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壳聚糖与羧甲基壳聚糖的抗菌性能研究

Antibacterial Action of Chitosan and Carboxymethylated Chitosan

XIAO FEI LIU,YUN LIN GUAN,DONG ZHI YANG,ZHI LI,KANG DE YAO

Research Institute of Polymeric Materials,Tianjin University,Tianjin300072,People’s Republic of China

Received23November1999;accepted19March2000

ABSTRACT: A series of chitosan with different molecular weights obtained by?-irradi-

ation depolymerization and another series of deacetylated chitosan were synthesized.

Several N,O-carboxymethylated chitosan and O-carboxymethylated chitosan were also

produced.The above samples were characterized by Fourier transform infrared spec-

troscopy(FTIR).Their antibacterial activities against E.coli were explored by the

optical density method.The antibacterial activity of chitosan is in?uenced by its

molecular weight,degree of deacetylation,concentration in solution,and pH of the

medium.Antibacterial activities were also found to be increased in the order of N,O-

carboxymethylated chitosan,chitosan,and O-carboxymethylated chitosan.Fluores-

cence of the FITC(?uorescein isothiocyanate)-labeled chitosan oligomers at the inside

of the E.coli cell was observed by a confocal laser scanning microscope.The antibac-

terial activity of chitosan oligomers seems to be caused mainly by the inhibition of the

transcription from DNA.?2000John Wiley&Sons,Inc.J Appl Polym Sci79:1324–1335,2001

Key words:chitosan;carboxymethylated chitosan;antibacterial activity;?uores-

cence

INTRODUCTION

As one of the most abundant natural biopolymers, and due to its unique polycationic nature,chi-tosan has a wide range of application?elds,such as in wastewater puri?cation,1coacervate forma-tion for cell entrapment,2and coating of seeds for improved yield.3

Interestingly,some antibacterial and antifun-gal activities have been described with chitosan and chitosan derivatives with quaternary ammo-nium.4–7Chitosan inhibits the growth of a wide variety of bacteria and fungi(cf.Table I).More-over,chitosan has several advantages over other types of disinfectants,that is,it possesses a higher antibacterial activity,a broader spectra of activity,a higher killing rate,and lower toxicity toward mammalian cells.8,9

Several mechanisms were proposed for the an-timicrobial activity by chitosan.10In one mecha-nism,the polycationic nature of chitosan inter-feres with the negatively charged residues of mac-romolecules at the surface.11Chitosan interacts with the membrane of the cell to alter cell perme-ability.12For example,fermentation in bakers’yeast is inhibited by certain cations,which act at the yeast cell surface to prevent the entry of glu-cose.13UV-absorption studies indicated that chi-tosan caused considerable leakage of protein-aceous material from Pythium oaroecandrum at pH5.8.14

The other mechanism involves the binding of chitosan with DNA to inhibit RNA synthesis.15It has been proposed that when chitosan is liberated from the cell wall of fungal pathogens by plant host hydrolytic enzymes chitosan penetrates the nuclei of fungus and interferes with RNA and

Correspondence to:Y.L.Guan.

Contract grant sponsor:National Natural Science Founda-tion of China.

Journal of Applied Polymer Science,Vol.79,1324–1335(2001)

?2000John Wiley&Sons,Inc.

1324

protein synthesis.The organism may be impaired by both its own chitosan and host phytoalexin induced by the liberated chitosan.16

Chitosan,however,shows its antibacterial ac-tivity only in an acidic range because of its poor solubility above pH6.5.Thus,water-soluble chi-tosan derivatives soluble to both acidic and basic physiologic circumstances may be good candi-dates for the polycationic biocide.

In this article,we prepared a series of chitosan that has various molecular weights(MW)and degree of deacetylation(DDA)and two types of carboxymethylated chitosan.Also,we examined whether their antibacterial activity against Esch-erichia coli(E.coli)depended on the MW,DDA, chitosan concentration(C),and pH of the me-dium.We observed?uorescence of the FITC(?u-orescein isothiocyanate)-labeled chitosan oli-gomers at the inside of the E.coli cell by a confo-cal laser scanning microscope. EXPERIMENTAL

Materials

The?rst type of chitosan was provided by Qing-dao Medicine Institute(Qingdao,Shangdong Province,China).One of this type had1.08?106M?calculated by the Mark–Houwink equation.17

[?]?KmM a,where Km? 1.81?10?3,a ?0.93,and the DDA was85%.The other of this type had2.74?105M?and a74%DDA.

The second type of chitosan was obtained from

the Wuxi University of Light Industry(Wuxi,

Jiangsu Province,China).One of this type had a

weight-average molecular weight(M w)of8000,a

molecular weight distribution(M w/M n)of1.80,

and a75%DDA,and the other of this type had a

M w of5000,an M w/M n of2.10,and73%DDA.

The E.coli was from the School of Biology

Science,NanKai University(Tianjin,China),and

was stored at4°C until it was used.Biochemical

reagents of beef extract,peptone,and agar powder

were purchased from the Tianjin Dongfang Bioma-

terial Co.(Tianjin,China).

Monoclonal anti-FITC(mouse IgG1isotype)

was Sigma Immuno Chemicals’s product No.

F-5636(Sigma Company,USA).All other re-

agents were analytical grade. Characterization

IR spectra of all the samples were recorded with a

Nicolet170SX IR spectrophotometer.After being

dried completely at50°C under a vacuum,the

sample could be used for analysis.

?-Irradiation Depolymerization of Chitosan

The?-irradiation of samples was carried out in a ?cell(Co-60source,Gamma400A,Tianjin Atomic Physical Research Institute,Tianjin,Chi-na).Chitosan samples in glass vials were irradi-ated with doses of up to100kGy under anoxia conditions.All irradiated vials were placed in a desiccator for at least1week prior to sample characterization.A series of chitosans with differ-ent MW was obtained(cf.Table II).

Deacetylation of Chitosan

To obtain different deacetylated chitosan(cf.Ta-ble III),chitosan(M??2.74?105)was re?uxed for10–55min at95°C with a40%(w/v)NaOH solution under nitrogen gas conditions.The sam-ples were washed with distilled water,re?uxed again under similar conditions,and washed with distilled water until neutral.18The DDA of the chitosan was determined by the FTIR baseline method as explained by Roberts and Domsey,19 using the relationship%DDA?(1?A1655/A3340?1/1.33)?100,where A is the logarithmic ratio of the absorption and transmittance at the given wavenumber.

Table I Antimicrobial Activities of Chitosan

Bacteria MIC a(ppm)

Agrobacterium tumefaciens100

Bacillus cereus1000

Corinebacterium michiganence10

Erwinia sp.500

Erwinia carotovora subsp.200

Escherichia coli20

Klebsiella pneumoniae700

Micrococcus luteus20

Pseudomonas?uorescens500

Staphylococcus aureus20

Xanthomonas campestris500

Fungi

Botrytis cinerea10

Fusarium oxysporum100

Drechstera sorokiana10

Micronectriella nivalis10

Piricularia oryzae5000

Rhizoctonia solani1000

Trichophyton equinum2500

a MIC:minimum growth inhibitory concentration.

ANTIBACTERIAL ACTION OF CHITOSAN1325

Preparation of N,O-Carboxymethylated Chitosan (N,O-CM-Chitosan)

Chitosan(M??2.74?105,20g)is suspended in isopropanol(200mL)under agitating,and so-dium hydroxide(50.4mL,10M)is added in six equal portions over a period of20min.The alka-line slurry is stirred for an additional45min,and the solid monochloroacetic acid(24g)is added in ?ve equal portions at5-min intervals.The reac-tion mixture is heated at60°C for3h.Then,cold distilled water(17mL)is incorporated into the mixture and its pH is adjusted to7.0with glacial acetic acid.The reaction mixture was?ltered and the solid product was washed with a70%metha-nol/water mixture(300mL)and then with anhy-drous methanol.The resultant N,O-CM-chitosan is dried in an oven at60°C under a vacuum.20The degree of carboxymethylation(DCA)is deter-mined by pH titration.21

Preparation of O-Carboxymethylated Chitosan (O-CM-Chitosan)

Chitosan M??2.74?105,10g)suspended in a 100mL NaOH solution(42%)is reacted with so-dium monochloroacetate obtained by adding monochloroacetic acid(11.7g)at0–30°C for5–24 h.The pH is adjusted to7with HCl and the solution is dialyzed for3days against deionized water.22The DCA is determined by pH titra-tion.21

Antibacterial Assessment

Antibacterial activities of the series of chitosan and its derivatives against E.coli were evaluated by using the optical density method as described.

A loopful of each culture was spread to give the single colonies on the nutrient agar(agar15g, peptone10g,beef extract3g,NaCl3g in distilled water1000mL;pH7.0)and incubated at37°C for 24h.A representative colony was picked off with a wire loop and placed in a nutrient broth(pep-tone10g,beef extract3g,NaCl3g in distilled water1000mL;pH7.0),which was then incu-bated at37°C overnight.Then,a culture where E. coli grew in a logarithmic growth phase was pre-pared for an antibacterial test.Two percent of them was inoculated to the medium containing different chitosans and their derivatives,which were dissolved in2M acetic acid,under shaken cultivation at37°C for24h.During incubation, the turbidity of the medium was measured at610 nm(by a756MC UV-vis spectrophotometer, Shanghai,China)every2h.

Fluorescence Observation on Antibacterial Activity of FITC-labeled Chitosan Oligomer

Preparation of FITC-labeled Chitosan Oligomer Chitosan,0.2g(M w?8000),was dissolved in20

mL of a H2O:C2H5OH?1:1mixed solution at pH 9.0and0.04%of FITC was reacted with the chi-tosan oligomer at an ice-cold temperature under stirring overnight.An FITC–chitosan oligomer was precipitated with ethanol and air-dried after extensive rinsing with ethanol.

Fluorescence Observation

Five percent of the culture medium adapting E. coli was inoculated in the medium containing

Table II Chitosan with Different MW for Antibacterial Activity Analysis

Samples Irradiation Dose

(kGy)M?(?104)DDA(%)M

Chitosan010885——Irradiated chitosan156585——Irradiated chitosan509.1686——Irradiated chitosan100 5.1188——Chitosan—27.474——Chitosan oligomer——758000 1.8 Chitosan oligomer——735000 2.1 Table III Properties of Chitosan and

Deacetylated Chitosan

Samples Re?ux

Time

(min)

DDA

(%)

(?104)

Chitosan07427.4 Deacetylated chitosan1075.626.1 Deacetylated chitosan2582.720.3 Deacetylated chitosan4089.916.9 Deacetylated chitosan559612.7 1326LIU ET AL.

0.01%of the FITC–chitosan oligomer under a shake culture at 37°C for 24h in the dark.E.coli cultured with the FITC–chitosan oligomer was collected by centrifugation (3000rpm for 15min)and rinsed extensively with physiological saline.The aggregated organisms were dispersed by ul-trasonication in the physiological saline and loaded on the ?uorescence free slide glass fol-lowed by air-drying.Then,a coverglass was set after treatment with 50%aqueous glycerol con-taining sodium azide and localization of the ?uo-rescence was observed by a Confocal laser scan-ning microscope (Model:TSC-NT 165123,US).23

RESULTS AND DISCUSSION

IR Spectra Analyses

Figures 1and 2show the FTIR spectra of chitosan with different viscosity-average molecular weights (M ?)and DDA.The main characteristic peaks of chitosan are at 3455(O O H stretch),2867(C O H stretch),1589(N O H bend),1154(bridge O stretch),and 1094cm ?1(C O O stretch).24From the spectra (cf.Fig.1),there is no qualitative

difference in peak locations between the irradi-ated chitosan and the started chitosan (cf.Table II),especially in the amino characteristic peak at about 1589cm ?1.For different deacetylated chi-tosan (cf.Table III),the higher the DDA of chi-tosan is,the weaker the acetyl group absorption (ca.1566and 1658cm ?1)is and the stronger amino peak is (cf.Fig.2).25

For N ,O -CM-chitosan (cf.Table IV),its spec-trum is different from the spectrum of chitosan (cf.Fig.3).The bands at 1597–1610and 1414cm ?1correspond to the carboxy group and O CH 2COOH group,respectively.26Compared with the peak of chitosan,the peak of N ,O -CM-chitosan at 1589cm ?1(N O H bend)decreases,whereas those at the peak,1066–1109cm ?1(C O O stretch),increase.These bands become more evident in that carboxymethyl has substi-tuted the amino and hydroxyl of chitosan.Figure 3also shows the IR spectra of O -CM-chitosan (cf.Table IV),and the 1730(O COOH),1080–1154

(O C O O O ),and 1624and 1516cm ?1(O NH 3?

)

bands appear.22

Therefore,that the carboxy-methyl simply substituted the hydroxyl of chi-tosan is

evident.

Figure 1IR spectra of the original chitosan:(A)M ??108?104),and the irradiated chitosans:(M ??(B)65?104;(C)9.16?104;(D)5.11?104).

ANTIBACTERIAL ACTION OF CHITOSAN 1327

Antibacterial Activities of Chitosan and CM-Chitosan

Chitosan,a cationic antibacterial agent,has been widely used,particularly for external disinfec-tion,and the target site of the cationic biocides is the cell envelope of bacteria.For Sudarshan et al.,the mechanism of antibacterial activities that the amino group of chitosan is bound to surface com-ponents of the bacteria and then inhibits their growth was developed.10They thought that at lower concentration chitosan may have bound to the negatively charged bacterial surface to dis-turb the cell membrane and cause cell death due to leakage of intracellular components;at high concentration,chitosan may have additionally

coated the bacterial surface to prevent leakage of intracellular components as well as to impede mass transfer across the cell barrier.

MW-dependent Antibacterial Activity

Figures 4and 5show the optical density versus the culture time for the chitosan with different MW against E.coli.Seven types of chitosan with MW ranging from 5000to 1.08?106were studied for their antibacterial activities.The smaller the optical density (OD)of the medium is,the higher is the antibacterial activity of the correspondent chitosan.According to Figure 4,compared to the OD of the medium without chitosan,the OD of that with chitosan was much lower,and it

de-

Figure 2IR spectra of different deacetylated chitosans:DDA%?(A)74;(B)75.6;(C)82.7;(D)89.9;(E)96.

Table IV DCA of CM-Chitosan

Sequence

Sample

N ,O -CM-chitosan

N ,O -CM-chitosan N ,O -CM-chitosan N ,O -CM-chitosan O –CM-chitosan O -CM-chitosan DCA (%)97.9

91.0

69.6

47.7

41.9

73.1

1328LIU ET AL.

creased gradually,with the MW varying from 5000to 9.16?104.This is evidence that chitosan has good antibacterial activity,and it was in-creased with the MW varying from 5000to 9.16?104.However,from Figure 5,the OD of the medium with the chitosan MW varying from 9.16?104to 1.08?106was increased gradually.This also meant that the antibacterial activity of chi-tosan decreased while its MW increased.

Sudarshan et al.argued that the growth inhib-itory activity of chitosan markedly increased with lengthening of the polymer,10while Groboillot et al.held that the crosslinked chitosan did not in-hibit bacterial growth,suggesting that only solu-ble chitosan is inhibitory.27They both think that water-soluble chitosan affects bacterial transport mechanisms through the cell walls by binding membrane macromolecules.

Summing up the result,it was seen that the antibacterial activity of chitosan,which is a poly-cationic compound due to a large amount of

O NH 3?

in the solution,may be depend on the concentration of the O NH 2of the polymer.Also,when the MW is under 9.16?104,the antibacte-rial activity of chitosan increases with increasing

of the O NH 2content (in other words,with in-creasing of the MW).When the MW exceeds about 9.16?104,amino groups of chitosan may be too many,which promotes a ?ctitious crosslinked structure through their strong intramolecular hy-drogen bondings and then they are no longer available to attach to bacteria surfaces.So,the antibacterial activity of chitosan decreased with increasing of its MW when it was above 9.16?104.

Antibacterial Activity Regulated by DDA

Figure 6shows the OD versus the culture time for the chitosan with different DDA against E.coli.The OD of the medium with ?ve types of different DDA chitosan was observed.The curves demon-strate that the OD decreased gradually with the percent of the DDA of chitosan,heightening from 74to 96.So,the antibacterial activity was in-creased respectively.

The results evidence that the heightening of DDA causes increase of the O NH 2concentration

and then increases the O NH 3?

numbers.Thus,

more O NH 3?

positive charges may have bound

Figure 3IR spectra of N ,O -CM-chitosans:DCA %?(A)47.7;(B)97.9;chitosan:(C)M ??27.4?104;and O -CM-chitosan:DCA%?(D)41.9;(E)73.1.

ANTIBACTERIAL ACTION OF CHITOSAN 1329

the negatively charged bacterial surface to cause agglutination.The study of Morimoto and Shige-masa about the inhibition of bacterial growth by deacetylated chitins with various DDA (from 66to 91%)also show that the antibacterial activity was increased with increase of the DDA.28This conclusion is in accordance with our study result

that the antibacterial activity of chitosan strongly depended on the DDA.

Dependence of Antibacterial Activity on Concentration (C )of Chitosan in Solution

Figures 7and 8show the OD versus the culture time for two kinds of chitosan with different

con-

Figure 4OD versus culture time for the chitosan with MW ranging from 5000to 9.61?104against E.coli (“no cs”means no chitosan in the

medium).

Figure 5OD versus culture time for the chitosan with MW ranging from 5000to 1.08?106against E.coli.

1330LIU ET AL.

centrations against E.coli.While some chitosan was added to the medium,its OD was obviously lower than that of the medium without chitosan.Moreover,with increase of the concentration of chitosan in the medium,which varied from 0.01%(w/v)to 0.1%(chitosan M ??5.14?104)and from 0.01to 0.5%(chitosan M w ?5000),their OD values decreased,respectively.In other words,the antibacterial activity of chitosan in the medium would increase if the concentration of chitosan had been increased.As the concentration of chitosan in the medium also indicates the

O NH 3?

concentration,the above result evidenced that the inhibitory effect of bacteria depended on

the amount of O NH 3?

and was strengthened

with the O NH 3?

concentration in the experi-mental range.However,we can not check the antibacterial activity of chitosan with a higher concentration because of the poorly solubility of

chitosan.

Figure 6OD versus culture time for the chitosan with %DDA ranging from 74to 96against E.

coli.

Figure 7OD versus culture time for the chitosan (M ??5.11?104)whose C in the medium is 0.01,0.05,and 0.10%against E.coli (“no cs”means no chitosan in the medium).

ANTIBACTERIAL ACTION OF CHITOSAN 1331

Effects of pH on Antibacterial Activity

Figure 9exhibits the OD versus the culture time for the chitosan dissolved in different pH’s of media against E.coli.In the same culture conditions,when the pH of the media was lower than 6.3,their OD values were larger than that of the medium with pH 6.3.So,when the medi-um’s pH ?6.3,the corresponding antibacterial activity of chitosan was lower than that of chi-tosan in medium with pH 6.3and decreased gradually with pH varying from 6.3to 4.0,then increased again with pH to 3.0.When a solu-tion’s pH ?6.3,their ODs were larger than that of the medium with pH 6.3,and the antibacte-rial activity of chitosan in it was clearly de-creased compared with that of chitosan in me-dium with pH 6.3.No antibacterial activity was observed in chitosan in media with pH 7.0or above,which may be due to chitosan’s poor sol-ubility in this

condition.

Figure 8OD versus culture time for the chitosan (MW ?5000)whose C in the medium is 0.01,0.05,0.10,and 0.50%against E.coli (“no cs”means no chitosan in the

medium).

Figure 9OD versus culture time for the chitosan (M ??5.11?104)solved in the medium whose pH ranges from 3to 8against E.coli.

1332LIU ET AL.

Chitosan is a polyelectrolyte,and its p K a is approximately 6.3.10In this condition,the O NH 2of chitosan was signi?cantly charged,existed as

O NH 3?

,and showed a good antibacterial activity.

With pH ?6.3,the amount of O NH 3?

decreased;moreover,the solubility of chitosan declined,so the antibacterial activity of chitosan diminished.

With pH ?6.3,the amount of O NH 3?

was not

varied,but the number of H ?

was increased.The two cationics competed in binding to the negatively charged bacterial surface,but only the polycationic can cause agglutination.So,the antibacterial activ-ity of chitosan in media with this pH decreased.

Antibacterial Activity of CM-Chitosan

Figure 10shows the OD versus the culture time for the chitosan,four kinds of N ,O -CM-chitosan,and two kinds of O -CM-chitosan against E.coli.The results indicated that the OD of N ,O -CM-chitosan whose DCA varied from 36.6to 97.7%was much larger than that of chitosan.The anti-bacterial activity of these N ,O -CM-chitosan was hardly observed compared with its original chi-tosan.However,the ODs of O -CM-chitosan (%DCA ?41.9and 73.1)were lower than that of chitosan,and the antibacterial activities were slightly enhanced.

N ,O -CM-chitosan is a product where O NH 2groups and O OH groups were substituted by O CH 2COOH groups.So,compared with chitosan,its O NH 2content was lower,then its antibacte-rial activity decreased,and no antibacterial activ-ity appeared.O -CM-chitosan is the substitution of chitosan with O CH 2COOH only to O OH;its number of O NH 2is not changed.Moreover,its

O COOH group may have reacted with the NH 2group intra-or intermolecularly and charged these NH 2groups.So,in the same condition,the

number of O NH 3?

groups of O -CM-chitosan is more than that of chitosan.Therefore,the antibacterial activity of O -CM-chitosan was increased.Fluorescence Observation on FITC–labeled Chitosan Oligomer

The ?uorescence micrographs of E.coli -accumu-lated FITC-labeled chitosan oligomers (MW ?8000and 5000)are displayed in Figures 11and 12.In these micrographs,the ?uorescence (bright)areas are FITC-labeled chitosan oligomers,and the elliptic area containing the bright ones is the E.coli cell.In Figure 11(a),the nine small pictures,left to right,then up and down,expressed a series of cross-sections of E.coli containing FITC-labeled chitosan oligomers from the outside to the inside to the out-side.It evidenced that the FITC-labeled chitosan oligomers were observed at the inside of the cell.Thus,permeated chitosan oligomers [MW ?8000and 5000;cf.Figs.11(b)and 12]were suggested to block the transcription from DNA to inhibit the growth of bacteria.As this ?uorescence study cor-responds closely to the results of antibacterial ac-tivity by chitosan oligomers,the antibacterial activ-ity of chitosan oligomers seems to be caused mainly by the inhibition of the transcription from DNA.23

CONCLUSIONS

To analyze the antibacterial activities of chitosan,one series of chitosans with MW ranging from 5.11?104to 1.08?106was obtained by ?

-irra-

Figure 10OD versus culture time for (A–D)N ,O -CM-chitosan (cf.Table IV),(E,F)O -CM-chitosan (cf.Table IV),and (G)chitosan M ??27.4?104)against E.coli.

ANTIBACTERIAL ACTION OF CHITOSAN 1333

diation depolymerization,and another series of different deacetylated chitosans whose DDA var-ied from 74to 89.9%was synthesized.Several kinds of N ,O -CM-chitosans and O -CM-chitosans were also produced to analyze the antibacterial activities of chitosan derivatives.

The study evidenced that the water-soluble chitosan has a good antibacterial activity against E.coli.The antibacterial activity was increased with the MW varying from 5000to 9.16?104and

decreased with MW varying from 9.16?104to 1.08?106.When the DDA or concentration in a solution of chitosan increased,the antibacterial activities of chitosan also increased.While the pH of the medium was lower than 6.3,its antibacte-rial activity of chitosan was lower than that of chitosan in pH 6.3.When the solution was pH ?6.3,its antibacterial activity of chitosan was clearly decreased compared with that of chitosan in pH 6.3.Also,no antibacterial activity was

ob-

Figure 11Fluorescence micrographs of FITC-labeled chitosan oligomer (MW ?8000)accumulated in the E.coli cell.[Color ?gure can be viewed in the online issue,which is available at https://www.sodocs.net/doc/ff10048893.html,.]

1334LIU ET AL.

served at pH 7.0or above because of chitosan’s poor solubility in this condition.Antibacterial ac-tivities were found to increase in the order of N ,O -CM-chitosan,chitosan,and O -CM-chitosan.Summing up these results,an identical conclu-sion can be drawn:The variety of the above effect factors on the antibacterial activity of chitosan means the varying numbers of NH 2groups of chi-tosan.The antibacterial activity of chitosan de-pended on the concentration of O NH 2of the poly-mer and increased with the addition of the O NH 2content.In the solution,chitosan is a polycationic

compound due to a large number of O NH 3?

,so the antibacterial activities of chitosan and carboxym-ethylated derivatives also depend on the effective

number of O NH 3?

groups.Also,a mechanism of antibacterial activities of water-soluble chitosan was that chitosan may have bound to the negatively charged bacterial surface to disturb the cell mem-brane and cause cell death.

The FITC-labeled chitosan oligomers (MW ?8000and 5000)were observed at the inside of the E.coli cell,and they show good antibacterial activities.Thus,another mechanism of the anti-bacterial activity of chitosan oligomers was that the permeated chitosan oligomer may have blocked the transcription from DNA and inter-fered with the RNA and protein systhesis.

The authors wish to thank the National Natural Sci-ence Foundation of China for ?nancial support of this research.

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1997,54(10),

621.

Figure 12Fluorescence micrograph of FITC-labeled chitosan oligomer (MW ?3000)accumulated in the E.coli cell.[Color ?gure can be viewed in the online issue,which is available at https://www.sodocs.net/doc/ff10048893.html,.]

ANTIBACTERIAL ACTION OF CHITOSAN 1335

羧甲基壳聚糖制备方法

羧甲基壳聚糖制备方法 (1)将壳聚糖溶于稀乙酸中，用过量的丙酮沉淀，得到壳聚糖乙酸盐，转入带有搅拌的反应瓶中，加入一定量的NaOH溶液和异丙醇，边搅拌边滴加氯乙酸的异丙醇溶液，控制反应温度为70℃，反应数小时，冷却至室温，用稀酸调pH值至中性，用85%甲醇洗涤，干燥，即得羧甲基壳聚糖。[2] (2)将纯化好的壳聚糖装入带有搅拌的反应瓶中，加入一定量的20%NaOH溶液和异丙醇，在室温下搅拌60min，然后滴加氯乙酸的异丙醇溶液，在室温下反应 5h，然后用稀盐酸中和至pH值为7，用丙酮沉淀产物，过滤，用85%甲醇溶液洗涤直至无氯离子，再用无水甲醇洗涤，60℃下真空干燥，即得产品。[2] (3)将2鲍壳聚糖加到200mL正丁醇中，室温搅拌溶胀20min，分6次加入 lOmol/L NaOH溶液，每次50mL, 40min一次，最后一次加完后再搅拌40rnin，得到碱性壳聚糖，然后把24g固体氯乙酸分5次加入，5min一次，在55~75℃ 搅拌反应3h，接着加入17mL水，用冰醋酸调pH值至7，抽滤，用70%甲醇 300mL分次洗涤，抽干后，再用300mL无水乙醇分次洗涤，于60℃真空二干燥，得产品。羧甲基化反应温度分别为55℃, 60℃, 65℃, 70℃和75℃，产量分别为31. 0g,33.8g, 36.58, 34.0g和33.2g, 65℃时最高。[2] (4)把甲壳素于一定温度下在40%~60%NaOH溶液中浸泡0. 5~5h，然后边搅拌边加入氯乙酸，再在0~70℃反应0. 5~5h，碱酸质量比控制在(1.2~1.6):1，在 0-80℃保温5~36h，然后用稀盐酸中和，分离产物，用75%乙醇溶液洗涤，于60℃干燥。这个方法也可制备羧甲基壳聚糖。[2] (5) 15g壳聚糖先在50%(w/w) NaOH溶液中碱化，然后加150mL异丙醇搅拌，加入18g氯乙酸，在65℃反应2h，用酸中和，70%甲醇多次洗涤，然后溶于水中，再用丙酮沉淀，过滤，用无水乙醇反复洗涤，过滤，真空干燥，得到精制的羧甲基壳聚糖。[2] (6) 3g粉状壳聚糖悬浮于100mL浓度分别为25%, 30%, 35%,40%的NaOH溶液中，加入5g氯乙酸与冰醋酸的混合液(摩尔比为1:1)，在30℃下反应，每隔1h加入5g氯乙酸与冰醋酸的混合液搅拌反应6h，最后用盐酸中和，过滤，用甲醇反复洗涤，干燥，得产物。[2] (7) 10g壳聚糖溶于1000mL 1%乙酸溶液中，加入200mL氯乙酸钠(氯乙酸用氢氧化钠溶液中和)及50%氢氧化钠溶液150mL，室温间歇搅拌反应4h，用酸中和停止反应，离心分离沉淀，溶于碱，过滤，滤液再中和，离心分离沉淀，用甲醇洗涤，干燥，得产物。[2] (8)超声波法制备羧甲基壳聚糖，可显著缩短反应时间，提高羧甲基的取代度。将0. 5g壳聚糖与5mL异丙醇、10ml 30 %NaOH溶液混合，再加入溶于10rnl异丙醇的氯乙酸(壳聚糖与氯乙酸的质量比为1：4~5)，在三角瓶中摇荡几分钟后，置于超声波清洗器中，用水作振荡介质，调节输出功率40W，升温到60℃反应3h，之后倾去上层清液，向粘状物中加入40rnL水，充分搅拌溶解，用1000盐酸中和到pH值为7,滤去不溶物，滤液中加入适量甲醇沉淀，过滤，无水乙醇洗涤，100℃烘干，即得产物。

壳聚糖抑菌性能研究

壳聚糖抑菌性能研究甲壳素－壳聚糖是一种极有前途的天然高分子聚合物，自20世纪60年代以来，人们对它们的研究、生产、应用变得十分活跃。特别是近几年，研究人员认识到它们的抑菌效能，通过深入研究，有些甲壳素－壳聚糖的抑菌产品已经问世。甲壳素脱乙酰基产物为壳聚糖。据研究，壳聚糖的抑菌作用可能有两种机理，一种是壳聚糖通过正电荷的－NH3吸附带负电荷的细胞壁，使壳聚糖吸附在细胞膜表面形成一层高分子膜，改变了细胞膜的选择透过性，阻止营养物质向细胞内的运输，致使细胞质流失、细胞质壁分离，从而起到抑菌杀菌作用；另外一种机理是壳聚糖通过渗入进细胞体内，吸附细胞体内带有阴离子的细胞质，并发生絮凝作用扰乱细胞正常的生理活动，从而杀灭细菌。近几年，随着对该特性认识的加深，人们不仅对能够影响其抑菌性能的机理进行了深入的研究，而且，也开始应用化学方法对其进行改性，从而提高壳聚糖的抑菌性能，最终达到扩大其应用范围的目的。目前，针对影响壳聚糖抑菌性能方面的研究主要有以下几个方面：分子量对壳聚糖抑菌性能的影响多数研究认为，寡聚糖和低分子量的壳聚糖的抑菌效果较好，随分子量上升效果逐渐下降。特别是对大肠杆菌，壳聚糖分子量越小，抑菌作用愈明显。例如：宋献周等就几种不同分子量的α－壳聚糖对几种常见菌（大肠杆菌、金黄色葡萄球菌、枯草杆菌、产气荚膜杆菌）的抑制研究表明，低分子量的α－壳聚糖的抑菌效果优于高分子量的α－壳聚糖。夏文水等采用E．coli作为试验菌株，测得分子量为1500的壳低聚糖抑菌效果最强。但是，也有一些研究利用不同的试验菌得出结论认为，壳聚糖分子量较大时，其抑菌能力更强。例如：Yousook等报导分子量为4万的壳聚糖在浓度为0．5％时，对S．taureus 和E．coli的杀灭率为90％：分子量为18万的壳聚糖在浓度为500PPM时，对S．taureus和E．coli的杀灭率为100％：分子量在30万以下时，壳聚糖对金黄色葡萄球菌的抑制作用随分子量减小而逐渐减弱。 pH值对壳聚糖抑菌性能的影响严钦等人研究认为，壳聚糖因为具有质子化铵，能与细菌大负电荷的细胞膜作用，干扰细菌细胞膜功能，造成细菌体内细胞质流失，扰乱细胞的正常生理代谢，从而达到杀菌的目的。而在pH为中性时，壳寡糖中的氨基没有被质子化，因而不能抑制细胞的生长，反而是作为一种糖被细菌利用。由此可见，通常在微酸条件下，壳聚糖具有明显的抑菌作用，但是当pH值为7时，壳聚糖不但没有抑菌效果，反而还有一定的促进细菌生长的作用。晶体形状对壳聚糖抑菌性能的影响甲壳质有3种晶型，即α、β和γ－壳二糖聚合物，目前，人们对壳聚糖的研究绝大多是针对α晶型，对其他两种研究甚少。蒋霞云等通过对比α－壳聚糖和β－壳聚糖的抑菌性能得出，具有高黏度和高脱乙酰度的β－壳聚糖的抑菌性能强于α－壳聚糖，从而填补了壳聚糖抑菌性能研究在该方面的空白。辐射对壳聚糖抑菌性能的影响目前，由辐射方法改变壳聚糖的抑菌性研究已经逐步深入进行，分别有金黄色葡萄球菌、酵母菌等多个菌种被测试，并分别得出了不同的作用效果及不同的作用浓度。王勇、张成刚等人将壳聚糖经 100Kgy60Coγ－射线辐射处理后，发现其对金黄色葡萄球菌抑菌效果最强，比为辐射前增加100倍，且最适作用浓度为0．01％。孟玲、张中泽通过对酵母菌的抑菌试验验证，经辐射处理后壳聚糖的抑菌活性明显增强，并且0．2g／L的辐射壳聚糖具有明显的抑菌活性。化学改性对壳聚糖抑菌性能的影响羧甲基化羧甲基壳聚糖是目前研究的较多一种物质，由于羧甲基化后其水溶性增强，因此大大拓宽了壳聚糖的应用范围。目前，羧甲基壳聚糖大多被用于食品保鲜方面。李治等人经实验证实，羧甲基壳聚糖在羧甲基化度小于0．6～0．8时，抗菌性均大于壳聚糖，当羧甲基化度0．3～0．6范围内，羧甲基壳聚糖具有较强的抗菌性，羧甲基化度大于或小于此范围，抗菌性均有所下降。磺化由于壳聚糖上具有较多的活泼集团，因此较容易进行各类集团的转化与连接。黎碧娜等对磺化壳聚糖的抑菌性能进行了研究认为，磺化壳聚糖对大肠杆菌、枯草杆菌、葡萄球菌、黑曲霉、假丝酵母都有抑制作用，并且浓度越高，抑菌效果越好。此外，磺化羟丙基壳聚糖和黄原酸壳聚糖也具有一定的抑菌性。

壳聚糖衍生物的抗菌性质

壳聚糖和壳聚糖衍生物的抑菌作用摘要：壳聚糖是一类有着广谱抑菌活性的天然多糖，其生物相容性好、易降解、无毒，因而作为一种可再生资源在抑菌领域受到了越来越多的关注。本文通过对壳聚糖来源、性质、壳聚糖衍生物的化学改性的方法和抑菌作用的分析，并对今后壳聚糖衍生物抑菌情况进行了初步的展望。为研制和开发新型的高抑菌活性的壳聚糖衍生物的开发提供理论参考。关键词：壳聚糖；衍生物；抑菌；机理引言壳聚糖是无毒、无污染，具有可再生、无毒副作用，生物相容性和降解性良好的天然氨基多糖。目前已被广泛应用于医药[1-2]、农业[3]、食品[4-5]等领域，并成为最近生物新材料研究的热点[6-7]。壳聚糖具有抗菌活性，对多种植物病原细菌和真菌均抑制作用[8]。但由于其不溶于水和大多数有机溶剂，只溶于稀酸，在很大程度上限制了其应用范围。壳聚糖通过化学改性，可以得到具有一定官能团的壳聚糖衍生物。与壳聚糖相比，这些衍生物的性能往往有较明显的改善。对于壳聚糖的化学修饰研究较多的有壳聚糖的酰基化、烷基化、羟基化、醛亚胺基化、硫酸酯化、羧甲基化、季铵化等，其中季铵化、羧甲基化和硫酸酯化的产物由于具有良好的水溶性而备受重视[9]。有关壳聚糖的结构修饰和构效关系的研究已成为研究热点[10],因此，研究开发具有更高抗菌活性的壳聚糖衍生物，对于改善人们的生活质量具有重要意义。 1壳聚糖的来源和性质 1.1壳聚糖的来源壳聚糖是自然界唯一的碱性天然多糖，壳聚糖的历史得追随到19世纪，当时Rouget 在甲壳素的天然聚合物中发现了其脱乙酰化的形式[11]。壳聚糖是白色或淡黄色无定型、半透明、略有珍珠光泽的固体。由于其原料和制备方法的不同，其分子量也有所不同，可以从数十万到数百万不等。甲壳素在浓碱中加热处理后，就可以脱去部分乙酰基，得到壳聚糖，反应路线如下。

羧甲基壳聚糖

羧甲基壳聚糖因为有良好的水溶性、保湿性和成膜性,安全无毒并具有抗菌、抑菌、乳化稳定作用,在日化、食品、造纸、制药等方面有重要的用途。 1保鲜剂壳聚糖是甲壳素脱乙酰基的产物,是一种天然的阳离子高分子多糖,它来源丰富,无毒无害,无污染及可降解,已广泛应用于化工、食品、化妆品、环保及医药等诸多领域。但壳聚糖仅溶于某些酸性介质,限制了其应用范围。对壳聚糖进行化学修饰即可得羧甲基壳聚糖,根据羧甲基的取代位置不同可以获得O-羧甲基壳聚糖、N-羧甲基壳聚糖和N,O-羧甲基壳聚糖三种产物。与壳聚糖相比,羧甲基壳聚糖在果,如水溶性、成膜性、吸湿保湿性、抗菌性、安全无毒性等,更适合于现代果蔬保鲜贮运的要求。羧甲基壳聚糖是一种天然的多糖涂膜保鲜剂,来源丰富,无毒无味,抑菌性强,在果实表面形成的膜具有很好的气体选择通透性,能有效地降低果蔬的呼吸强度和蒸腾作用,从而保持果蔬的新鲜度,延长果蔬的贮藏寿命。研究表明羧甲基壳聚糖对金黄色葡萄球菌、大肠杆菌、枯草杆菌这三种常见的食品腐败菌有较强的抑制作用,其中对金黄色葡萄球菌的抑制效果最好,其最小抑制浓度为0·1%,对大肠杆菌、枯草杆菌最小抑制浓度均为0·2%。羧甲基壳聚糖对酵母菌群、黄曲酶素、黑曲霉等也有明显的抑制作用。（羧甲基壳聚糖在果蔬保鲜中的应用研究进展吴伟,林宝凤） 2对铅离子的吸附壳聚糖是甲壳素脱乙酰基后的产物其自然资源非常丰富是性能优良的金属离子吸附剂在工业废水处理贵重金属离子回收[3]等方面具有广阔的应用前景制备水溶性壳聚糖及其衍生物引入其它功能性基团改善它的溶解性及功能拓宽其应用范围是当前研究开发甲壳素和壳聚糖的重要课题羧甲基壳聚糖是壳聚糖经化学改性得到的水溶性衍生物由于羧基的引入使其结合金属离子能力大大提高可广泛应用于水处理贵重金属离子富集回收等方面进入人体健康者血铅的正常范围为0.483~1.45μmol/L当血铅含量达2.72~3.84μmol/L时即可发生铅中毒铅中毒可直接损伤人和动物的甲状腺功能还可损伤生殖细胞及降低性功能本文将初步研究羧甲基壳聚糖CMCS对铅离子吸附的基本特性以期为含铅废水的处理提供新的途径及理论依据。羧甲基壳聚糖与壳聚糖水溶性低聚壳聚糖相比对铅离子具有更强的吸附能力且吸附能力随着羧甲基取代度的增大而增大羧甲基壳聚糖吸附铅离子的行为遵循单分子层吸附机理符合动力学方程t/Qt=t/Qeq+M/KCM影响吸附过程的因素主要有时间pH值离子强度温度等为羧甲基壳聚糖在处理含铅的工业废水方面提供了一定的理论依据。（羧甲基壳聚糖对铅离子的吸附性能研究林友文陈伟罗红斌） 3降脂作用壳聚糖及其衍生物的调节血脂作用日益受到人们重视，关于降脂机制目前尚无定论。有人认为壳聚糖结构中含有氨基，作为聚阳离子可与胆酸、胆固醇结合并随粪便排出体外，能阻止消化系统吸收胆固醇和甘油三酷从而发挥降脂作用。（壳聚糖、梭甲基壳聚糖的降脂及抗氧化作用林友文林青郑景峰蒋智清） 4在农业上的应用羧甲基壳聚糖易溶于水，具有植物生理调节功能。Cuezo研究表明，用其处理番茄可提高叶片中叶绿素的含量；如用羧甲基壳聚糖处理玉米开花期的果穗和种子，可提高玉米籽粒中蛋白质的含量。玉米是低蛋白作物，因为玉米在氮代谢过程中，谷氨酰胺合成酶和谷氨酸脱氢酶往往受到抑制，NH离子补偿能力下降，使得贮藏蛋白含量较低。师素云以羧甲基壳聚糖处理玉米开花期果穗，发现发育籽粒中的谷氨酰胺合成酶、谷氨酸脱氢酶和谷丙转氨酶活性均明显增强，而蛋白水解酶活性下降，其中谷氨酰胺合成酶活性比对照组高20%以上，谷氨酸脱氢酶在处理后10、15、和25天时分别比对照组高30%、40%和50%以上，谷丙转氨酶活性高20%以上，而蛋白水解酶活性下降了30%以上；羧甲基壳聚糖对作物生长和营养代谢具有调节功能。师素云等用羧甲基壳聚糖水溶液处理玉米种子，其种子发芽率、幼苗

壳聚糖的改性及抑菌性研究_宋玉民

第46卷2010年第3期西北师范大学学报(自然科学版) Vo l 146 2010 No 13 Journal o f N ort hw est No rmal U niver sity (N atural Science) 收稿日期:2009O 10O 13;修改稿收到日期:2009O 12O 23 基金项目:甘肃省自然科学基金资助项目(0710JRZA 108);甘肃省高分子材料重点实验室基金资助项目作者简介:宋玉民(1951)),女,甘肃张掖人,教授,博士研究生导师.主要研究方向为生物无机化学. E O ma il:song ym@nwnu 1edu 1cn 壳聚糖的改性及抑菌性研究宋玉民1,常彩萍1,达文燕2,栾尼娜1 (1.西北师范大学化学化工学院,甘肃兰州 730070;2.西北师范大学生命科学学院,甘肃兰州 730070) 摘要:在氮气保护下,以硝酸铈铵为氧化还原引发剂,冰乙酸为溶剂,通过壳聚糖(CS)与盐酸聚六亚甲基胍盐衍生物(PH GH)的接枝共聚反应,制备出了具有较好水溶性的CS/PHGH 材料.采用红外光谱、元素分析对该材料进行了表征,并对其抑菌性实验进行了研究.结果表明,合成的水溶性CS/PHGH 对真菌、革兰氏阳性菌与革兰氏阴性菌的抑制作用均优于壳聚糖,尤其对真菌的抑菌效果最灵敏,其最小抑菌浓度(M IC)为0139mg #mL -1,抑菌圈直径为21mm.关键词:壳聚糖(CS);盐酸聚六亚甲基胍(P HG H);接枝共聚;抑菌性中图分类号:O 63611 文献标识码:A 文章编号:1001-988ú(2010)03-0071-04 M odification of chitosan and its antibacterial SONG Yu -min 1,CH ANG Ca-i ping 1,DA Wen -yan 2,LU AN N-i na 1 (1.Colleg e of Chem istry and Chemical Eng ineering ,No rthw est No rmal U niversity,L anzhou 730070,Gansu,China; 2.Colleg e of L ife Science,N or thwest N or mal U niv ersity ,Lanzho u,730070,Gansu,China) A bstract:Grafting guanidine polymer(PH GH )onto chitosan(CS)is conducted by free -radical polymerization using ceric amm onium nitrate as an initiator in the acetic acid so lution under nitrog en atm ospher e and the CS/PH GH of new material is pro duced.The FT -IR,elem ent analy sis of new mater ial is tested.And the antibacterial o f the new material is investigated.T he results sho w that the antibacterial activity of CS/PH GH is better than that of CS for fung us,g ram -positive bacteria and gram -neg ative bacteria,especially fo r fung us.T he minim al inhibitory concentration(M IC)is 0139mg #mL -1 fo r fungus,the diameter of the inhibition zo nes is 21mm. Key words:chitosan(CS);poly hex am ethylene guanidine hy dro chloride(PH GH );graft copolym er;antibacterial 随着生活质量的提高以及现代科学技术的飞速发展,人们不可避免地接触到各种各样的细菌、真菌等微生物.这些微生物在特定条件下迅速繁殖,并通过各种接触方式传播疾病,影响人们的健康,故抗菌材料的研究引起学者们的广泛关注.壳聚糖(CS)是甲壳素的衍生物,是唯一的碱性天然多糖,具有许多独特的物理、化学性质和生物功能 [1] . 然而通常情况下,CS 仅溶于酸或酸性溶液,直接限制了它在各方面的应用.由于CS 分子内存在游离的氨基、羟基,可进一步改性,其衍生物不但溶解性大大增加,且抑菌效果也显著增强.有关CS 的抑菌性及生物相溶性,国内外均有报道[2,3] ,杜予民等报道了一种壳聚糖胍盐衍生物的制备方法[ 4] ,但对改性后壳聚糖的溶解性,对细菌真菌的最小抑菌浓度、抑菌圈直径、温度与时间对菌种生长的影响均未见报道.笔者针对壳聚糖难溶于水的特点,通过甲基丙烯酸缩水甘油酯与盐酸聚六亚甲基胍的反应引入乙烯基,以硝酸铈铵为氧化还原引发剂,与壳聚糖反应制备出水溶性较好的壳聚糖胍盐衍生物,并研究了新材料的抑菌性质. 71

不同取代羧甲基壳聚糖的制备及其结构测定

不同取代羧甲基壳聚糖的制备及其结构测定 * 陈浩凡潘仕荣王琴梅中山大学附属第一医院人工心脏研究室,广州　510089 摘要　目的　制备不同取代的羧甲基壳聚糖并测定其结构。方法　通过不同反应条件得到不同位置取代和取代度的羧甲基壳聚糖,并用物理和化学方法进行分子结构表征。结果　在O 位和(或)N 位发生了羧甲基化反应,产物为不同取代度的N ,O-羧甲基壳聚糖(N ,O -CM C),N -羧甲基壳聚糖(N -CM C)和O-羧甲基壳聚糖(O-CM C)。结论　胶体滴定法是测定羧甲基壳聚糖取代度的优选方法;壳聚糖羧甲基化后水溶性极大地改善,应用前景广泛。关键词　羧甲基壳聚糖;　取代度;　电位滴定法;　胶体滴定法中图法分类号　R318.08 S ynthesis of Carboxymethyl Chitosan and Determination of Substitution Degree Chen Haofan ,Pan Shirong ,Wang Qinm ei T he First H osp ital Af f iliated to Sun Yat -sen University of M ed ical Sciences ,Guangz hou 510089Abstract Objective T o prepare and determine carbox ymethyl chitosan w ith different substitution .Methods A series of carboxy methy l chitosan having various degrees and positions of substitution w ere ob-tained by controlling reaction conditions,and characterized by chemical and physical methods.Results Car-boxy methylation occurred at hydrox yl groups and (or)amino group.The products of N,O-carbox ymethyl chi-tosan (N ,O -CM C )with different degree of substitution ,N -carboxym ethyl (N -CMC )and O -carboxym ethyl (O-CM C)were obtained.Conclusion Colloid titration is the optimized method to determine substitution de-gree of carboxym ethyl chitosan.T he product has superior w ater-soluble property and broad prospect of applica-tion . Key words carboxym ethy l chitosan ;　substitution degree ;　electrolytic titration ;　colloid titration * 　广东省自然科学基金资助项目(No .20001398)和广东省科技攻关项目(N o.K B02902G )陈浩凡,男,1972年生,博士研究生甲壳素(chitin )大量存在于甲壳动物(如蟹、虾)的甲壳中,是地球上数量最多的含氮有机物。由于甲壳素不溶于普通溶剂,故难以应用。壳聚糖(chitosan)是甲壳素的N-脱乙酰基产物,能溶于酸性溶液,与人体细胞有很强的亲和性和相容性,并具有良好的吸湿性、纺丝性和成膜性,且无毒副作用,因而成为优良的生物医学材料。羧甲基壳聚糖(carbox ymethyl chitosan ,CM C )是壳聚糖羧甲基化后的产物,由于它既保留了壳聚糖的优点,又极大地改善了水溶性,因而具有更广泛的用途,在众多甲壳素衍生物中,倍受关注。取代度是壳聚糖在生产、研究和应用中一个重要的技术指标。壳聚糖分子C 2氨基上的氢原子、C 3和C 6羟基上的氢原子均可以被羧甲基取代,但以医用材料为标准,研究不同取代位置、取代度羧甲基壳聚糖的制备,并对其结构进行测定,目前尚缺少系统报道。本实验以甲壳素和壳聚糖为原料,合成N ,O -羧甲基壳聚糖(N ,O-CMC),N-羧甲基壳聚糖(N-CMC)和O-羧甲基壳聚糖(O-CMC),通过物理和化学方法,测定不同取代羧甲基壳聚糖的分子量、取代位置、取代度及其结构特征,为研究其结构与防止手术后粘连作用之间的构效关系奠定基础并提供有关技术参数。1　材料和方法 1.1　材料和仪器甲壳素和壳聚糖(脱乙酰度87%,江苏南通双林生物制品有限公司);乙醛酸(德国Merk - 第32卷第2期第152页2003年4月华中科技大学学报(医学版) J Huazhong U niv Sci T ech [Heal th Sci]Vol.32　No.2　P.152 Apr.2003

羧甲基壳聚糖衍生物的制备

羧甲基壳聚糖衍生物的制备 1、实验原理壳聚糖是由氨基-D-葡萄糖单体通过β-1,4-糖苷键连接起来的直链糖，是天然多糖中惟一的碱性多糖，具有许多特殊的物理化学性质和生理功能。但壳聚糖只能溶于一些稀酸中，不能直接溶于水中，这在很大程度上限制了它的应用。因此，对壳聚糖进行化学改性，提高其溶解性能，尤其是水溶性，对拓展壳聚糖的应用领域具有重要意义。壳聚糖的化学改性是壳聚糖研究的一个重要领域，旨在通过在壳聚糖的-NH 2和-OH 上引入新的官能团而改善其溶解性及其他物理化学及生物学性能。壳聚糖的改性研究较多的有：酰基化、烷基化、羧基化、羟基化、接枝共聚、季铵盐化等。在迄今所报道的600余种壳聚糖衍生物中，羧甲基壳聚糖（Carboxymethyl chitosan ，CMC ）是研究较多的一种，是壳聚糖最重要的的衍生物之一。CMC 在日化、食品、造纸、医药、化妆品等方面都有着重要的用途，此类衍生物具有良好的水溶性、表面活性、成膜性、吸湿保湿性、安全无毒性、抗菌、抗氧化等生物性能，在化妆品、食品、生物医药等方面呈现出广阔的应用前景。羧甲基壳聚糖反应方程式如下： O CH 2OH OH NH 2H O n 2COOH O CH 2OH OH NHCH 2COONa H O n Et 3N 壳聚糖分子中的氨基和氯乙酸发生取代反应，得到N-羧甲基壳聚糖，三乙胺的作用为吸收反应释放的盐酸，促进反应的发生。 2、实验药品和玻璃仪器壳聚糖，氯乙酸、氢氧化钠、异丙醇、乙醇、醋酸等；三口瓶、回流冷凝管、恒温加热搅拌器等。 3、实验内容 3.1 N-羧甲基化反应在烧杯中把8g 氯乙酸[1]溶解在30ml 水中，氢氧化钠溶解在20ml 水中，在半个小时内磁力搅拌下，用胶头滴管把氢氧化钠溶液滴加到氯乙酸的水溶液中，使溶液的pH 调到8[2]，滴加完后，把混合溶液和2g 壳聚糖放人三口烧瓶中，然后加入2ml 缚酸剂三乙胺，升温到90度，水浴回流，磁力搅拌反应3h-4h 。反应结束后，向烧瓶中加入50ml 水[3]，转入烧杯中，磁力搅拌下用碱液调节溶液的pH 到7-8[4]，然后离心分离除去不溶物，离心后的清液倒入烧杯中，慢慢加入二倍量的乙醇，沉淀[5]，并磁力搅拌洗涤5分钟，产品抽滤，滤渣用乙醇水混合溶剂洗涤10分钟[6]，抽滤，最后用无水乙醇洗涤10分钟[7]，105度烘干。四、实验注释 [1] 氯乙酸为强烈的腐蚀性产品，称量时应小心。 [2] 氢氧化钠的量应计算好。 [3] 加水的目的充分溶解水溶性的羧甲基壳聚糖。 [4] 可以采用10%氢氧化钠调节，注意混合溶液的pH 应慢慢调。 [5] 加入乙醇的目的为破坏羧甲基壳聚糖在水中的溶解度，有利于羧甲基壳聚糖的析出。 [6] 乙醇和水的比例为8：2，可以把混合溶剂倒入烧杯中，放入羧甲基壳聚糖产品，磁力搅拌10分钟，目的为除去沉淀产品中夹杂的无机盐等杂质。

壳聚糖抗菌剂研究进展

Bioprocess 生物过程, 2017, 7(4), 41-48 Published Online December 2017 in Hans. https://www.sodocs.net/doc/ff10048893.html,/journal/bp https://https://www.sodocs.net/doc/ff10048893.html,/10.12677/bp.2017.74006 Research Progress on Chitosan Antimicrobial Maotao Wu SunRui Marine Environment Engineering Co., ltd, Qingdao Shandong Received: Nov. 20th, 2017; accepted: Dec. 1st, 2017; published: Dec. 7th, 2017 Abstract Chitosan is a nature macromolecule. With the investigation, its applications are broad. The article summarizes the research and application of chitosan as an antimicrobial, the mechanism and the infective factors, and the development foreground of the chitosan antimicrobial is prospected. Keywords Chitosan, Antimicrobial, Mechanism, Prospect 壳聚糖抗菌剂研究进展吴茂涛青岛双瑞海洋环境工程股份有限公司，山东青岛收稿日期：2017年11月20日；录用日期：2017年12月1日；发布日期：2017年12月7日摘要壳聚糖是一种天然的高分子，随着研究的深入发展，应用范围越来越广泛。本文概述了壳聚糖在抗菌剂领域的研究应用情况，归纳总结了其抗菌机理及其影响因素，同时展望了壳聚糖抗菌剂的发展前景。关键词壳聚糖，抗菌剂，机理，展望

壳聚糖及其衍生物抗菌性能进展

中国实用口腔科杂志2011年7月第4卷第7期甲壳素（chitin）是N-乙酰基-D-葡萄糖胺以β-l，4键结合而成的多糖，是蟹、虾等甲壳类、甲虫等的外骨骼及蘑菇等菌类的细胞壁成分，广泛存在于自然界。壳聚糖（chitosan）是甲壳素脱去乙酰基的产物，安全无毒具有良好的生物兼容性，与人体细胞有良好的亲和性，无免疫原性，具有抗癌和抗肿瘤的作用。壳聚糖及其衍生物因其特有生物活性对多种细菌、真菌具有广谱抗菌的功能，在口腔抗微生物方面的应用逐渐得到重视。本文就壳聚糖及其衍生物抗菌性能方面研究现状进行综述。 1壳聚糖的抗菌活性 1.1壳聚糖对细菌的抗菌作用壳聚糖具有广谱抗菌作用。近年来研究发现，壳聚糖可抑制大肠杆菌、沙门菌属、金黄色葡萄球菌、绿脓杆菌、李斯特单核细胞增生菌、小肠结肠炎耶尔森菌、链球菌、霍乱弧菌、志贺痢疾杆菌、产气单胞菌属及某些真菌等的生长［1］。邓婧等［2］采用纸片药敏试验法，在pH6.5时对不同浓度壳聚糖进行抑菌实验，发现其对变形链球菌、金黄色葡萄球菌、白色念珠菌、幽门螺杆菌、牙龈卟啉单胞菌均有抑制作用。2％壳聚糖对变形链球菌、金黄色葡萄球菌的抑制效果最好，1.5％、1.0％、0.5％对变形链球菌、金黄色葡萄球菌、白色念珠菌的抑制效果优于幽门螺杆菌和牙龈卟啉单胞菌。有研究发现，在pH5.5时，1.0%壳聚糖（脱乙酰度为88.7%）对金黄色葡萄球菌、大肠埃希菌有强抑制作用［3］。由于壳聚糖良好的成膜性和独特的抗菌性，它能有效抑制2种牙周致病菌——伴放线放线杆菌和牙龈卟啉菌的生长。Ikinci等［4］将壳聚糖凝胶或膜与洗必泰联用，证明壳聚糖对牙龈卟啉菌有一定的抑制作用，可避免洗必泰的不良反应，既可延长其作用时间，也能够明显抑制细菌生长。壳聚糖对促进血链球菌生物膜脱落有显著作用，且小分子量壳聚糖的作用效果最佳。壳聚糖对几种常见口腔致病菌不仅有抑制作用，而且经高温处理后其作用也很稳定，所以在治疗口腔感染方面壳聚糖将是有效药物［2］。1.2壳聚糖对真菌的抑制作用壳聚糖还具有抗真菌活性。壳聚糖可有效抑制皮肤浅表真菌的生长。刘晓等［5］研究壳聚糖凝胶对皮肤浅表真菌的抑制作用，发现壳聚糖凝胶剂对红色毛癣菌、断发毛癣菌均有较强抑菌作用，抑菌质量浓度为2.5～5g／L。Rhoades等［1］使用脱乙酰度为89%、质量浓度为1g／L的天然壳聚糖对念珠菌和白色隐球菌进行抑菌实验，发现其对2log cfu／mL念珠菌有明显的抑制作用，而对白色隐球菌却无抑制作用。Muhannad 等［6］在pH5.0条件下，使用0.5%壳聚糖（脱乙酰度92%）的乳剂对白色念珠菌的抗菌效果进行观察，发现24h后能使白色念珠菌数量减少达99%、黑曲霉菌减少达90%。可见壳聚糖对真菌也有很广泛的抑制作用，且作用效果与抗细菌作用类似。作者单位：中国医科大学口腔医学院牙体牙髓科，沈阳110001 通讯作者：于静涛，电子信箱：Yjtao555@https://www.sodocs.net/doc/ff10048893.html, 综述壳聚糖及其衍生物抗菌性能研究进展刘扬，于静涛，孙莹莹，宋雪莲文章编号：1674-1595（2011）07-0437-03中图分类号：Ｒ78文献标志码：Ａ提要：壳聚糖由天然多糖甲壳素经脱乙酰化处理而成，是生物相容性和水解性较好的低聚糖，具有较好的广谱抗菌性。近年来，壳聚糖及其衍生物的抗菌性是医药、保健、食品和化妆品等领域的研究热点，本文就壳聚糖及其衍生物抗菌性能方面研究进行综述。关键词：壳聚糖；壳聚糖衍生物；抗菌性；抗菌机制 Research on antibacterial action of chitosan and chitosan derivatives.LIU Yang，YU Jing-tao，SUN Ying-ying，SONG Xue-lian.Department of Endodontics，School of Stomatology，China Medical University，Shenyang 110001，China Summary：Chitosan，made by dehydration of natural polysaccharide chitin，is a biocompatible and soluble oligosaccha?ride and a good broad-spectrum antimicrobial.In recent years，antibacterial activity of chitosan and its derivatives is of special interest of research in the field of medicine，health，food and cosmetics，etc.This paper is a review on anti-bacte?rial performance of chitosan and its derivatives. Keywords：chitosan；chitosan derivatives；antibacterial action；antibacterial mechanism 437

N-辛基-N-O-羧甲基壳聚糖制备及表面活性研究

n- 辛基-n,o- 羧甲基壳聚糖制备及表面活性研究摘要本论文以天然高分子壳聚糖为原料，对其进行化学改性，制备出了一系列取代度不同的n- 辛基-n,o-羧甲基壳聚糖基表面活性剂。通过ftir 、ea、tg等对产物进行了表征，表明成功合成了目标产物；产物的羧甲基取代度为79.4%，辛基取代度分别为 3.47%，17.11%，26.82%，辛基的引入使得壳聚糖的结晶性能下降；改性后壳聚糖溶解性增强。采用芘荧光探针法以及悬滴法分别测定了壳聚糖基表面活性剂的临界胶束浓度以及表面张力，结果表明羧甲基取代度为79.4%，辛基取代度分别为 3.47%，17.11% ，26.82%时临界胶束浓度分别为 0.7879mg/ml 、0.2609mg/ml 、0.0592mg/ml ；产物能显著降低水的表面张力，最低值为 39.2mn/m,且辛基取代的越大、临界胶束浓度越低，降低水表面张力的效率越高。。其生物官能性和相容性、安全性、血液相容性、微生物降解性等优良性能被各行各业广泛关注，广泛应用于食品、化妆品、医药、农业及环保等诸方面[5] 。 1.2 壳聚糖的改性壳聚糖以其独特的生物相容性、生物降解性、抗菌性、无毒性、生物活性和物理化学性质引起人们的重视,在化工、纺织、印染、造纸和医药等领域有广泛的应用前景。然而由于分子内、分子间的氢键作用,使其呈紧密的晶态结构,所以不溶于水和大多数有机溶剂。只有当脱乙酰度为50%左右时,二次结构破坏最大,结晶度降低,才能较好地溶于水。溶解性差成为限制壳聚糖应用的主要因素因此,有必要对壳聚糖进行改性,以达到利用其生物活性和生理活性的目的。壳聚糖的分子结构中含有活性功能基：c3-oh、c6-oh、c2-nh2，特别是c2-nh2 的存在,可以通过引入功能基团,改善壳聚糖的物理化学性能,拓宽其应用范围。壳聚糖的化学改性方法有多种,其中包括：羧甲基改性、酰化改性、季铵化改性、烷基化改性、羟烷基改性、接枝反应、交联反应、偶联反应等等。本论文重点研究壳聚糖的羧甲基化改性与烷基化改性。 1.2.1 羧甲基壳聚糖羧甲基壳聚糖是以一氯乙酸为主要改性原料的重要的水溶性壳聚糖,可溶于中性、碱性和弱酸性水中,其成膜性、保湿性也十分优异,在日化、食品、医药、医用生物材料等领域中具有广泛的应用前景[6] 。羧甲基壳聚糖包括n- 羧甲基壳聚糖(n-cmc) , o- 羧甲基壳聚糖(o-cmc) 和n,o- 羧甲基壳聚糖(n,o-cmc) [7] ,可以通过选择反应物和反应条件来控制产物的类型。对壳聚糖进行羧甲基化改性可以改善壳聚糖的水溶性。黄攀等[8] 以壳聚糖、乙醛酸为原料, 制备了羧化度在25?78%勺水溶性n-cmc，并发现其在62.5卩g/ml?5000卩g/ml浓度范围内与小鼠成纤维细胞株l929 具有良好的细胞相容性。lin 等[9] 以2-羧基苯甲醛与壳聚糖通过席夫碱反应并还原得到n-苄氧羰基壳聚糖，用戊二醛交联制得ph响应性的水凝胶。柯仁怀等[10] 以甲壳素为原料,采用连续操作、不分离中间产物的方法合成了羧甲基取代度为 1.08 的水溶性n,o- 羧甲基壳聚糖,并通过重构插层法制备羧甲基壳聚糖/mg2al 双层氢氧化物复合物。除此之外,羧甲基壳聚糖亦能应用于其他领域,例如絮凝剂、抗菌剂、药物载体等。刘红娅等[13] 以甲壳素为原料采用两步微波法制备了o- 羧甲基壳聚糖,产物具有良好的絮凝性能, 可作为处理模拟染料废水及实际印染废水的絮凝剂。ramchandra 等[14] 制备了n,o- 羧甲基壳聚糖与锌的络合物以及壳聚糖与锌的络合物,并用革兰氏阳性菌和革兰氏阴性菌做了抗菌性能测试,结果表明n,o- 羧甲基壳聚糖与锌的络合物的抗菌性要优于壳聚糖与锌的络合物。 anitha等[15]利用离子交联法用tpp和cacl2制备了壳聚糖、o-羧甲基壳聚糖和n,o-羧甲基壳聚糖纳米粒,并对材料的细胞毒性和抗菌性进行了检测,结果表明三种材料对乳腺癌细胞的毒性很小, 而n,o- 羧甲基壳聚糖纳米粒拥有三者中最强的抗菌性。目前羧甲基壳聚糖的制备工艺已经相当成熟。riccardo 等［16］用乙醛酸和壳聚糖通过席夫碱反应以及硼氢化钠还原反映制备出不同取代度的n-cmc。张贵芹等［17］以壳聚糖与氯乙酸在氢氧化钾-异丙醇介质中，在壳聚糖与氯乙酸、氢氧化钾与氯乙酸质量比分别在2:1 及 2.3:1 时，室温下反应 5 h 制到取代度较高的o-cmc。

O-羧甲基壳聚糖的研制与结构分析

1 本科毕业论文( 设计) O - 二级学院专业班级学生姓名张三

诚信声明我声明，所呈交的毕业论文（设计）是本人在老师指导下进行的研究工作及取得的研究成果。据我查证，除了文中特别加以标注和致谢的地方外，论文（设计）中不包含其他人已经发表或撰写过的研究成果，也不包含为获得其他教育机构的学位或证书而使用过的材料。我承诺，论文（设计）中的所有内容均真实、可信。样本2

O- O-CMC）是壳聚糖的羧甲基化衍生物，在医药、化妆品等多种领域有着广泛的应用前景。本实验通过使用氯乙酸与壳聚糖反应制备了O-羧甲基壳聚糖，即在碱性条件下，以甲壳素为基本原料，以异丙醇作为溶胀剂，采用氯乙酸途径制备方式，通过控制不同的反应条件（反应路线、时间、温度、碱的浓度和投料比等），佳工艺路线。…… -

样本4 ，one of the derivatives of chitosan properties including biocompatibility, Retention Capacity, has a promising applicable perspective for its chitosan. ......

目 1.前言. 2.结构鉴定 (2) 2.1.红外图谱（IR） (2) 3.羧甲基壳聚糖取代度及分子量的测定 (3) 3.1.取代度的测定――胶体滴定法 (3) 3.1.1.羧甲基壳聚糖氨基含量的测定 (3) 3.2.羧甲基壳聚糖取代度、分子量测定结果 (3) …… 6.结论 (4) 6.1.影响产物的条件分析 (4) 6.1.1.反应介质碱性强度的影响 (4) 参考文献 (5) 致谢 (6) 附录A 1/f频谱图 (7) 样本5

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