Precise quantification of the antibacterial activity of chitosan by NB medium neutralizer

doi:10.1016/j.jmst.2020.09.004

Abstract

Abstract:

In the present research, nutrient broth (NB) medium was identified to be able to neutralize the antibacterial activity of chitosan and its derivatives. Therefore, an improved test method independent of NB medium was proposed to precisely quantify the antibacterial effectiveness and efficiency of chitosan. The minimum bactericidal concentration (MBC) of chitosan was 60 μg mL ^-1 against S. aureus and E. coli, and 0.01 % (w/v) chitosan could kill 100 % of bacteria within 3 min. From another point of view, the neutralizing efficiency of NB could be tripled by adding 25 g L ^-1 of sodium chloride. Then the neutralizing mechanism of NB medium was ascribed to flocculation between chitosan and protein. Adding extra sodium chloride could significantly reduce the size of floccules, and smaller floccules would lose the ability of binding with bacteria directly, showing higher neutralizing rate on the macro scale.

Key words: Chitosan, Antibacterial activity, Neutralizer, Flocculation

Mengyang Wang, Shichao Bi, Jianhui Pang, Zhongzheng Zhou, Di Qin, Honglei Wang, Xiaojie Cheng, Xiguang Chen. Precise quantification of the antibacterial activity of chitosan by NB medium neutralizer[J]. J. Mater. Sci. Technol., 2021, 70: 224-232.

Figures/Tables 11

Table 1 Composition of each group in neutralizer identification and evaluation test (μL).

Group	Water	Bacteriostat	Neutralizer	Diluted bacterial suspension	Total volume
1	4900	0	0	100	5000
2	4850	50	0	100	5000
3	350	50	4500	100	5000

Fig. 1. Antibacterial rates of 0.01 % (w/v) CSA, CS/Ac, TMC, CMCS and HBC solution against S. aureus and E. coli (a) and the neutralizing rates of NB neutralizers (b). Error bars indicates standard deviation (n = 3 repeats), and different letters above bars indicate significant differences between samples (All results were analyzed using two-way ANOVA followed by a post-hoc Tukey test, p < 0.05).

Fig. 2. Neutralizing rates of adding different volumes of NB neutralizer in the reaction system. Results were expressed as mean ± SD (n = 3).

Fig. 3. Images of S. aureus and E. coli colonies survived on the plates after treatment with different concentrations of CSA. Survived colonies are circled in red.

Fig. 4. Time-killing rate curves of CSA against S. aureus and E. coli. Results were expressed as mean ± SD (n = 3).

Fig. 5. Neutralizing rates of NB’s single and combinatorial composition (a) and neutralizing rates of NB neutralizer with different final concentrations of NaCl (b). Results were expressed as mean ± SD (n = 3).

Fig. 6. Images of neutralizing product solution reacted by FITC-chitosan acetate and NB neutralizer.

Fig. 7. Fluorescent images of neutralizing product solution reacted by FITC-chitosan acetate and NB neutralizer with 5 g L-1 (a), 7.5 g L-1 (b), 10 g L-1 (c), 15 g L-1 (d), 20 g L-1 (e) and 25 g L-1 (f) of NaCl.

Fig. 8. Schematic diagrams of neutralizing mechanism of standard NB neutralizer (a) and high salt NB neutralizer (b).

Fig. 9. Bright field and fluorescence micrographs of neutralizing product solution reacted by Calcein-AM stained bacteria, chitosan acetate and NB neutralizer with 5 g L-1, 15 g L-1 and 25 g L-1 of NaCl. Blue arrows indicated free bacteria, red arrows indicated floccules without bacteria and yellow arrows indicated floccules with bacteria adsorption.

Fig. 10. Neutralizing rates of neutralizing product and neutralizing product filtrated by 0.22 μm membrane filter. Number in the brackets means the volume of neutralizer added in the reaction system. Results were expressed as mean ± SD.

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