Effect of MW and concentration of chitosan on antibacterial activity

of Escherichia coli

Nan Liu a,b, Xi-Guang Chen a,*, Hyun-Jin Park b, Chen-Guang Liu a, Cheng-Sheng Liu a,

Xiang-Hong Meng a, Le-Jun Yu a

a College of Marine Life Science, Ocean University of China, 5 Yu Shan Road, Qingdao 266003, Peoples’ Republic of Chinab Graduate School Biotechnology, Korea University, 1, 5-Ka, Anam-Dong, Sungbuk-Ku, Seoul 136-701, South Korea

Received 3 March 2005; received in revised form 5 September 2005; accepted 27 October 2005

Available online 19 January 2006

Abstract

Different molecular weight (MW) chitosans (5.5!104 to 15.5!104 Da) with the same degree of deacetylation (80%G0.29), were obtained by

the method of acetic acid hydrolysis. The effect of antimicrobial activities of chitosan and acetic acid against Escherichia coli were investigated.

All of the chitosan samples with MW from 5.5!104 to 15.5!104 Da had antimicrobial activities at the concentrations higher than 200 ppm. The

growth of E. coli was promoted at concentration lower than 200 ppm. The antibacterial activity of chitosan had relationship to the MW at the

concentration range from 50 to 100 ppm. The antibacterial activity of low MW chitosan is higher than that of the high MW samples. But the

chitosan sample with the middle MW (9.0!104 Da) could promote the growth of bacteria. In the different stages of cultivation, the earlier

chitosan was added the greater effect it did. And the mechanism of antibacterial activity was that E. coli was flocculated.

q 2005 Elsevier Ltd. All rights reserved.

Keywords: Chitosan; Antibacterial activity; Molecular weight; Concentration; Time sensitivity; Mechanism

1. Introduction

Chitosan is an abundant natural biopolymer obtained from

the exoskeletons of crustaceans and arthropods which is a

nontoxic copolymer consisting of b-(1,4)-2-acetamido-2-

deoxy-D-glucose and b-(1,4)-2-anaino-2-deoxy-D-glucoseunits. As its unique polycationic nature, chitosan has been

used as active material such as antifungal activity (Ben-

Shalom, Ardi, Pinto, Aki, & Fallik, 2003; Hirano & Nagano,

1989; Kendra, Chiristian, & Hadwiger, 1989; Roller & Covill,

1999; Uchida, Izume, & Ohtakara, 1989) antibacterial activity

(Choi et al., 2001; Chung, Wang, Chen, & Li, 2003; Helander,

Nurmiaho-Lassila, Ahvenainen, Rhoades, & Roller, 2001; Jeon

& Kim, 2000; Liu, Guan, Yang, Li, & Yao, 2001) and

antitumor activity (Koide, 1998; Mitra, Gaur, Ghosh, &

0144-8617/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.carbpol.2005.10.028

Abbreviations MW, molecular weight; DD, degree of deacetylation; FTIR,

Fourier transform infrared spectroscopy; MIC, minimum inhibitory concen-

tration; DMSO, dimethylsulfoxide; MTT, 3-(4, 5-dimethylthizao-2-yl)-2, 5-

diphenyl-tetrazolium bromide.* Corresponding author. Tel./fax: C86 532 203 2586.

E-mail address: xgchen@ouc.edu.cn (X.-G. Chen).

Maritra, 2001; Qin, Du, Xiao, Li, & Gao, 2001; Qin et al.,

2004; Suzuki et al., 1986).

The main factors affecting the antibacterial activity of

chitosan are molecular weight (MW) and concentration. There

are some reports that chitosan is more effective in inhibiting

growth of bacteria than chitosan oligomers (No, Park, Lee, &

Meyers, 2002; Uchida et al., 1989) and the molecular weight of

chitooligosaccharides is critical for microorganism inhibition

and required higher than 10,000 Da (Jeon & Kim, 2000). The

minimum inhibitory concentration (MIC) of chitosans ranged

from 0.005 to 0.1% depending on the species of bacteria and

MWs of chitosan (No et al., 2002) and was varied depending

upon the pH of chitosan preparation (Liu et al., 2001).

Chitosan cannot dissolve in water but in acetic acid solution.

As we all know, acetic acid has the antimicrobial activity. This

property cannot be ignored as the solvent of chitosan in the

experiment of investigation the antimicrobial activity of

chitosan. On the other hand, bacteria in different growth stages

have different sensitivity to chitosan. All these require further

investigation.

In this paper, a series of chitosan samples with different

MWs were prepared. The effect factor such as chitosan MW,

chitosan concentration, acetic acid and time sensitivity of

Carbohydrate Polymers 64 (2006) 60–65

www.elsevier.com/locate/carbpol

N. Liu et al. / Carbohydrate Polymers 64 (2006) 60–65 61

bacteria to the antibacterial activation of chitosan against

Escherichia coli were investigated.

2. Material and methods

2.1. Materials

Chitosan, from a crab shell with a molecular weight (MW)

of 500 kDa and deacetylation degree (DD) of 80%, was made

in our laboratory. Ethanol, hydrochloric acid, acetic acid,

dimethylsulfoxide (DMSO), 3-(4,5-dimethylthizao-2-yl)-2,5-

diphenyl-tetrazolium bromide (MTT), Coomassie Brilliant

Blue G-250, sodium hydroxide, peptone, sodium chloride,

and agar were of analytical grade and supplied by Sigma

Company (Sigma Co., St Louis, USA). Stock solution of

chitosan (1%) was prepared in 1% acetic acid with pH being

adjusted to 5.4 with NaOH.

2.2. Cultivation of the microorganism

Escherichia coli: ATCC 25992 was incubated overnight at

37 8C in nutrient broth (peptone 1%, beef extract 0.5%, NaCl

0.5%, pH 6). The cultures obtained were diluted with

autoclaved nutrient broth to obtain cell suspensions which

was adjusted to an absorbance of 0.2 at 610 nm. It was used for

antibacterial activity experimentation.

2.3. Different MW chitosan preparation

Chitosan was degraded by the method of acetic acid

hydrolyzes referenced from Chen et al. Chitosan (10 g, 100

mesh power) was dissolved in 190 ml of 5% aqueous acetic

acid, incubated at 50 8C for 2, 4, 6, 9, 12, 15, 25, 37, 121 and

145 h, respectively, and then centrifuged (5000 g) for 20 min.

The supernate was added to 4 N aqueous NaOH to pH 7–9. The

sediment was filtered and sequentially rinsed in water and

ethanol and dried at 50 8C. The samples obtained from the

reactions were named from A to J. The degree of deacetylation

was determined by the method of acid–base titration

(Sekiguchi et al., 1994) and FT-IR-spectrum. The viscosity

change was investigated by using an Ubbelohde Viscosimeter.

Table 1

Chitosan samples with different degradation time

Samples Degradation

time (h)

Molecular weight

(!104 Da)

DDA (%)

O 0 50 80.0

A 2 15.5 79.7*

B 4 14.5 80.4*

C 6 14 79.5*

D 9 9.6 79.8*

E 12 9.0 80.2*

F 15 8.8 79.6*

G 25 7.0 80.3*

H 37 6.5 80.1*

I 121 6.0 79.9*

J 145 5.5 80.1*

*PO0.05. Compared with sample O (original chitosan).

The viscosity molecular weight was calculated based on Mark

Houwink equation ([h]Zkma), with KZ1.64!10K30!DD14

and aZK1.02!10K2C1.82 (Chen & Hwa, 1996) here, DD is

the degree of the deacetylation of chitosan expressed as the

percentage, which was shown in Table 1.

2.4. Effect of acetic acid to E. coli

Tests were conducted in two sets: a test set with acetic acid

and a control set without acetic acid. In the test set, six

concentrations of acetic acid (20, 50, 100, 200, 500 and

1000 ppm) were used. Each concentration of acetic acid was

prepared with autoclaved nutrient broth to 100 ml. E. coli was

inoculated into each mixture with optical absorbance of 0.2 at

610 nm and was incubated with shaking at 37 8C for 48 h. The

effect of acetic acid to E. coli was monitored by spectropho-

tometer per 2 h.

2.5. Effect of the MW and concentration of chitosan against

E. coli

Ten different MW chitosan samples (A–J) and six different

concentrations (20, 50, 100, 200, 500 and 1000 ppm) were used

to evaluate the effects of the MW and concentration of chitosan

against E. coli. The same volume of water was added to the

control group. These samples were added at initial of

cultivation. The mixtures were incubated with shaking at

37 8C for 48 h were then mensurated at A610.

2.6. Time sensitivity assay

The time sensitivity assay was determined by the method of

optical density. Chitosan sample was added at different stage of

culture such as lag phase (0 h), initial stage of log phase (14 h),

middle stage of log phase (18 h), final stage of log phase (22 h)

and stationary phase (26 h). And the changes of OD value were

monitored at 610 nm per 2 h.

2.7. Measurement viable bacteria

The amount of viable bacteria was measured by the method

of MTT. Bacteria were cultivated as above and 0.5 ml 1%

chitosan acetic solution was added to 100 ml culture and

incubated at 37 8C for 2 h. The aliquots (1000 ml) of the culturewere pipetted into 1.5 ml EP tube which contained MTT

(100 ml), and reacted at 40 8C for 4 h, then centrifuged at room

temperature for 10 min at 1000 g. DMSO of 1000 ml was addedto dissolve the formazan crystals. The dissolvable solution was

jogged homogeneously about 15 min by the shaker. The optical

density of the formazan solution was read on an ELISA plate

reader (ELX 800, Bio-tek) at 490 nm. Each assay was

performed at least three times.

2.8. Determination of protein

The protein content in the culture medium was determined

by the method of Bradford (1976). Bacteria were cultivated to

N. Liu et al. / Carbohydrate Polymers 64 (2006) 60–6562

A610 of 0.2 and 0.5 ml 1% chitosan acetic solution was added to

100 ml culture and incubated at 37 8C for 2 h. Then centrifuged

(2000 g) for 20 min. To supernate (2 ml) was added into 5 ml

of Coomassie Brilliant Blue (100 mg of Coomassie Brilliant

Blue G-250 was dissolved in 50 ml 95% ethanol and 120 ml

H3PO4 (85%) was added. The solution was diluted to a final

volume of 1 l). After 2 min of incubation, the absorbance was

measured by spectrophotometer (UNIC 7200, UNIC apparatus

Co. Ltd, Shanghai) at 595 nm. The culture medium with

bacteria was used as control group. All experiments were

repeated three times.

3. Result and discussion

3.1. Chitosan preparation

Different MW chitosan samples A to J were obtained by the

acetic acid hydrolyzes; all the samples were white powders.

The MW of the chitosans were ranged from 5.5!104 to 15.5!104 Da and the degree of deacetylation of the chitosans have no

obvious difference between the samples from A to J (80%G0.29, PO0.05) shown in Table 1. Fig. 1 shows the FT-IR

spectrums of the original chitosan (O) and the different MW

chitosan samples A–J. There were strong amino characteristic

peaks of chitosan at around 3420, 1655, and 1325 cmK1, and

the peaks assigned to the saccharide structure were at

1152 cmK1 (C–H stretch), 1154 cmK1 (bridge-o-stretch), and

Fig. 1. IR spectra of the original chitosan and the degraded chitosan. (O)

Original chitosan 50!104 Da; (A) 15.5!104 Da; (B) 14.5!104 Da; (C)

14.0!104 Da; (D) 9.6!104 Da; (E) 9.0!104 Da; (F) 8.8!104 Da; (G) 7.0!

104 Da; (H) 6.5!104 Da; (I) 6.0!104 Da; (J) 5.5!104 Da.

1094 cmK1 (C–O stretch). The spectrums of the chitosan

samples A to J had no obvious difference with the original

chitosan, and no differ between each of the samples. The results

showed that chitosan samples made from the method of acetic

acid hydrolyze had no obvious change in the DDA and

molecular structure. The similar result had been reported by

Chen, Zheng, Wang, Lee, and Park (2002).

3.2. Antibacterial activity of acetic acid

The efficient antibacterial concentration of acetic acid was

investigated in detail in this paper. Fig. 2 was the antibacterial

activity of the acetic acid with different concentrations against

E. coli. At the concentrations range from 20 to 50 ppm, the

optical absorptions at 610 nm were no difference between the

experiment groups to control group. When the concentration of

acetic acid was 100 ppm, it was little lower than the control set.

And when the concentration was higher than 200 ppm, acetic

acid has shown its bacterial activity obviously. When the

concentration achieved 500 and 1000 ppm, almost all the E.

coli had been killed. The results showed that the antibacterial

activity of acetic acid has relationship to concentration. At low

concentrations (below 200 ppm), acetic acid had no anti-

bacterial activities. At middle concentration (200 ppm), it had

the obviously antibacterial activity. At high concentrations

(over 200 ppm), it could kill all of the E. coli.

3.3. Effect of chitosan concentration

The effect of concentration to the antibacterial activity of

chitosan against E. coli was shown in Fig. 3. At the

concentration of 20 ppm, all of the chitosan samples had

the stimulative effect on the growth of E. coli. When the

concentration was 50 ppm, the antibacterial activities of three

chitosan samples with low MW (samples H, I and J) had

exceeded the action of acetic acid. When the concentration was

100 ppm, all the samples had exceeded the action of acetic acid

except sample E. When the concentrations were over 100 ppm,

chitosan and acetic acid had efficacious antibacterial activities

against E. coli. The results showed that all of the samples at the

concentration of 20 ppm and seven samples (A, B, C, D, E, F

Fig. 2. Effect of acetic acid to the antibacterial activation of chitosan.

0

0.2

0.4

0.6

0.8

1

20 50 100 200 500 1000

The concentration of chitosan(ppm)

OD

610

nm A BC DE FG HI JHAC Control

Fig. 3. Effect of concentration to the antibacterial activation of chitosan.

N. Liu et al. / Carbohydrate Polymers 64 (2006) 60–65 63

and G) at the concentration of 50 ppm could promote the

growth of E. coli. And with the increase of the concentration,

the antibacterial activations of the chitosan samples had

increased. When the chitosan concentrations higher than

200 ppm, it could almost kill all of the bacteria and the effects

were same with the acetic acid at the same concentration.

0.7

0.8

0.9

Control

1

3.4. Effect of chitosan MW

Molecular weight relationships of antibacterial activity by

chitosan have been reported by various investigators. No et al.

(2002) reported that chitosan of 746 kDa appeared most

effective against E. coli. The results were little different from

ours. The antibacterial activities of chitosan samples with

different MWs and concentrations could be seen in Fig. 4. At

high concentration (over 200 ppm), the antibacterial activities

of each chitosan sample were almost same and all of the

0

0.2

0.4

0.6

0.8

1

A B C D E F G H I J

Chitosan samples

OD

610n

m

20 50 100 200 500 1000

Fig. 4. Effect of MW to the antibacterial activation to chitosan.

bacteria could be killed. At low concentration (20 ppm), there

was no antibacterial activities and could promote the growth of

E. coli. But at the middle concentration (50–100 ppm), there

were some differences between different MW chitosan in the

antibacterial activation. The high MW chitosan samples (A, B,

C and D) had the same antibacterial activity against E. coli. But

the antibacterial activity of sample E with the MW of 9.0!104 Da declined. And with the decrease of MW, the

antibacterial activities were increased. The results showed

that at the high concentrations (200, 500 and 1000 ppm) and

low concentration (20 ppm), the antibacterial activity of

chitosan had no relationship to the MW. But at the

concentration from 50 to 100 ppm, the antibacterial activities

of chitosan with different MWs were difference. The

antibacterial activity of chitosan was affected by MW only at

the concentration range from 50 to 100 ppm. It was very

similar with other reports that chitosan could inhibited the

growth of E. coli at high concentration.

3.5. Mechanism

Normally, bacteria have the different sensitivity at different

culture stage. Figs. 5 and 6 showed the time sensitivity of E.

coli with chitosan sample E (9.0!104 Da) and J (5.5!104 Da)

at the concentration of 100 ppm. Sample E (Fig. 5) made the

optical density play down when it was added in the culture

medium which contented bacteria. With different added time

the effects were differences. It made the lag phase extend when

chitosan was added at initial culture time, made the optical

density play down markedly when added at log phase and made

little decline of optical density when added at stationary phase.

But whenever sample E was added in the optical density would

exceed the control group. The earlier sample E was added the

0

0.1

0.2

0.3

0.4

0.5

0.6

0 8 16 24 32 40 48

Culture Time (h)

OD

610

nm

2

3

4

5

Fig. 5. Time sensitivity of E. coli to the sample E at concentration of 100 ppm.

(1) Chitosan sample was added at lag phase; (2) Chitosan sample was added at

initial stage of log phase; (3) Chitosan sample was added at middle stage of log

phase; (4) Chitosan sample was added at final stage of log phase; (5) Chitosan

sample was added at stationary phase.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 8 16 24 32 40 48 Culture time (h)

OD

610n

m

Control 12345

Fig. 6. Time sensitivity of E. coli to the sample J at concentration of 100 ppm.

(1) Chitosan sample was added at lag phase; (2) Chitosan sample was added at

initial stage of log phase; (3) Chitosan sample was added at middle stage of log

phase; (4) Chitosan sample was added at final stage of log phase; (5) Chitosan

sample was added at stationary phase.

N. Liu et al. / Carbohydrate Polymers 64 (2006) 60–6564

greater effect had. The results showed that sample E could

make flocculation in the culture medium and inhabit the growth

of E. coli temporarily and at last it could promote the growth of

E. coli.

The time sensitivity of E. coli with chitosan J (5.5!104 Da)

at the concentration of 100 ppm was shown in Fig. 6. It also

made the optical density play down and the lag phase extend.

But different from sample E it could inhibit the growth of

E. coli enduringly. And the later sample J was added the

weaker effect had.

Fig. 7 showed the change of amount of bacteria and protein

content after the chitosan sample had been added into the

culture medium for 2 h. Comparing with the control groups, the

protein content had little change and the amount of bacteria

declined obviously (about 0.8) made by the chitosan except

sample E. The results show that the flocculation made by

chitosan was almost bacteria and the bacteria could be killed by

chitosan. At low concentration chitosan could not flocculate all

Fig. 7. Comparing of the protein content and amount of bacteria.

the bacteria and kill them in the culture medium and the

survival would go on reproducing (Figs. 4–6).

4. Conclusion

Different molecular weight chitosans with same degree of

deacetylation were obtained by the method of acetic acid

hydrolysis. As the solvent of chitosan, acetic acid with the

concentration over 200 ppm had the antibacterial activity

against E. coli. All of the chitosan samples with the MW from

5.5!104 to 15.5!104 Da had the good antimicrobial activities

at high concentrations (over 200 ppm). And all of the samples

at low concentration (20 ppm) could promote the growth of E.

coli. Sample E (9.0!104 Da) at the concentrations of 50 and

100 ppm still could promote the growth of E. coli. Other

chitosan samples could inhibit the growth of E. coli at the

concentration range from 50 to 100 ppm. And the antibacterial

activity was affected by the MW of chitosan. The antibacterial

activity of lowMW chitosan is higher than that of the high MW

samples. In the stage of culture, the earlier chitosan sample was

added into medium the greater effect it did. The mechanism of

antibacterial activity of chitosan was that it could make the

bacteria flocculate and kill it.

Acknowledgements

The authors are indebted to the financial support from

National Natural Science Foundation of China (No. 30370344)

and The Scientist Encouragement Foundation of Shandong

Province (2004BS7001).

References

Ben-Shalom, N., Ardi, R., Pinto, R., Aki, C., & Fallik, E. (2003). Controlling

gray mould caused by Botytis cinerea in cucumber plants by means of

chitosan. Crop Protection, 22, 285–290.

Chen, R. H., & Hwa, H. D. (1996). Effect of molecular weight of chitosan with

same degree of deacetylation on the thermal, mechanical, and permeability

properties of the prepared membrane. Carbohydrate Polymers, 29,

335–358.

Chen, X. G., Zheng, L., Wang, Z., Lee, C. Y., & Park, H. J. (2002). Molecular

affinity and permeability of different molecular weight chitosan

membranes. Journal of Agricultural and Food Chemistry, 50, 5915–5918.

Choi, B. K., Kim, K. Y., Yoo, Y. J., Oh, S. J., Choi, J. H., & Kim, C. Y. (2001).

In vitro antimicrobial activity of a chitooligosaccharide mixture against

Actinobacillus actinomycetemcomitans and Streptococcus mutans. Inter-

national Journal of Antimicrobial Agents, 18, 553–557.

Chung, Y. C., Wang, H. L., Chen, Y. M., & Li, S. L. (2003). Effect of abiotic

factors on the antibacterial activity of chitosan against waterborne

pathogens. Bioresource Technology, 88, 179–184.

Helander, I. M., Nurmiaho-Lassila, E.-L., Ahvenainen, R., Rhoades, J., &

Roller, S. (2001). Chitosan disrupts the barrier properties of the outer

membrane of gram-negative bacteria. International Journal of Food

Microbiology, 71, 235–244.

Hirano, S., & Nagano, N. (1989). Effects of chitosan, pectic acid, lysozyme and

chitinase on the growth of several phytopathogens. Agricultural and

Biological Chemistry, 53, 3065–3066.

Jeon, Y. J., & Kim, S. K. (2000). Production of chitooligosaccharides using an

ultrafiltration membrane reactor and their antibacterial activity. Carbo-

hydrate Polymers, 41, 133–144.

N. Liu et al. / Carbohydrate Polymers 64 (2006) 60–65 65

Kendra, D. F., Chiristian, D., & Hadwiger, L. A. (1989). Chitosan oligomers

from Fusarium solani/pea interactions: Chitinase/beta-glucanase digestion

of sporelings and fungal wall chitin actively inhibit fungal growth and

enhance disease resistance. Physiological and Molecular Plant Pathology,

35, 215–230.

Koide, S. S. (1998). Chitin–chitosan: Properties, benefits and risks. Nutrition

Research, 18, 1091–1101.

Liu, X. F., Guan, Y. L., Yang, D. Z., Li, Z., & Yao, K. D. (2001). Antibacterial

action of chitosan and carboxymethylated chitosan. Journal of Applied

Polymer Science, 79, 1324–1335.

Mitra, S., Gaur, U., Ghosh, P. C., & Maitra, A. N. (2001). Tumour targeted

delivery of encapsulated dextran-doxorubicin conjugate using chitosan

nanoparticles as carrier. Journal of Controlled Release, 74, 317–323.

No, K. H., Park, N. Y., Lee, S. H., &Meyers, S. P. (2002). Antibacterial activity

of chitosans and chitosan oligomers with different molecular weights.

International Journal of Food Microbiology, 74, 65–72.

Qin, C. Q., Du, Y. M., Xiao, L., Li, Z., & Gao, X. H. (2001). Enzymatic

preparation of water-soluble chitosan and their antitumor activity.

International Journal of Biological Macromolecules, 31, 111–117.

Qin, C. Q., Zhou, B., Zeng, L. T., Zhang, Z. H., Liu, Y., Du, Y. M., et al. (2004).

The physicochemical properties and antitumor activity of cellulase-treated

chitosan. Food Chemistry, 84, 107–115.

Roller, S., & Covill, N. (1999). The antifungal properties of chitosan in

laboratory media and apple juice. International Journal of Food

Microbiology, 47, 67–77.

Sekiguchi, S., Miura, Y., Kaneko, H., Nishimura, S. L., Nishi, N., Iwase,

M., & Tokura, S. (1994). Molecular weight dependency of

antimicrobial activity by chitosan oligomers. In K. Nishinari, & E.

Doi (Eds.), Food hydrocolloids: Structures, properties, and functions

(pp. 71–76).

Suzuki, K., Mikami, T., Okawa, Y., Tokoro, A., Suzuki, S., & Suzuki, M.

(1986). Antitumor effect of hexa-N-acetylchitohexaose and chitohexaose.

Carbohydrate Research, 151, 403–408.

Uchida, Y., Izume, M., & Ohtakara, A. (1989). Preparation of

chitosan oligomers with purified chitosanase and its application. In G.

Skjak-Bræk, T. Anthonsen, & P. Sandford (Eds.), Chitin and chitosan:

Sources, chemistry, biochemistry, physical properties and applications (pp.

373–382). London: Elsevier.