Carbohydrate Polymers 63 (2006) 385–392

www.elsevier.com/locate/carbpol

Novel method for preparation of b-cyclodextrin/grafted chitosan

and it’s application
Khaled El-Tahlawy *, Mohamed A. Gaffar, Safaa El-Rafie
Textile Research Division, National Research Center, Cairo, Egypt
Received 8 August 2005; accepted 18 August 2005
Available online 3 February 2006

Abstract

A novel technique for preparation of b-cyclodextrin-grafted chitosan was carried out by reacting b-cyclodextrin citrate (b-CD citrate) with
chitosan. b-Cyclodextrin citrate was synthesis by esterifying b-cyclodextrin (b-CD) with citric acid (CA) in presence or absence of sodium
hypophosphite as a catalyst in a semidry process. Different factors affecting preparation of b-CD citrates were studied to obtain b-CD citrate with
high carboxyl content, such factors include reaction temperature, citric acid concentration, material to liquor ratio and duration.
b-Cyclodextrin/grafted chitosan was prepared by coupling b-CD citrate with chitosan dissolved in different formic acid solutions having
different concentrations. The reacting ingredients were subjected to various reaction conditions to attain the optimum condition.
b-Cyclodextrin/grafted chitosan were evaluated by measuring the nitrogen content of both chitosan and grafted chitosan. Chitosan and
b-cyclodextrin/grafted chitosan, having different molecular weights, were evaluated as antimicrobial agents for different microorganisms such as,
Bacillus Megaterium, Pseudomonas Fragi, Bacillus Cereus Staphylococcus Aureus, Escherichia Ecoli and Aeromonas hydra.
q 2005 Elsevier Ltd. All rights reserved.

1. Introduction area of applications such as deodorant, aroma, antimicrobial,

insect repellent, mite repellent finishes that have recently
Cyclodextrins (CDs) are cyclic oligosaccharides. They are become popular and in treating effluents.
produced by enzymatic degradation of starch and were first Chitin is one of the most abundant naturally occurring
obtained by Villies 1891. There are three main types of polysaccharide next to cellulose. Chitin consists mainly of
cyclodextrin,a, b and g of six, seven and eight cyclic maltose, b-(1–4)-2-acetamido-2-deoxy-D-glucose units. Despite much
respectively (Kobayashi et al., 1981; Cox et al., 1984; Bender recent research into its utilization, its tightly intermolecular
and Komiyama, 1978; Deratani and Poepping, 1995; Seo et al., hydrogen bonding and poor solubility to common organic
1987; Mizobuchi et al., 1980). The cyclodextrin consists of solvents have so far prevented widespread utilization of chitin
tours like macrocyclic ring. All hydroxyl groups are located at (George, 1992). Chitosan is N-deacetylated form of chitin that
the top and bottom of the tours. Thus the hydrophobic cavity of is obtained by alkaline treatment of chitin (50% of aqueous
cyclodextrins is capable of including a variety of hydrophobic NaOH) at high temperature. Chitosan and its derivatives have
compounds via host-guest complexation (Takashima et al., become useful polysaccharides in the biomedical area because
2004). This property has been extensively exploited in the past of its biocompatible, biodegradable, and non-toxic properties
to change physiocopharmacetical properties of lipophilic drugs (Lee et al., 1997).
such as water solubility, bioavailabilty, improved stability and The antimicrobial and antifungal activities of chitosan and
effectiveness (Sortino et al., 2001). chitosan derivatives (Devlieghere, Vermeulen, & Debevere,
Many attempts to utilize CD and CD derivatives in textile 2004; Lim, Sang-Hoon; Hudson, &Samuel, 2004a,b) have
applications were carried out in the last decade (Szejtli, & been described, since chitosan inhibits the growth of a wide
Jozsef 2003; Buschmann, Knittel, & Schollmeyer., 2001). This variety of bacteria and fungi. Moreover, chitosan has several
was brought about by the recognition that the inclusion advantages over other types of disinfectants, that is, it
complex formation capability of CD can be applied to different possesses a higher antibacterial activity, a broader spectra of
activity, a higher killing rate, and lower toxicity toward
mammalian cells.
* Corresponding author. Many attempts were carried out to prepare cyclodextrin-
0144-8617/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. grafted chitosan in the literature. Zhang et al. prepared two new
doi:10.1016/j.carbpol.2005.08.057 adsorbents by reacting of b-CD and sulfonated b-CD with
386 K. El-Tahlawy et al. / Carbohydrate Polymers 63 (2006) 385–392

epoxy-activated chitosan and chitosan, respectively (Zhang, 2.3. Preparation of b-cyclodextrin/grafted chitosan
Wang, & Yi, 2004). Wu Wen-Teng et al. immobilized b-CD to
chitosan beads by crosslinking with1, 6-hexamethylene Linking of b-CD citrate onto chitosan was taken place by
diisocyanate (Chiu, Chung, Giridhar, & WuChiu, 2004). reacting the pendant free carboxyl groups of b-CD citrate with
Michel Morcellet studied the reaction of cyclodextrin the amino groups of chitosan. A definite volume of water
monochlorotriazinyl derivative with chitosan (Martel et al., containing different b-CD citrate concentrations was introduced
2001), and Farusaki et al. (Furusaki et al., 1996) coupled into a solution containing chitosan dissolved in different formic
carboxymethyl derivative of b-CD to chitosan. acid concentrations (0–0.4 ml/1 g chitosan). The reaction
In our pervious work, b-CD/grafted chitosan was prepared mixture was then magnetically stirred and heated at different
by graft copolymerization of b-CD itaconate onto chitosan reaction temperatures (80–140 8C) for 3 h using different
using ceric ammonium nitrate as a redox initiation system material-to-liquor ratios (1:10–1:25). At the end of the reaction,
(Gaffar et al., 2004). Cyclodextrin itaconate has been prepared the products were precipitated by adding 100 ml of NaOH
by esterifying b-CD with itaconic acid using a semidry process. solution (0.2 N). The samples were thoroughly washed with
As continuity, efforts are made in this work to prepare distilled water till neutral (pH 7) to ensure the removal of un-
cyclodextrin/grafted chitosan with high CD-content meanwhile reacted b-cyclodextrin citrate. Finally, the samples were washed
it is easily and completely soluble in organic acid solutions. with acetone and oven dried at 60 8C for 24 h.
This was achieved by reacting of b-CD with citric acid at
different reaction conditions to prepare b-CD citrate, as a 2.4. Antibacterial spectrum
reactive CD, of high carboxyl content. b-CD citrate was
allowed to react with chitosan of three different molecular Tryptone soya agar and broth were used for estimation the
weights at various reaction conditions. antibacterial spectrum of chitosan and cyclodextin/grafted
chitosan, having three different molecular weights, on several
2. Experimental spoilage and pathogen strains namely B. cereus B. megaterium,
Pseudomonas fragi, S. aureus, Aeromonas Hydra, and
2.1. Materials Escherichia coli
Chitosan and cyclodextin/grafted chitosan samples of
b-Cyclodextrin was kindly supplied by Cerestar Co. (USA). different concentrations (12.5–100 mg) were added prior
Chitosan, having three levels of molecular weight, e.g. 50,000; pouring. All treatments were incubated at 37 8C for 24 h. All
30,000; and 1500 was purchased Korea Chitosan Co., Ltd and incubated media were visually observed to find out whether the
other suppliers. Citric acid (CA), sodium hypophosphite strain had grown or not. Besides, the minimum inhibition
monohydrate (SHP), formic acid, and sodium hydroxide were concentration (MIC) of each strain was determined.
laboratory grade chemicals.
Bacillus megaterium 744 were obtained in lyophilized form, 2.5. Testing and analysis
from Northen Regional Research Center, Illionion (USA).
While Pseudomonas fragi NRRL B-727 was obtained from The carboxyl content of b-CD citrate was determined using
National Center for Agricultural Utilization Research (USA). acid base titration method according to a reported method
Bacillus cereus ATCC 11778 was obtained from American (Yang & Wang, 2000).
type culture collection (ATCC) (USA). While strain Staphy- The grafting efficiency of b-CD citrate onto chitosan was
lococcus aureus (No. 315) was obtained from US Food Drug calculated mathematically via determining the nitrogen % of
Administration (FAD) Microbiology Lab (USA). chitosan before and after modification according to Kjeldal
Tryptone Soya Broth and Tryptone Soya media were method (Vogel, 1966).
obtained from Oxoid Ltd, UK The minimal inhibition concentration (MIC) of bacteriocin
was qualitatively determined for several bacterial strains
2.2. Synthesis of b-cyclodextrin citrate according to the reported method (Davidson & Parish, 1989).

b-Cyclodextrin citrate (b-CD citrate) was prepared using a 3. Results and discussion
semidry reaction method by mixing of 2 g of b-CD with
definite amount of water containing different citric acid 3.1. Tentative mechanism
concentrations (1–4 mole/1 g CD) in presence and absence of
SHP. The reaction mixture was allowed to react in a circulating Esterification of polysaccharides (e.g. cellulose (El-
air oven at different reaction temperatures (80–140 8C) for Tahlawy, 1999; Voncina & Le Marechal, 2005), starch (Xueju
specific times. The cured samples were purified by washing & Qiang, 2004)) can be prepared by the reaction of poly-
with isopropanol using a soxhlet for 6 h in order to remove un- carboxylic acid with hydroxyl groups of these polymers in the
reacted components as well as any soluble fragments or by- presence of alkaline or amphoteric catalyst under curing
products, followed by drying at 60 8C for 24 h. After drying, conditions via reactive cyclic anhydride intermediate mechanism.
the b-CD citrate was kept over P2O5 for at least 48 h before Preparation of b-CD citrate was carried out by the reaction of
analysis. b-CD with citric acid, in presence and absence of SHP, via the
 K. El-Tahlawy et al. / Carbohydrate Polymers 63 (2006) 385–392 387

formation of a reactive cyclic anhydride intermediate by the Given below are the results obtained along with their
dehydration of two adjacent carboxyl groups (Lim, Sang-Hoon, appropriate discussions (Fig. 1).
Hudson, & Samuel 1997) under curing conditions. Since the
primary hydroxyl groups of b-CD are the most basic, they are
4.1. Effect of sodium hypophosphite concentration
more favorable for esterification than secondary one.
The mechanism of esterification reaction would be
Fig. 2 shows the effect of sodium hypophosphite concen-
accompanied by several possibilities as in Scheme 1. The
tration (SHP) (0–0.5 mole/mole CA) as a catalyst versus the
dominant of which may be as follows:
carboxyl content of b-CD citrate, when b-CD was treated with
FTIR- spectra of b-CD citrate shows strong peak at
1730 cmK1. This peak indicates the formation of carboxylic citric acid at different reaction temperatures (80–140 8C) using
ester of cyclosextrin citrate esters. material to liquor ratio 1:0.6. It is clear from the data that,
increasing SHP concentration from 0 to 0.5 mole/mole CA is
accompanied by increase in the carboxyl content of b-CD
4. Preparation of b-cyclodextrin citrate citrate, such increase in the carboxyl content could be
attributed to the enhancement in the esterification efficiency
Heating of citric acid is accompanied by formation of of CA to react with b-CD even at low temperature (80–100 8C).
anhydride, when b-CD is present in the reaction mixture, the The carboxyl content of b-CD citrate prepared by esterifying of
anhydride can react with b-CD to form b-CD citrate adduct. b-CD with CA in absence of SHP gave us a promising results,
Major factors affecting esterification of CD with CA in since the formed b-CD citrate is easier in the purification step.
presence of/or absence of SHP as a catalyst were studied. The data (Fig. 2) indicates also that the reaction temperature

Scheme 1. The reaction possibilities of citric acid with cyclodextrins.

388 K. El-Tahlawy et al. / Carbohydrate Polymers 63 (2006) 385–392

Carboxyl content (meq. /100 g CD citrate)

70
350
60
300

250
40 200
%T

150 Temperaure °C
80
20 100 100
50 120

0
0 0.5 1 1.5 2 3
4000 3000 2000 1000 400 Reaction Duration (hours)
Wave number [cm–1]
Fig. 3. Effect of reaction duration on the the carboxyl content of CD citrate at
Fig. 1. Representation of the FTIR spectra of b-CD citrate. different temperatures. [CA], 2 mole/1 mole CD; M/L—ratio 1:0.6. [SHP], 0.

plays an important role during the esterification process. It is at 3 h, and (b) rate and extent of reaction increases by raising
clear from the data that, regardless of SHP concentration used, the reaction temperature within the range studied.
increasing the reaction temperature from 80 to 120 8C is
accompanied by a significant increase in the carboxyl content
4.3. Effect of material to liquor ratio
of b-CD citrate. Further increase in the reaction temperature
beyond 120 8C is accompanied by marginal increase in the Fig. 4 shows the effect of material to liquor ratio (1:0–1:1.2)
carboxyl content. This behavior could be attributed to the on the carboxyl content of b-CD citrate, when CA was allowed
favorable effect of raising the reaction temperature (1) to esterifies b-CD using CA; 2 mole/1 mole CD, reaction time;
dehydration of CA resulting in the formation of the reactive 2 h at two different reaction temperature (100 and 120 8C). It is
anhydride intermediate, and (2) esterification process as a well know that, the material to liquor ratio plays an important
whole. Since our target is preparation of b-CD citrate with high function during the esterification process especially in a
carboxyl content without affecting the CD-ring. It could be semidry state, since the amount of water is responsible for
concluded that 100 8C is the optimum reaction temperature for homogenization of the reacting ingredients as well as
preparation of b-CD citrate. increasing the availability of CA molecules in the vicinity of
b-CD. Also it is well established that the esterification process
4.2. Effect of reaction duration is carried out through (a) dehydration of water, (b) formation of
anhydride intermediate and finally (c) the esterification
The relation between the reaction duration of esterification reaction. Keeping the above in mind, the effect of material to
reaction and the carboxyl content of b-CD citrate at three liquor ratio started from zero water content to 1.2 ml of water
containing 2 mole/1 mole b-CD was studied. It is clear from
different reaction temperatures (80, 100, 120 8C) is shown in
the data (Fig. 4) that, increasing the liquor from 0 to 0.6-ml
Fig. 3. The esterification reaction was carried out using CA,
water/1 g b-CD is accompanied by significant improvement in
2 mole/1 mole CD and material to liquor ratio 1:0.6. It is clear
the carboxyl content. Further increase in the water content
from the data that, (a) the carboxyl content of b-CD citrate is
above this limit is accompanied by a decrease in the carboxyl
increasing gradually till reaches the maximum of the reaction
content of b-CD citrate. Increasing water content from 0 to 0.6
leads to an increase in the homogeneity of the reactants and
Carboxyl content (meq/100 g CD- citrate)

350
consequently enhancing the reactivity and accessibility of CA
to esterify b-CD. Further increase in the water content above
0.6 ml caused a dilution of the reaction medium which needs
either elevation of the reaction temperature from 100 to 120 8C
300

Temperaure °C
Temperaure°C
100
(meq/100 g CD citrate)

80
120
250
Carboxyl content

100
400
120 200
140 0
1:00 01:00.3 01:00.6 01:00.9 01:01.2
200 Material - to - liquor ratio
0 0.125 0.25 0.5
SHP concentration (mole/ 1 mole CA)

Fig. 2. Effect of SHP concentration on the the carboxyl content of CD citrate at Fig. 4. Effect of material-to-liquor ratio on the carboxyl content of CD citrate at
different reaction temperature [CA], 2 mole/1 mole CD; M/L—ratio 1:0.6. different temperatures. [CA], 2 mole/1 mole CD, time; 2 h, [SHP], 0.
 K. El-Tahlawy et al. / Carbohydrate Polymers 63 (2006) 385–392 389
Carboxyl content (meq/100 g CD- citrate )

20
500
450
400 15

Graft Yield %
350
300
250 10
200
150
Temperaure °C 5
100 100
50 120
0
1 2 3 4 0
CA concentration (mole/1mole CD) 0 0.1 0.2 0.3 0.4 0.5
Formic acid concentration ( ml / 1g chitosan )
Fig. 5. Effect of [CA] on the the carboxyl content of CD—citrate at two
different reaction temperatures. Time, 2 h; M/L, ratio; 1:0.6, [SHP]; 0, [CD], Fig. 6. Effect of [formic acid] on the graft yield % of CD/grafted chitosan CD—
1 mole. citrate; 0.15 g (1.34 mequiv free carboxyl); [Chitosan], 0.54 g (6.7 mequiv free
amino), M/L, ratio; 1:20, M.Wt of chitosan; 50.000, temperature; 100 8C, time,
or prolonging the reaction time to evaporate the water from the 3 h.
reaction medium.
associated with (1) greater solubility of chitosan, thereby
increasing surface area of the chitosan molecules to react with
4.4. Effect of CA concentration b-CD-citrate, (2) increasing the homogeneity of the reacting
ingredient, and (3) higher condensation efficiency.
The effect of CA concentration on the extent of
esterification (expressed as mequiv carboxyl/100 b-CD citrate)
is illustrated in Fig. 5. The esterification reaction was carried 5.2. Effect of b-CD citrate concentration
out using material-to-liquor ratio 1:0.6, reaction time; 3 h at
two different reaction temperatures (100 and 120 8C). It is Fig. 7 shows the effect of b-CD citrate concentration
obvious that as a general, increasing CA concentration in the (carboxyl content 273 mequiv/100 g) incorporated in the
reaction medium is accompanied by a noticeable increase in reaction medium (0.05–0.6 g, i.e. 0.446–5.36 mequiv car-
the carboxyl content of the obtained b-CD citrate. This could boxyl) on the graft yield percent at two different formic acid
be associated with higher availability of CA molecules in concentration (0 and 0.4 ml/1 g chitosan) using 0.54 g chitosan
vicinity of CD molecules at higher concentration, leading to (6.7 mequiv free amino groups) at 100 8C for 3 h using material
increasing the extent of esterification of b-CD. It could be to liquor ratio of 1:20. It is clear from the data that, increasing
concluded that, the optimum condition for preparation of b-CD b-CD citrate concentration incorporated in the reaction
citrate is CA; 2 mole/1 mole CD, reaction time; 2 h, reaction medium is accompanied by increasing the graft yield %, such
temperature; 100 8C, and material-to-liquor ratio 1:0.6. increase in the G.Y. % may be due to increasing the number of
available b-CD citrate molecules to react with chitosan. At the
5. Preparation of cyclodextrin/grafted chitosan same time, increasing b-CD citrate concentration incorporated
in the reaction medium in the presence and absence of formic
b-CD citrate with carboxyl content of 273 mequiv/100 g acid (0 and 0.4 ml/1 g chitosan) shows two different values,
was prepared according to the optimum conditions. The since G.Y % at 0.4 ml of formic acid is higher than that in
prepared sample was used as a reactive CD to react with
20
amino groups of chitosan for preparation of b-CD/grafted
chitosan. Different parameters affecting the grafting reaction
were studied briefly. 15
Graft Yield %

5.1. Effect of formic acid concentration 10

Fig. 6 shows the effect of formic concentration that is used

for dissolving chitosan, as a function of graft yield %. It is 5
Undissolved chitosan
evident from the data (Fig. 6) that, increasing formic acid Dissolved chitosan
concentration from 0 to 0.4 ml/1 g chitosan is accompanied by 0
a significant increase in the graft yield % and reached 0 1 2 3 4 5 6
maximum value at 0.4 ml formic acid/1 g chitosan at which CD citrate concentration
chitosan is completely soluble in water. The enhancement in Fig. 7. Effect of [CD—citrate] on the graft yield of CD/grafted chitosan.
the grafting reaction, expressed as graft yield %, by increasing [Chitosan]; 0.54 g (6.7 mequiv free amino), M/L, ratio; 1:20, temp.; 100 8C;
the formic acid incorporated in the reaction medium could be time, 3 h; M.Wt of chitoasn, 50.000, [Formic acid]; 0.4 ml/1 g chitosan.
390 K. El-Tahlawy et al. / Carbohydrate Polymers 63 (2006) 385–392

absence of formic acid, this could be associated with the 35

condensation reaction was carried out on the surface of
chitosan molecules, in absence of formic acid, while in the 30
presence of formic acid, the chitosan molecules are completely

Graft yield %
soluble in water which increase the number of accessible amino 25
groups to react with the carboxyl groups of b-CD citrate.
20
5.3. Effect of reaction temperature
15
It is well known that condensation reaction between the
carboxyl groups of b-CD citrate and the amino groups of 10
chitosan is highly dependent on the reaction temperature. Fig. 8 1:10 1:15 1:20 1:25
Material - to - Liqur Ratio
shows the effect of reaction temperature as a function of
grafting percent, expressed as G.Y. %, when the grafting Fig. 9. Effect of material-to-liqur ratio on the graft yield % of CD/grafted
reaction was carried out in the presence and absence of formic chitosan. [CD-citrate]; 0.6 g (5.36 mequiv free carboxyl); [Chitosan], 0.54 g
acid. In the absence of formic acid, the grafting reaction is (6.7 mequiv free amino), M.Wt. of chitosan; 50.000, [Formic acid]; 0.4 ml/1 g
directly proportional to the reaction temperature, since raising chitosan; temperature, 100 8C; time, 3 h.
the reaction temperature from 80 to 140 8C is accompanied by the data (Fig. 9) that, using material to liquor ratio of 1:15
a significant increase in the G. Y. % from 1.72 to 14.84%, such represents the optimum condition for the grafting reaction.
increase in the G. Y. % may be due to (1) increasing the extent Using liquor ratio higher than 1:15 leads to a decrement in the
of diffusion of b-CD citrate molecules onto the vicinity of graft yield %, which could be ascribed to the dilution of the
chitosan molecules, (2) increasing the swellability of chitosan reaction medium.
molecules and (3) higher condensation efficiency. While in the
presence of formic acid, raising the reaction temperature from
80 to 100 8C is accompanied by increasing the G.Y %. Further 5.5. Effect of molecular weight of chitosan
increase above this limit is followed by a decrease in the G.Y.
%. This behavior could be attributed to formation of N-chitosan The effect of the molecular weight of chitosan molecules
formate rather than reaction with b-CD citrate according to the incorporated in the reaction medium on the graft yield percent
following equation: is shown in Fig. 10, when the grafting reaction was carried out
using chitosan, 0.54 g (6.7 mequiv free amino group) of three
O100 C
RH3 NCKOOCH ÿÿÿ
/ÿ RKNHCOH C H2 O different molecular weights (50,000, 30,000, 1500), b-CD
citrate, 0.6 g (5.485 mequiv carboxyl), 0.4 ml of formic acid/
1 g chitosan using material to liquor ratio 1:15 at 100 8C for
5.4. Effect of material to liquor ratio
3 h. It is obvious from Fig. 10 that, the molecular weights of
The relation between material to liquor ratio and the grafting chitosan play an important role in the grafting process. The
reaction, expressed as G.Y. %, was observed in Fig. 9, when extent of the grafting (G.Y %) is inversely proportional to the
the grafting reaction was carried out using [chitosan], 0.54 g molecular weight of chitosan, i.e. decreasing the molecular
(6.7 mequiv Free amino group), b-CD citrate, 0.6 g weight of chitosan is accompanied by a significant increase in
(5.485 mequiv carboxyl) at 100 8C for 3 h. It is clear from the graft yield %, since the extent of the grafting % is obeying

80
20
70

15 60
Graft yield %
Graft Yield %

50

10 40

30

5 20
Undissolved chitosan
10
Dissolved chitosan
0 0
80 100 120 140 0 10000 20000 30000 40000 50000
Temperature °C Chitosan Molecular Weight

Fig. 8. Effect of temperature on the graft yield % of CD/grafted chitosan [CD— Fig. 10. Effect of molecular weight of chitosan on the graft yield % of
citrate]; 0.6 g (5.36 mequiv free carboxyl); [Chitosan], 0.54 g (6.7 mequiv free CD/grafted chitosan. [CD-citrate]; 0.6 g (5.36 mequiv free carboxyl);
amino), M/L, ratio; 1:20, M.Wt. of chitosan; 50.000, [Formic acid]; 0.4 ml/1 g [Chitosan], 0.54 g (6.7 mequiv free amino), M/L ratio; 1:15, [Formic acid];
chitosan; time, 3 h. 0.4 ml/1 g chitosan; temperature, 100 8C; time, 3 h.
 K. El-Tahlawy et al. / Carbohydrate Polymers 63 (2006) 385–392 391

Table 1
Minimal inhibition concentration of recorded pathogenic bacterial strains

Kind of microorganism Strain Minimal inhibition concentration MIC, mg CD-citrate

M.wt of chitosan M.wt of chitosan linked CD
50,000 30,000 1500 50,000 30,000 1500
Gram negative A. hydra 25 25 12.5 12.5 12.5 12.5 R
E. coli 50 50 12.5 25 12.5 12.5 R
P. fragi 50 50 12.5 25 12.5 12.5 R
Gram positive S. aureus 25 25 12.5 12.5 12.5 12.5 R
B. megaterium 25 25 12.5 12.5 12.5 12.5 R
B. cereus 25 25 12.5 12.5 12.5 12.5 R

R: resistance up to 100 mg.

the following trends: 1500O30,000O50,000. The increase coupling with b-CD citrate improves its antimicrobial activity,
in the graft yield % by decreasing the molecular weight of expressed in the decrement in the MIC, compared with
chitosan chains could be attributed to (1) lower viscosity, (2) chitosan. This behavior appears especially in chitosan of
higher mobility of chitosan molecules, (3) less sterric higher molecular weights. This behavior could be attributed to
hindrance, and (4) higher accessibility of chitosan molecules (1) enhancing the dissolution of chitosan and (2) the creation of
to react with b-CD citrate. carboxyl groups along with chitosan molecules serve in the
dissolution of phospholipids area and causing leakage
the intercellular components and finally the death of the
5.5.1. Utilization of b-cyclodextrin-grafted chitosan as a new
antimicrobial agent microorganisms (Adams & Hall, 1988). The above finding
Chitosan, a cationic antimicrobial agent (Lim & Hudson, illustrated too why CD/grafted chitosan is has antimicrobial
2003; Lim & Hudson, 2004), has been widely used, particularly activity against both gram positive and gram negative bacteria,
for external disinfection and the target site of the cationic while chitosan is a good antimicrobial agent for gram positive
biocides is the cell envelope of bacteria. only.
Table 1 shows the antimicrobial activity, expressed as
minimal inhibition concentration (MIC), of chitosan and b-CD/
grafted chitosan against two kind of mcro-organisms, gram 6. Conclusions
positive, such as S. aureus, B. megaterium, B. cereus and gram
negative, such as A. hydra, E. coli and P. fragis. We have synthesized b-cyclodextrin-grafted chitosan using
The data indicated that, regardless of the chitosan used, the a reaction between b-cyclodextrin citrate and chitosan for
minimum inhibition concentration (MIC) decreases as the preparation of a new antimicrobial agent. b-CD citrate was
molecular weight of chitosan decrease. The mechanism behind prepared by esterifying b-CD with citric acid in the presence/or
the antimicrobial activity of chitosan can be summarizing as absence of SHP as a curing catalyst using a semi dry reaction
follow: (1) the cationic nature of chitosan binds with sialic acid method. It could be concluded that the optimum condition for
in phospholipids, consequently restraining the movement of preparation of a reactive b-CD-citrate is CA; 2 mole/1 mole
microbiological substance. (2) Chitosan molecules penetrate CD, reaction time; 2 h, reaction temperature; 100 8C, and
into the cells of micro-organisms and prevent the growth of the material-to-liquor ratio 1:0.6. b-CD citrate ‘having carboxyl
cells by prohibiting the transforming of DNA to RNA. Based content of 273 mequiv/100 g ‘ was allowed to react with
on the above knowledge, the antimicrobial activity is generated chitosan via a condensation reaction. It is also concluded that
from the free amino groups in chitosan in an aqueous acidic the molecular weight of chitosan, reaction temperature, and
environment. It’s well known that, the MIC of chitosan to formic acid concentration are representing the major affecting
impart antimicrobial properties depends upon the molecular factors for the condensation reaction.
weight, degree of deacetylation, concentration of chitosan and
kind of functional groups introduced to the chitosan backbone References
by chemical modification. From the above finding, the
enhancement in the antimicrobial activity of chitosan of Adams, M. R., & Hall, C. J. (1988). International Journal of Food Science and
Technology, 23, 287–292.
lower molecular weight could be attributed to the penetration
Bender, M. L., & Komiyama, M. (1978). Cyclodextrin chemistry. Berlin:
ability of chitosan to the cell wall of microorganisms and Springer.
consequently enhancing the accessibility of the amino groups Buschmann, H.-J., Knittel, D., & Schollmeyer, E. (2001). Journal of Inclusion
of chitosan to prohibit the growth of microorganism. This Phenomena and Macrocyclic Chemistry, 40(3), 169–172.
behavior reflects the decrement in the MIC by decreasing the Chiu, S.-H., Chung, T.-W., Giridhar, R., & Wu, W.-T. (2004). Food Research
International, 37(3), 217–223.
M.Wt of chitosan.
Cox, G. S., Turro, N. J., Yang, N. C., & Chen, M. (1984). Journal of the
The data indicates also that, b-CD citrate has no Chemical Society, Chemical Communication, 106, 422.
antimicrobial effect against the studied microorganisms. On Davidson, P. M., & Parish, M. E. (1989). Journal of Food Technology ,
the other hand, chemical modification of chitosan via it’s 148–155.
392 K. El-Tahlawy et al. / Carbohydrate Polymers 63 (2006) 385–392

Deratani, A., & Poepping, B. (1995). Macromolecular Chemistry and Physics, Mizobuchi, Y., Tanaka, M., & Shono, T. (1980). Journal of Chromatography,
196, 343. 194, 153.
Devlieghere, F., Vermeulen, A., & Debevere, J. (2004). Food Microbiology, Seo, T., Kajihara, T., & Iijima, T. (1987). Macromolecular Chemistry and
21(6), 703–714. Physics, 188, 2071.
El-Tahlawy, Kh. F. (1999). Colourage, 46(5), 21–26. Sortino, Salvatore, Giuffrida, Salvatore, Fazio, Sandro, & Monti, Sandra (2001)
Furusaki, E., Ueno, Y., Sakairi, Y., Nishi, N., & Tokura, S. (1996). . New Journal of Chemistry, 25(5), 707–713.
Carbohydrate Polymers, 20, 29. Szejtli, Jozsef (2003). Starch/Staerke, 55(5), 191–196.
Gaffar, Mohammed A., El-Rafie, Safaa M., & El-Tahlawy, Khaled F (2004). Takashima, Yoshinori, Nakayama, Tomofumi, Miyauchi, Masahiko, Kawa-
Carbohydrate Polymers, 56(4), 387–396. guchi, Yoshinori, Yamaguchi, Hiroyasu, & Harada, Akira (2004).
George, A. F. (1992). Chitin Chemistry , 64–75. Chemistry Letters, 33(7), 890–891.
Kobayashi, N., Ueno, A., & Osa, T. (1981). Journal of the Chemical Society, Vogel, A. I. (1966). Elementary practical organic chemistry: Part 3,
Chemical Communication, 340.
quantitative organic analysis (2nd ed.). London: Longman Group Ltd. p.
Lee, K. Y., Park, W. H., & Ha, W. S. (1997). Journal of Applied Polymer
652.
Science, 63, 425.
Voncina, B., & Le Marechal, A. (2005). Journal of Applied Polymer Science,
Lim, Sang-Hoon, & Hudson, Samuel.M. (2003). Journal of Macromolecular
96, 1323–1328.
Science, Polymer Reviews, C43(2), 223–269.
Lim, Sang-Hoon, & Hudson, Samuel.M. (2004a). Carbohydrate Polymers, Xueju, Xie, & Qiang, Liu (2004). Starch/Staerke, 56(8), 364–370.
56(2), 227–234. Yang, C. Q., & Wang, D. (2000). Textile Research Journal, 70(7), 615–620.
Lim, Sang-Hoon, & Hudson, Samuel M. (2004b). Carbohydrate Research, Yang, Charles. Q., Wang, Xilie, & Kang, In-Sook (1997). Textile Research
339(2), 313–319. Journal, 67(5), 334–342.
Martel, B., Michael, D., Gregorio, C., Weltrowski, M., Bourdonneau, M., & Zhang, X., Wang, Y., & Yi, Y. (2004). Journal of Applied Polymer Science,
Morcellet, M. (2001). Journal of Applied Polymer Science, 39(1), 169–176. 94(3), 860–864.