antibacterial activity of chitosan chemically modified with new technique

Antibacterial Activity of Chitosan 121

Antibacterial Activity of Chitosan Chemically Modified with New Technique

M.S. Mohy Eldin1*, E.A. Soliman1, A.I. Hashem2, T.M. Tamer1

1Polymer Materials Research Department,Advanced Technologies and New Materials Research Institute (ATNMRI),Mubarak City for Scientific Research and Technology Applications (MUCSAT),New Borg El-Arab City 21934, Alexandria, Egypt.2Organic Chemistry Department,Faculty of Science, Ain-Shams University,Cairo, Egypt.*Corresponding author: [email protected]

Received 01 May 2008; Accepted 25 November 2008; published online 23 December 2008

The antibacterial activity of chitosan was chemically modified by introducing further amino groups to the backbone of chitin using parabenzoquinone (pBQ) as activation agent and ethylene di amine (EDA) as amino groupsource. The aminated chitin was further deacetylated to obtain finally chemically modified chitosan with highercontent of amine groups. Factors affecting both activation process, and amination process amine grafting,have been studied through following its effect on the amount of introduced amine groups. The success ofgrafting process has been confirmed using FT-IR and TGA analysis. The antibacterial activity of the modifiedchitosan was tested on four different bacterial strains; two gram negative (Escherichia coli, pseudomonasaeruginosa), and two gram positive (Bacillus cereus, Staphylococcus aureus). It was found that the antibacterialactivity of the modified chitosan is better than the native one, and increases by increasing the amount ofintroduced amine group (degree of the grafting), the degree of deacetylation. Lowering the molecular weightwas found of negative effect. On the other hand, The Cytotoxicity activity test using Caco 2 cell line shows asafety result. As chitosan, modified chitosan show potential bactericidal activity along all strain examinedspatially on the gram negative bacteria (Escherichia coli, pseudomonas aeruginosa). Finally, the modifiedchitosan shows higher solubility, almost double, at pH range from 5 to 6 comparing to chitosan it self. © Societyfor Biomaterials and Artificial Organs (India), 20080501-18.

Trends Biomater. Artif. Organs, Vol 22(3), pp 125-137 (2008) http://www.sbaoi.org

Introduction

Microorganisms play an important role in ourlife it was dispersed in air, water and soil .Someof these organisms are human friendly and wasuseful in several fields, in the other hand, theother type was danger and may cause severalproblems in various fields.

In order to promote the general health andinhibit the infection of these bad organismsseveral materials was being employed asantimicrobial agents.

One of the most interesting substance ischitosan, chitosan that attract the attention of

the scientist in last few years this because ofits special properties including biodegradability,biocompatibility and non toxicity, it was a naturalpolysaccharide composed of linear poly β-(1-4)-2 deoxy-D glucopyranose (1-3).

Chitosan has a broad rang application onseveral fields such as water treatment, spill oilremoval, drug delivery, tissue engineering,wound healing, food preservation, enzymeimmobilization.

Chitosan exhibits an antimicrobial activityagainst abroad range of microorganisms.There are several mechanisms which besuggested to explain this activity one of them

126 M.S. Mohy Eldin, E.A. Solima, A.I. Hashem, T.M. Tamer

was related this antimicrobial activity to thebasic nature of the polymer and the aminecontent. Chitosan is a positively chargedmolecule and the target of its antimicrobialaction is the negatively charged cell wall ofbacteria, where it binds and disrupts the normalfunctions of the membrane, e.g. by promotingthe leakage of intracellular components andalso by inhibiting the transport of nutrients intothe cells (4-6). The antimicrobial activity ofchitosan is well known against a variety ofbacteria and fungi coming from its polycationicnature (7).In another suggesting, chitosan wasbinded with DNA and inhibition of mRNAsynthesis occurring via the penetration ofchitosan into the nuclei of the microorganismsand interfering with the synthesis of mRNA andproteins in the final inhibit the bacterial growth.Furthermore, chitosan is a biomaterial widelyused for effective delivery of manypharmaceuticals (8). Accordingly, chitosan maybe suitable for incorporating other antipyreticfor the preparation of long-acting antibacterialwound dressing.

The antimicrobial activity of chitosan increaseswith decreasing pH (5, 9-12). This is due to thefact that the amino groups of chitosan becomeionized at pH below 6 and carry a positivecharge. Unmodified chitosan is notantimicrobially active at pH 7, since it does notdissolve and also since it does not contain anypositive charge on the amino groups) (13, 14).The antimicrobial activity of chitosan alsoincreases with increasing degree ofdeacetylation, due to the increasing number ofionisable amino groups (14). Severalapproached was done to increase theantimicrobial activity of chitosan by introduceamino group, on the primary amino groups ofthe back bone of the chitosan polymer chainsbut it was failed (15). The authors explained theobtained results to the position of the introducedamine groups.

In this work, we try to increase the antimicrobialactivity of chitosan via increase the amino groupon the polymer back bone by attaching aminogroup directly on the hydroxyl group ofpolysaccharide. A new technique has been usedto avoid the consumption of the original aminogroups of the chitosan as sites of grafting, so

chitin was first grafted with amino groups inseparate step, then it was deacetylated to havethe aminated chitosan.

Materials and Methods

Materials

Sodium chloride (Purity 99.5%, M.wt.58.44) wasobtained from Sigma-Aldrich Chemicals Ltd.(Germany).Acetic acid (Purity 99.8%,M.wt.60.05) was obtained from Sigma- AldrichChemicals Ltd. (Germany).Chitin from crabshells, practical grade was obtained fromSigma- Aldrich Chemicals Ltd. (Germany).ParaBenzoquinone (Purity 99%, M.wt.108) wasobtained from Sigma- Aldrich Chemicals Ltd.(Germany).Sodium hydroxide pellets (Purity 99-100 %, M.wt.40) was obtained from Sigma-Aldrich Chemicals Ltd. (Germany).Ethylene diamine (Purity 99%, M.wt.60) was obtained fromAlfa aesar, (Germany). Coomassie Brilliant blueG250,Ortho Phosphoric acid (Purity 85%,M.wt.98) ADWIC, (Egypt)Dehydrate alcohol(Purity 99.9%, M.wt.46.07). International co forsupp&med. Industries, (Egypt)Tryptone powderBacteriolgical Grade, Biobasic inc.(Canda).Agarbacteriological Grade, Gibcobri,(Scotland)YEAST EXTRACT, bacteriologicalGrade, Biobasic inc. (Canda).

Microorganism

Four bacteria were tested for the antimicrobialactivity of chitosan and chitosan modified. theseincluded two gram negative bacteria(Escherichia coli, pseudomonas aeruginosa )and anther two gram positive bacteria (Bacilluscereus, Staphylococcus aureus ). Bacteria wasincubated overnight at 37 oC in nutrient broth(peptone 1%, yeast extract 0.5 %, NaCl 1% andpH 5.5)

Methods

Preparation of Modified chitosan

The modification of chitosan was performedinto three steps namely; chitin activation,amination and finally deacetylation

In the first step, chitin activation,4 gm of chitinwas dispersed in 50 ml of distilled water atdefined pH, dissolved in it pBQ and stirred for 6


hr. the activated chitin (AC ) was separated andwashed well with distilled water .

In the second step, chitin amination, the (AC)was dispersed in 50 ml of distilled waterdissolved in it ethylene di amine and stirred for6 hr. the aminated modified chitin (AMch) wasseparated and washed well with distilled water.

The last step, aminated chitin deacetylation,was performed According to Rigby and Wolfarnmethod (17, 18), the aminated chitin derivative

chitin and modified chitosan were carried outusing Fourier Transform InfraredSpectrophotometer (Shimadzu FTIR - 8400 S,Japan) to confirm occurrence of activation andamination reactions.

Thermal gravimetric Analysis (TGA)

Analysis by TGA of chitin, activated chitin,aminated chitin and modified chitosan werecarried out using Thermogravimetric Analyzer(Shimadzu TGA –50, Japan) to evidencechanges in structure as a result of activationand amination reactions.

Scanning Electron Microscopic Analysis (SEM)

Scanning of chitin, activated chitin, aminatedchitin and modified chitosan were carried outusing Analytical Scanning Electron Microscope(Joel Jsm 6360LA, Japan) to explore changesin the morphology, resulted from activation andamination processes of the polymer matrixsurface.

Solubility test

Solubility test of the sample was performed bydissolving a weighted sample in 2% acetic acidand stirred at room temperature at the desiredtime, and then the sample was filtrated, driedand weighted. The solubility was determinedby the following equation (1):

Preparation of reagent

Coomassie Brilliant Blue G-250(100 mg) wasdissolved in 50 ml 95% ethanol; to this solution100 ml 85 % (w/v) phosphoric acid was added.The resulting solution was diluted to a finalvolume of 1 liter. Final concentrations in thereagent were 0.01 % (w/v) Coomassie BrilliantBlue G-250, 4.7 % (w/v) ethanol and 8.5 % (w/v)phosphoric acid.

Bio evaluation of modified chitosan

Antimicrobial activity (21, 22)

The measurement of the antimicrobial activity

was treated with 40 % aqueous solution ofNaOH at 120-150 oC for 6 hr. The obtainedaminated chitosan (AMC) was separated andwashed well with distilled water.

Preparation of different MW of aminated chitosan

Aminated chitosan was degraded by the methodof acetic acid hydrolyzes referenced from Chenet al (19). Aminated chitosan was dissolved in5 % aqueous acetic acid incubated at 50 oC for0, 3, 6, 16, 24 and 48 hr and then the centrifuged(5000 rpm) for 20 min. the supernatant wasadded to 4N aqueous NaOH. The sedimentwas filtrated and sequentially rinsed in waterand ethanol and dried at 50 oC.

Characterization of modified chitosan

Qualitative determine of -NH2 group, TheBradford assay (20)

This method is based on the observation thatthe Coomassie Brilliant Blue G-250 exists intwo different color forms, red (365 nm) andblue(595 nm) the red form is converted to blueform upon binding of the dye to the auxochromegroup –NH2 .

Changes in the amine groups content wasmonitored as follow: 100 µl of 1% of chitosan ormodified chitosan solution in the acidic acidsolution (2%v/v) was added to 900 µl of theBradford reagent in a small test tube. Theabsorbance of the mixture was measured at595 nm

Infrared Spectrophotometric Analysis (FT-IR)

Analysis by FTIR Spectroscopic investingstructure for chitin, activated chitin, aminated


of the chitosan and modified chitosan was doneaccording to (21, 22). Briefly, the bacteria wereinoculated in a L.B medium (1 % peptone, 0.5%yeast extract and 1% NaCl) the inoculation wasconducted at 37 oC for 24 hr with shaking theobtained bacterial suspension was diluted withthe same peptone medium solution to 100times.

0.1 ml of diluted bacteria suspension wascultured in 5 ml liquid peptone medium contain0.1 ml of chitosan solution 1% has beensterilized under 121 oC for at least 20 min, theinoculated medium was maintained at 37 oCfor 18 hr with shaking. The no of bacteria wascounted by the ultraviolet absorbance of culturemedium at 620 nm.

Bactericidal activity (23)

The bactericidal activity of chitosan and itsderivatives were measured by enumeration ofviable organism, the bacteria was growth innutrient broth (peptone 1%, yeast extract 0.5%,NaCl 1%, pH 5.5) incubated overnight at 37 oC.the cultures obtained were diluted withautoclaved nutrient. One milliliter of the cellsuspension was added to 1 ml of 1 % chitosanderivatives that has been autoclaved at 121 oCfor 20 min, the samples were removed after 0,1, 2, 3, 4, 5, 6hr respectively. Portion were spreadon triplicate nutrient agar plates, which wereincubated at 37 oC for 24 h, and the numbers ofcolonies were counted.

Cytotoxicity test (24)

The evolution of the Cytotoxicity test was donewith the direct contact method. The culture ofmouse fibroblasts, with Ca 0.6 x 10 5 cells eachwere established on Petri-dishes on contact ofthe sample. After 24 hr the old culture mediumis removed and a new one will added. The cellwill be recorded after 120 hr and the viability willbe calculated.

Results and Discussion

A- Preparation of modified chitosan

The structure of chitosan is useful to thesynthetic chemist interested in site selectivemodifications. Such modifications have resulted

into several derivatives of chitosan with distinctproperties and applications. The presence ofmultiple nucleophilic groups within the chitosanbackbone requires suitable synthetic protocolin order to obtain the desired selectivity. Thesynthetic transformation steps performed areoften relatively simple, exploiting the differencein the nuleohilicities of primary amino group (atC-2) versus the two hydroxyl groups (at C-3 andC-6). From the literature it was found that thegreater reactivity of amino groups rather thanhydroxyl groups. However, the degree ofselective substitutions varies greatly upon thereaction conditions. Several derivatives ofchitosan was prepared to increase the aminecontent of chitosan via grafting it on the primaryamino groups of the back bone of the chitosanpolymer chains it self but the antibacterial activityof the product was decrease (15). In this workwe will introduce external amino group to thechitosan on the hydroxyl groups rather thanamino groups starting from chitin using paraBenzoquinone (pBQ) as activating agent andEthylene Di Amine (EDA) as source of theintroduced amino groups. As shown in thefollowing schema (figure 1)

Activation step

In this part we was activated the hydroxyl groups(C-3and C-6) starting from chitin using pBQ.

Effect of the para Benzoquinone concentration

The effect of the pBQ concentration on theheterogeneous attraction of the hydroxyl groupof chitin to the carbonyl group of the pBQ wasinvestigated. The molar ratio of chitin relative topBQ ranged from 1:1/16 to 1:2 (Figure 2). Fromthe figure it was shown that increase the numberof introduced amine groups by increase themolar ratio reached to 1:1/4 and then relativelystable by further increase of the molar ratio. Thehydrophobic nature of chitin may be explainedthis trend which indicated occurring of theactivation reaction only on the surface.

Effect of reaction’s pH on the activation step

The pH of the reaction medium of activationstep was studied from 7.5 to 11 (figure 3). Itwas found noticable increase of the potential of


the activation step by increasing the pH. Therises of the pH increase the electrohilicity ofhydroxyl groups which attack the carbonyl group.The reaction is electrophilic substitutionprocess which activated in the basic medium,and for this reason the authors ignore the acidicpH during the test.

Effect of reaction’s temperature on the activationstep

The effect of the reaction temperature wasstudied from 20 to 60 oC. It was observed thatthere is no effect on the activation step by raisingthe temperature from 20 oC to the 40 oC. Slightincrease at the 50 oC has been noticed. Again,these results paid our attention to the possibilityof occurring the reaction on the surface of chitindue to its hydrophobic nature.

Effect of reaction’s time on the activation step

The effect of the time of the activation step wasstudied from 30 minute to 6 hr (figure 4). It wasfound increase the amine content on thepolymer from 30 minute to the four hour andthen leveling off With further increase of reactiontime. This trend could be referred to theconsumption of hydroxyl groups on the surfaceof the activated chitin within four hours reactiontime.

Amination step

In this step external amine groups wereintroduced to the structure of activated chitin (AC)

by grafting ethylene di amine, as a source ofamine groups, to the second carbonyl group ofthe pBQ as indicated in figure (1).

Effect of ethylene di amine concentration on theamination step

The effect of the molar ratio of the ethylene diamine relative to the para Benzoquinone attachéto activated chitin (AC) was studied in the rangefrom 1:1/8 to 1: 2 (figure 5). It was observed asignificant increase in the grafting of aminegroup by increasing the molar ratio till 1:1. Thisbehavior could be attributed to increase thepossibility of reaction between amine groupsin the EDA and the carbonyl groups of the pBQ.Further increase of molar ratio over 1:1 leads todecrease the introduced amine groups. Crosslinking ability of EDA between two pBQ activationcenters could be an explanation to reduce theterminal induced amine groups.

Effect of reaction’s temperature on theamination step

The temperature profile of the reaction wasstudied in the range from 30 to 60 oC (figure 7).The amine content was found dependent onthe reaction temperature, reversing to theactivation step with pBQ, which the introducedamount increased gradually with temperature.Almost 40% of amine content increment hasbeen obtained by increasing the temperaturefrom 30 to 60oC. This behavior could beexplained by the difference of the polymer matrixsurface nature which is hydrophobic in the

Fig 1: Schematic diagram for synthesis of aminated chitosan


activation step with pBQ and turns to behydrophilic in the amination step. Thishydrophilic nature of the surface affected byrising of temperature, during the reaction with

EDA through increasing its rate of diffusion andconsequently the rate of reaction with pBQactivated centers.

Effect of reaction’s time on the amination step

The reaction time was tested from 30 min to 4hr (figure 6). It was found that increase theamine content of the polymer from 30 minuteto the first hour and then decrease with furtherincrease of reaction time. This trend could bereferred to the consumption of most pBQactivated centers on the surface of the activatedchitin in the first hour. Now the surface iscrowded with introduced amine groupscreating sterric hindrance and increasing thepossibility of the reaction between the freeterminal amine groups and the left pBQ freecenters on the surface. This behavior leadsfinally to reduce the free terminal amine groupsintroduced to activated chitin.

0.4

0.45

0.5

0.55

0.6

0.65

0.7

0.75

0 2 4 6 8 10 12pBQ (mM)

abso

rban

ce

Figure 2: Effect of pBQ concentration on theintroduced amine groups reaction. (Reactiontemperature 20oC and pH 10 for 6hr)

0.40.450.5

0.550.6

0.650.7

0.750.8

7 8 9 10 11 12Reaction pH

abso

rban

ce

Figure 3: Effect of the pH of the activation stepon the introduced amine groups reaction. (4mMof pBQ at 20 oC for 6 hr)

0.20.25

0.30.35

0.40.45

0.50.55

0.60.65

0 2 4 6 8Reaction time (hr)

abso

rban

ce

Figure 4: Effect of the activation process timeon the introduced amine groups reaction(4mM of pBQ at 20 oC and pH 10)

Table 1: the Effect of the degree of amination on the antimicrobial activity of modified chitosanagainst different microorganisms

Maximum inhibition pBQ :chitin

ratio

Bradford

absorbance E.coli Bacillus pseudomonas stapylococcus

Chitosan control 0.095 0.626298 0.35689 0.6873178 0.67047076

0.0935 0.105 0.774394 0.462014 0.8053936 0.69258203 0.187 0.114 0.798616 0.573322 0.8622449 0.7318117 0.374 0.251 0.831834 0.582155 0.8717201 0.85805991 0.545 0.351 0.887889 0.623675 0.9234694 0.94293866 0.747 0.355 0.907958 0.767668 0.9613703 0.97360913


C. Evaluation of the antimicrobial activity of themodified chitosan

Amine groups of chitosan play an essential roleon its antimicrobial activity. This role wasobserved as increase the potential inhibition ofchitosan as increase the degree ofdeacetylation of it. The increase of the aminogroup substitution on the chitosan chainsincreases the positively cationic nature ofchitosan in acidic solutions which lead toincrease the chance to an interaction betweenthe chitosan and the negatively charge on thecell walls of the microorganisms. This

Figure 5: Effect of ethylene di amineconcentration on the introduced aminegroups reaction (30 oC for 6 hr)

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 1 2 3 4EDA (mM)

abso

rban

ce

Figure 6: Effect of reaction time on theintroduced amine groups reaction. (1.8 mM ofEDA at 30 oC)

0.3

0.35

0.4

0.45

0.5

0.55

0.6

0.65

0 1 2 3 4 5Reaction time (hr)

abso

rban

ce

Table 2 : Effect of molecular weight of modified chitosan on itsantimicrobial activity against different microorganisms

Maximum inhibition vicusity no

E.coli Bacillus pseudomonas stapylococcus 5.42262 0.863 0.9567 0.6895 0.6861626 7.744904 0.74 0.8154 0.60714 0.668331 9.586797 0.713 0.6025 0.4949 0.6184023 15.3021 0.617 0.5972 0.49708 0.5920114 16.48861 0.482 0.4761 0.41472 0.4835949 17.06351 0.412 0.3595 0.33455 0.3951498

Table 3: Effect of the media pH on the antimicrobial activity of modifiedchitosan against different microorganisms

Maximum inhibition media pH E.coli Bacillus pseudomonas stapylococcus 8 0.009554 0.031802 0.01312 0.03495 7 0.100318 0.099823 0.100583 0.096291 6 0.681529 0.541519 0.50656 0.474322 5 0.762739 0.660777 0.701895 0.616976 4 0.98328 0.749117 0.915452 0.674037

Table 4: the solubility percent of the chitosanand amino chitosan in different pH

pH Chitosan

solubility

Modified chitosan solubility

4 97.2 99 5 41.7 81.1 6 17.9 29.6 7 0 3.9 8 0 0


observation stimulate Jukka Holappa (15) toincrease the amine content of chitosan viagrafting with quternized betainates. But the newmodified chitosan doesn’t give the requiredresult .this was explained by the consumptionof the more reactive amine group with anther

less reactive one. From this assay it wasconfirmed on the importance of the aminegroups attached to backbone of the polymerchain on its antimicrobial activity.

Figure 7: Effect of reaction’s temperature onthe introduced amine groups reaction (1.8 mMof EDA at for 6hr)

0.20.25

0.30.35

0.40.45

0.50.55

0.60.65

0.7

20 30 40 50 60 70Reaction temerature

abso

rban

ce

Figure 8: Effect of the degree of deacetylationon the antimicrobial activity of modifiedchitosan against different microorganisms,♦♦♦♦♦the Bradford absorbance of NH2 group, %Escherichia coli , % Bacillus cereus , *pseudomonas aeruginosa, “ Staphylococcusaureus

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

3 5 7 9 11time of deacetylation

max

inhi

btio

n

0

0.05

0.1

0.15

0.2

0.25

amin

e ab

sFigure 9: Effect of type of diamine used on theantimicrobial activity of chitosan and aminatedchitosan with EDA (7x) and HMDA (11x)againstdifferent microorganisms (¨ the Bradfordabsorbance of NH2 group % Escherichia coli, %Bacillus cereus, * pseudomonas aeruginosa, “Staphylococcus aureus).

00.20.40.60.8

11.2

0 7x 11xno of separated atom

max

miu

m in

hibt

ion

0

0.05

0.1

0.15

0.2

amin

e ab

s

Figure 10: FT-IR spectra of : (a) chitin (b) activatedchitin, (c) aminated chitin and (d) modified

Table 5 : Effect of solvent type on antimicrobial activity of aminatedchitosan against different microorganisms.

Maximum inhibition solvent E.coli Bacillus pseudomonas staphylococcus ascorbic 0.737873 0.699548 0.566563 0.564677 cetric 0.919776 0.875113 0.787152 0.766998 formic 0.964552 0.976471 0.902477 0.922886 acetic 0.951493 0.928507 0.883901 0.876451


Effect of the introduced amine groups amounton the antimicrobial activity

The degree of chitosan modification withexternal amine groups was studied (table1). Itwas found that antimicrobial activity of the

modified chitosan increased as a result ofincreases grafted amine groups. This leadsconsequently to increase the positive chargeson the polymer. The selective grafting of thisexternal amine groups on the hydroxyl groupsof chitosan, increased its amine content in

Figure 11: Scan electron microscope of (a) chitin, (b) aminated chitin, (c) chitosan and (d) aminatedchitosanTable 6: Bacteriocidal activity of chitosan againstE. coli, Bacillus cereus, P. aeruginosa, and S.aureus

Number of colonies Mixing time (hr) E.coli Bacillus Pseudomonas S.aureus

0 561 602 460 496 1 126 415 44 245 2 85 360 25 148 3 38 338 12 106 4 27 255 4 78 5 23 185 3 73

Table 7: Bactericidal activity of modified chitosanagainst E. coli, Bacillus cereus, P.aeruginosa,and S.aureus

Number of colonies Mixing

time (hr) E.coli Bacillus Pseudomonas S.areus

0 494 373 263 451 1 45 102 33 145 2 22 63 26 106 3 5 25 7 69 4 3 21 6 55 5 1 20 4 37


addition to its effective original amine groups.In microbial profile, the powerful effect ofmodified chitosan on the gram negative ratherthan the gram positive bacterium was explainedby the difference on the pathologicalcomposition of the cell wall.

Gram negative bacteria has thick layer ofphospholipids rather than the peptidoglycancomparing to the gram positive which has thicklayer of peptidoglycan. The negative charges ofthe phospholipids enhance the adhesion powerof poly cationic polymer on the cell wall.

Effect of the degree of deacetylation of themodified chitosan on its antimicrobial activity

Effect of the degree of deacetylation of theaminated chitosan on its antimicrobial activityagainst gram negative and positive bacteriawas studied (figure 8). It was found that theantimicrobial activity of the aminated chitosanincreases with increase deacetylation time.This attributed to liberate more amine groupsalong the polymer chain which leading to

Figure 12: TGA analysis of (a) chitin (b) activatedchitin, (c) aminated chitin and (d) amino chitosan

increase the positive charges resulted from theprotonation amino groups in acidic conditions.

Effect of molecular weight of the modifiedchitosan on its antimicrobial activity

Inhibition effect of the molecular weight of theaminated chitosan was studied on differentbacterial species (table 2). The molecularweight of chitosan, as the amino groups, playsa great role in the antimicrobial activity. It wasfound that the antimicrobial activity effect ofaminated chitosan decreased by decrease themolecular weight on both the gram positive andnegative bacteria. In General, the decrease ofthe molecular weight of chitosan increases theantimicrobial activity but this increase waslimited to certain molecular weight, after it theactivity was declined.

This phenomenon was explained as decreaseof the molecular weight of chitosan improvesthe movement of polymer chains in the solutionby decrease the viscosity. Theoretically,Chitosan polymer was plugged the cell wallchannels and inhibited normal metabolism ofthe cell. As far polymer molecular weightdecreases, it escaped through the channel intothe cell.

Effect of the media pH on the antibacterialactivity of modified chitosan

The surrounding pH of the modified chitosanplays an essential role on its applications. Thisacidic pH responsible for solubility by formingthe poly cationic character of the polymer. Basedon this idea, the pH of the culture media wasstudied (table3). It was found that increase ofthe media pH decreased the antibacterialactivity and this activity was limited in neutraland basic pH. This attributed to the solubility ofthe polymer. The solubility of chitosan and the

viability % Total cells X 105 Dead cell

X 105 Live cells

X 105 sample

89.9 7.95 1.2 6.75 Control 86.9 6.15 0.8 5.35 Chitosan 76.9 6.5 1.5 5 aminated Chitosan

Table 8: Cytotoxicity result of chitosan and modified chitosan. (0.6 x 105 cellsincubated for five days)


aminated chitosan was measured as in table4. The improving of the solubility of aminatedchitosan over chitosan it self was attributed tografted amino groups. The aminated chitosansolubility at pH 5.0 to 6.0 is almost double of itsunmodified chitosan counter part.

Effect of the type of amine on the aminationprocess of chitosan

The effect of the separation distance betweenthe amine group and the backbone of thepolymer on the antimicrobial activity was studiedfigure (9). In this way different type of theaminated chitosan was prepared using ethylenedi amine and hexamethylene di amine assources of the induced amine groups. Fromthe figure it was found that the increase thereactivity from chitosan to the modified chitosan(using ethylene di amine) which direct result toincrease the amine groups along the polymerchain. By replacing the ethylene di amine withhexamethylene di amine the antimicrobialactivity decrease slightly as a result of increasethe distance, the chain polymer backbone thisbehavior could not be referred to the amount ofintroduce amine groups science it has not beaffected

This study showed that increase the distancebetween the amine groups and the backbone(in case of hexamethylene di amine) decreasedthe effectively of the amine group. These resultsrepresent the role of the pyranose ring on themechanism of action. It is also confirmed thepowerful activity of the original amine groupswhich attached directly on the chain’s backboneover other amine groups. In addition, it explainsthe decrease of the antibacterial activity of theN-grafted chitosan because thesemodifications replaced the more active aminegroups with less active ones.

Effect of solvent type on the antibacterial activityof modified chitosan

The effect of the aminated chitosan solvent onits antimicrobial activity was studied using fourdifferent organic acids namelly acetic, formic,citric and L.ascorbic acid (table 5). It was foundthat the potential of the activity was decreasedin order formic acid > acetic acid > citric acid >>

L.ascorbic acid. Increase the potential of theformic rather than acetic at the sameconcentration attributed to decrease themolecular weight and size which enable it toseparate the polymer chain and increased itssolubility other than acetic acid. In the other handthe bivalent character of citric acid decrease itssolubility in the case of L. ascorbic acid itenhance the metabolism of the microorganismand soften the effect of potential activity ofchitosan

Bactericidal activity of modified chitosan

The bactericidal activity of chitosan andaminated chitosan was studied on gramnegative and gram positive, table 6 and 7. Thedecreases of the number colonies by timeindicate that they are not only stopping thegrowth of the bacteria but also killed it. Fromtables it was observed a higher killing potentialof aminated chitosan over chitosan it selfaccording to the rate of declining of the coloniesnumber.

It was found that increase the inhibition of gramnegative rather than gram positive bacteriawhich attributed to the physiological structureof difference the cell wall of the two strains.

This result confirmed the rapture of the cell wallmechanism rather than nuclear proteininteraction mechanism. The interaction of theamine groups of modified chitosan with the cellwall decrease its selective permeability whichleds to leakage of the intracellular substance,such as electrolytes,UV-absorbing material,protein, amino acids, glucose, and lacticdehydrogenase. As a result, chitosan andmodified chitosan inhibit the normalmetabolism of microorganisms and finally leadto death of this cell.C

Cytotoxicity test of modified chitosan

Cytotoxicity of Chitosan and aminated Chitosanwas tested using caco 2 cell line with the directconnect method and the data are shown in table(8 ).

several search was tested and measured thecytotoxicity of Chitosan. Although Chitosanexhibits some concentration dependent


cytotoxicity at high degree of deacetylation, thisvalue still in the save area. In the following testthe viability of the live cell was decreased byincrease the amine group substitution, however,the toxicity of aminated chitosan is negligible.

Table 8 : Cytotoxicity result of chitosan andmodified chitosan . (0.6 x 105 cells incubatedfor five days)

Polymer characterization

FT-IR analaysis

The FT-IR spectrum of the modified chitosanand intermediates were obtained using FTIR-8400S SHIMDZU. Japan. As shown in figure 10,the major difference are the wide peaks at 3431cm-1, (I) corresponding to the stretchingvibration of –NH-2 and OH groups become moresharp at modified chitosan result fromsubstitution of –OH groups with –NH2 groups.

Absorption band intensity at 1560, 1649 cm-1(II) corresponding to amide bands have beenincreased in (AC )via introduce further carbonylgroups of pBQ (curve b)then return to normalintensity at aminated chitin (curve c) as a resultof reaction the carbonyl groups with aminegroups of EDA during amination step. Finally,peaks have been reduced after deacetylationas a result of removal the acetyl groups inmodified chitosan (curve d).

Scan electron microscope (SEM micrograph)

As we observed in the SEM micrographs in(figure 11), the surface of the chitin becamerougher after amination. This attributed tografting process on the surface hydroxyl groupsof chitin particles. Also an increase of aminatedchitosan roughness than chitosan has beenobserved. The increase of the surface

roughness is automatically combined byincrease of the surface area which enhancedits adhesion with microorganism cell wall.

TGA analysis

Thermal Gravimetric Analysis (TGA) of modifiedchitosan and the intermediate products werecarried out using TGA-50 SHIMADZU Japan. Asshown in figure 12, the first weight loss attemperature 118 oC result from the evaporationof the water from the sample indicating theincrease of the water content in the samples9.459, 9.939, 10.384, 12.248% of chitin andactivated chitin, aminated chitin and modifiedchitosan respectively. This confirmed theincrease of the hydrophilic properties of chitinthrough modifications. The second weight lossof samples was start at 256 oC. The decreaseof the weight loss percent of the modifiedchitosan rather than chitin could be attributedto the introduced amine groups.

Conclusion

The antibacterial activity of chitosan wasincreased by grafting amino groups to itsbackbone chains. It was found that theantimicrobial activity of chitosan was dependanton the degree of grafting, degree ofdeacetylation, the molecular weight and the pHof the tested media. The modified chitosan hasstronger effect on the gram negative bacterialrather than the gram positive one. Also thebactericidal activity was shifted to right byintroducing amino groups. In conclusion , theintroducing of more amino groups directly onthe back bone of chitosan, in addition to itsoriginal amine groups has successed inincreasing its antibacterial activity and doesn’taffect its cytotoxicity.

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