Japan, famous for i ts snowcrabs and associated seafood processing facil i t ies

that generated such shellf ish wastes. I t is probab ly no coincide nce that Professo r

Shigehiro H irano of Tottori Universi ty (now retired) becam e an eminent pioneer in

chit in research since the late 1960's. This exam ple duplicated in similar si tuations

around the world from the 1950's onward no doubt has changed the course, or at least

fast-forw arded the chit in story outcom e. Eventually, this resulted in the increased

unde rstanding of the science and techno logy o f chit in that culminated in the first

"mi les tone" as i t were , wi th Professor Mu zzarel l i ' s landm ark book. 7



Ne xt bega n the giant leap in the s tudy and applicat ion o f chi t in and ch itosan ar is ing

from tw o fortui tous events . Firs t, industr ial interest turned into activi ty that establ ished

the regular avai labi l i ty of the biopo lym er for research and com mercial ex ploi tat ion.Today , p roducers o f ch i tin and ch i tosan span the g lobe and a re found in Nor th

Am erica, Eu rope and Jap an and increasing ly from China, Ind ia and So uth East Asia. 8

The qua l i ty o f the product i s var ied . The predom inant ch i t in and ch i tosan products a re

produced and con sumed m ain ly in Japan . Increas ingly , Korea is p roducing a mix of

products a im ed a t an expor t marke t .

Second, the m om entum genera ted by the se r ies of In te rna t iona l C hi t in /Chi tosan

Co nferen ces begin ning w ith the f irs t a t M IT, US A in 1977 and w ith the 8 h recently

he ld in Yam aguchi , Japan in 2000. The European C hi tin Soc ie ty and the Japanese

Chit in and Chitosan Society also further s t imulated act ivi ty with their own meetings

and the latest , The Asia-Pacif ic Chit in/Chitosan Symposium series , begun in the mid-

1990s, adds to the on-going internat ional forum on the biopolymer.

A p p l i c a t i o n a r e a

W ater t rea tment

Agr icu l ture

Text i le and p aper

B io t echno logy

Food /H ea l t h

Supp lemen t s

Cos me t i c

Biomedica l

Specif ic u s e

Coagula t ing / f loccula ting agents for po l lu ted was te

waters

Rem oval / recovery of meta l ions f rom aqu eous was te

water

Plant el ici torAnt imicrobia l agents

Plant seed coat ing

Fert i l izer

Fibers for text i le and woven fabrics

Paper and f i lm

Chr oma tog r aphy pack ing

Enzyme immobi l iz ing mater ia l s

Natural thickeners

Food addit ives including pet food

Foo d proce ssing (e .g. in sugar ref ining)Fil t rat ion and clar if icat ion

Hypocholes te ro lemic agent ( s l imming agents )

Ingredients for hair and skin care (condit ioners)

Burns and wou nds dress ings for hum ans and

animals

Biomaterial (e .g. absorbable sutures)

Ant icoagulan t o r an t i th rombog enic m ateria ls

(as sulfated-chit in derivat ives)

Hemostat ic agents (as chi tosan)

Drug del ivery, gene del ivery

Table 1 .1 : Ap plicat ions of chi t in, ch i tosan and their derivat ives 9



The driving force for much of the excitement su rrounding chit in and chitosan are the

potential a pplicatio ns that the materials can be used for. Table 1.1 lists som e examp les

of the known and potential applications for chit in, chitosan and their derivatives that

have caugh t the imagination of scientists , raw material producers and man ufacturers

and users alike.

Waste water treatm ent u sing chitosan to chelate metal ions was one of the f irst

applications o f chitin. Various formulations for hair care products such as shampoos

and condit ioners followed suit . Over the past 20 years the other fields of applications

have co me on stream, key amo ng them b eing the use of chitosan as

hypo choleste rolemic agents. Figure 1.3 shows a collage of some of these products.

Figure 1.3: Represen tat ive products of chit in and chitosan such as soaps,

socks, weight-control pi l ls and health food supplements

[By permission of the Korea Chitosan Com pany, Ltd]

The market size estimate as listed in Table 1.2 illustrates the increasing potential and

versati l i ty of this biopolymer. I t is notewo rthy that biomedical applications have the

potential to be the largest revenue gen erators.

Therefo re, the f lurry of activi ties com men cing in the late 1970's report ing the

"discovery" o f this "new" and excit ing biopo lymer can be looked on as the watersh ed

for chitin. From virtual anonym ity, many products containing chitin and chitosan are

now k now n and more are forthcoming. These activi ties have in turn lead to the need



for a better unders tanding of the production, science and technology and uti l ization of

chitin, sustaining the research cycle that is so necessary for the future applications and

scientific k now ledge o f chit in .

I n d u s t r y

- .Agriculture

J a p a n M a r k e t

(106 U S$/y ear) l~

Bio-fertilizer 21 -

- 11 0ood and beverages

Cosm etics and toile tr ies

i Chi topack KQ 8025

_Waste and dr inking water treatment

T o t a l

W o r l d M a r k e t

(10 6 U S$ /yea r) 11

2.3

90

Biom edical 2000 1250 ,

Imm obilized Cell and culture " - 45

3

- 14 0

2 0 2 4 1 9 1 5

Table 1.2" Potential size of the market for chitin, chitosan and their derivatives

Today the exciting promise of chit in has been delivered and more importantly, is set to

play a significa nt role in the biom edical field as a biom aterial o f the 21 st Century.

Perhaps chitin may even one-day surpass i ts older and better-known sibling, cellulose.

1.5 B I O M E D I C A L A P P L I C A T I O N S

Tow ards the close of the 20 h Century, the chitin c om m unity has becom e increasingly

enthusias tic over the biomedical opportunities for this mater ia l . W hy is this so? A

plethora of processing methods including i ts chemistry and character ization together

with a more in-depth knowledge of biomedical applications including cell / t issueinteractions , have em erged. As a result , the breadth of biomedical applications spanned

by chitin continues to expand, making chitin a mater ia l increasingly impossible to

ignore. The possibil i t ies appear endless . The surge of chit in in the biomed ical

direction is rooted f rom the po stulation that there wo uld be better acceptance o f the

mater ia l by the human biological system due to i ts natural or igins and close analogy to

body consti tuents . Together with the potentia l of limitless supply of this renewable

mater ia l , these major inf luences have perpetually thrust chit in to the forefront as a

biomater ia l . Will this "gold-mine" o f opportunity be realized?

The aim of this book is to address these biomedical applications of chitin . Each cr it ical

aspect that can shed light on these issues will be put forward. In the ensuin g chapters,

the foremost biomedical applications that have been proposed for chit in , wound

dressings, blood-interaction mater ia ls , or thopedic applications , t issue engineer ing

scaffolds and drug delivery will be surveyed. This will be followed by an assessment

of whether chit in can and will meet the diverse biomedical applications proposed by



care fu l ly cons ider ing the requ i rements govern ing them. Survey ing and p lac ing in

context a l l the relevant biocompatibi l i ty s tudies that has been reported for chi t in so far

and asks what e lse needs to be done.

For ch i t in to beco me a biom ater ia l of the 21 st Century, the im pact o f the sou rces and

product ion of chi t in must a lso be careful ly considered s ince they have impact on the

biopo lym er 's avai labi l i ty for chi t in to escalate in ut il i ty as a biom ater ia l . Eq ual ly

important wil l be how the propert ies of chi t in can be inf luenced by the mode of

ma nipulat io n. The role of the many var ied chem istr ies of chit in and their re levanc e in

chi t in exploi ta t ion wil l a lso be touched on. Final ly a s ta tus report wh ere chi t in is on the

regulatory road to approval wil l be discussed and a comparison with some of i ts

competi tors wil l conclude this explorat ion of chi t in fulf i l l ing i ts biomater ia ls role .

1 .6 R E F E R E N C E S

G.O. Aspinal l , Polysacch arides . Perga mo n Press , Oxford, UK, 1970

Encyc loped ia o f po lymer sc ience and technology , Volume 3 , H .F . Mark , N .G.

Gaylord, N.M. Bikales , eds . , John Wiley & Sons Inc. , New York, N.Y. , 1965

C. Jeuniaux, A br ief survey of the ear ly contr ibut ion of European scient is ts to

chi t in know ledge. Adv ances in Chit in: 1 , A. Dom ard, C. Jeuniaux, R.

M uzzarel l i , G. Robe rts , eds . , Jacques Andre Publ ishers , Lyon, France, 1996. 1-9

R.A .A. Muzzarel l i , Chi t in . Perga mo n Press Ltd, Oxford, UK, 1977. 87

K. Kuri ta , Chit in and chi tosan der ivat ives , in Desk Referenc e of Funct ion al

Po lymers . Syn thes i s and Appl ica t ions , R . Arshady , ed . , Amer ica Chemica l

Soc ie ty , W ash ing ton D.C. , USA, 1996 . 239-259

M.Falk, D.G. Smith, J. McLachlan, A.G. McInnes, Studies on chitan (13(1--->4)-

l inked 2-ace tamido-2-deoxy-D-g lucan) f ibe rs o f the d ia tom T h l a s s i o s i r a

f l u v i a t i l i s h u s t e d t , I I : Proton magnet ic resonance, infrared and x-ray s tudies .

Canadian J . Chemistry 44 (1966) 2269-2281

R.A.A. Muzzarel l i , Chi t in , Pergamon Press Ltd, Oxford, UK., 1977.

Chit in and ch i tosan: A n expan ding range o f markets await ex ploi ta t ion, 3 r~

Edi t ion . John W i ley and Sons Inc , Ne w York , N .Y. , 1998

M .F.A. Goose n, S. Hirano. in Ap plicat ions of chi t in and chi tosan, M FA G oosen,

ed. , Tec hno mic Pub lishing Co. , Inc., Lancaster , PA. 1997. Cha pters 1 and 2.Liu 1994 Food Industry 26(1): 26-36 ( in Chinese) . Cou rtesy of RH Ch en,

Ta iwan , 2000

D. Knorr , Recovery and ut i l izat ion of chi t in and chi tosan in food processing

was te managem ent . Food Techno logy 45 (1991) 114-122

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Chapter 1 - The Relevance of Chitin