Growth inhibitory effect on bacteria of chitosan membranes regulated with deacetylation degree

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Abstract

Antibacterial activity of chitosan membranes was investigated by a conductimetric assay using a Bactometer. The purpose of this investigation was to produce a practical, high-performance membrane for separation engineering. The antibacterial activity of powdered chitosan membrane was evaluated by the minimal inhibitory concentration (MIC). The MIC for Escherichia coli was almost 200 (mg-chitosan/ml-bacterial suspension), and for Staphylococcus aureus it was 40 (mg-chitosan/ml-bacterial suspension). Growth of the gram-positive sample (S. aureus) was more strongly inhibited by chitosan than the gram-negative sample (E. coli). This inhibitory effect was recognized as a bactericidal effect. Antibacterial activity was also observed and depended on the shape and the specific surface area of the powdered chitosan membrane. The influence of the deacetylation degree (DD) of the chitosan on inhibiting the growth of S. aureus was investigated by two methods: incubation using a mannitol salt agar medium, and a conductimetric assay. By both methods, chitosan with a higher DD successfully inhibited growth of S. aureus. Our findings regarding the dominant role of the DD of chitosan will be useful for designing long-life, hygienic, membrane-based processes.

Introduction

For years, material development focused on growth inhibition of bacteria was expected to lead to long-life, hygienic, membrane-based processes. The growth of microorganisms and the accumulation of colloids or organic compounds were major causes of membrane fouling, generally called “biofouling” [1]. Membrane fouling negatively influenced the permeation flux and some aspects of membrane performance (e.g., reduced salt rejection and elevated operational pressure [2], [3], [4], [5]).

The development of membrane materials with antibacterial activity is important from both the economic viewpoint and for the hygienic management of practical membrane processes. Chitosan produced from crustacean shells is an attractive material for reducing biofouling. The authors previously reported a typical molecular characteristic of chitosan membranes, namely, the water permeability of these membranes when used to control the deacetylation degree (DD) [6]. An examination of the practical aspects of using chitosan membranes is necessary for developing membrane processing techniques.

Chitosan and its resolvent inhibit the growth of mold with plant pathogenicity [7], [8]. In contrast, chitin does not inhibit the growth of mold [9]. The effect of chitosan concentration on the growth of mold (Fusarium solani, Fusarium oxysporum) was investigated. Mold growth was completely inhibited by 0.1% chitosan. A higher DD has a stronger growth inhibitory effect on mold [10].

According to Uchida [10], chitosan inhibited bacterial growth not only of gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa) but also of gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus). For example, after 4 days of incubation, bacterial growth was completely inhibited in broth containing a chitosan concentration of 0.02% at pH 6.0. Lower viscosity (i.e., lower molecular weight) chitosan had a stronger growth inhibitory effect on bacteria.

According to No et al. [11], antibacterial activities of six chitosans and six chitosan oligomers with different molecular weights were examined against four gram-negative (including E. coli) and seven gram-positive bacteria (including S. aureus). Chitosans showed higher antibacterial activities than chitosan oligomers, and markedly inhibited growth of most of the bacteria tested. Chitosan generally showed stronger bactericidal effects towards gram-positive bacteria than gram-negative bacteria. In addition, antibacterial activity of chitosan was inversely affected by pH (in the range tested, pH 4.5–5.9), with higher activity at lower pH values. These results were reported within the context of potential applications of liquid-soluble chitosan.

Various rapid examination techniques for antibacterial activity have been suggested, including turbidimetry [12], an ATP assay [13], calorimetry [14], an impedance assay [15], and conductimetric assays [16], [17], [18], [19]. The present study employed the conductance method to evaluate the antibacterial activity of chitosan membranes; the method was based on the detection time of electric conductance.

Bacteria are generally classified according to gram dyeing. E. coli 745 (gram-negative) and S. aureus 9779 (gram-positive) were employed to evaluate the antibacterial activity of chitosan. These bacteria are representative of the bacterial species that are important in public sanitation and food hygiene. The antibacterial activity of chitosan membranes as a solid system for regulating the deacetylation degree was investigated by a direct conductimetric assay using a Bactometer. In this paper, chitosan was examined as a solid, not as a solute in solution as in previous papers. This approach will contribute to the design of a biopolymer membrane with high performance and long life for use in practical separation processing applications.

Section snippets

Preparation of chitosan membranes

Chitosan (low molecular weight, Sigma–Aldrich Japan K.K., Tokyo) and polyethylene glycol (MW7500, Wako, Osaka) were dissolved in 10% acetic acid. The chitosan solution was diluted to 2% (w/w) with methanol. Acetic anhydride (97.0%, Wako, Osaka) was added to the chitosan solution after vacuum filtration. The resultant casting solution was dried in a petri dish for 12 h at 333 K and subsequently gelled by immersing it in 4% NaOH. The resultant product was washed with distilled water, and the

Blank test for initial electrical conductivity

Blank tests are necessary for judging the reproducibility and reliability of the conductimetric assay using the Bactometer employed in this paper. Table 1 presents the blank initial conductance values for various experimental well conditions in the modules. The electrical conductivity for the initial 48 h in bacteria-free systems was almost constant. Table 2 presents the conductivity for different DDs of chitosan. The conductivity did not depend on the DD of chitosan. These blank tests indicate

Conclusion

Direct conductimetric assays using a Bactometer clearly demonstrated the antibacterial activity of chitosan membranes differing in their deacetylation degree. The influence of chitosan membranes on E. coli and S. aureus, which are food hygiene index bacteria, was investigated. Chitosan inhibits the growth of S. aureus more strongly than it inhibits the growth of E. coli. Chitosan inhibited the growth of gram-positive samples more strongly than that of gram-negative samples, and the effect was

Acknowledgements

The authors express sincere gratitude to Professor Mikio Kikuchi of the Kanagawa Institute of Technology for conducting the bacterial experiments and for useful discussions. The authors sincerely appreciate the Keyence Corporation for their photographing technique through the Scanning Electron Microscope Model VE-7800.

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1

Present address: Department of Industrial Chemistry, Faculty of Engineering Division 1, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan.

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