Abstract
In order to study the antibacterial activity of chitosan metal chelates prepared in magnetic effect, the antibacterial activities of these chelates on Staphylococcus aureus were investigated by the agar diffusion paper method. The minimum inhibition concentrations of chitosan-metal chelates were measured. With different degrees of substitution, the inhibition efficiency of the chitosan-metal chelates is different. The inhibition of chitosan on S. aureus increased with the chitosan concentration. Among the chitosan-metal chelates, the inhibition efficiency of CS-Cr is the best. The inhibition efficiency of chitosan-metal chelates prepared in the magnetic field of 400 kA/m on S. aureus is higher than the inhibition efficiency of chitosan-metal chelates prepared without the magnetic field enhanced. The minimum inhibitory concentrations are, respectively, as CS-Cu: 12.5 mg/mL, CS-Pb: 6.25 mg/mL, CS-Cr: 3.125 mg/mL. It is well known from the results that chitosan-metal chelates maybe applied in antibacterial process.
Introduction
Many of the metal ions are broad-spectrum fungicides and have better antibacterial activities on Escherichia coli, Staphylococcus aureus, and others (1-2-3). For example, antibacterial mechanisms of Ag+ and Cu2+ are that metal ions hampered electronic delivery systems and damaged cell membranes. Chitosan (CS), a linear polysaccharide, (1, 4)-linked 2-amino-2-deoxy-b-D-glucose and 2-acetamido-2-deoxy-b-D-glucose units, is one of the most promising biomaterials in the world, not only because of its abundance but also due to its various properties. CS and its derivatives have excellent biological activities and have been widely used in chemical industry, food, cosmetics, environmental protection, Chinese medicine, and many other areas (4-5-6-7). At the same time, CS can react with many substances. Amido and the neighboring hydroxyl in the molecule react with many metal ions such as Ag+, Cu2+, Pb2+, etc. to form stable chelates (8). Assuming that CS has antibacterial activities, we suppose that these chelates may be used as secondary energy. In order to verify this supposition, the inhibition activities of chitosan metal chelates (CS-Cu, CS-Pb, CS-Cr), formed by the chitosan reacting with metal ions: lead, copper and chromium, on S. aureus have been studied and the effect of magnetic field on the antibacterial activities of chitosan metal chelates on S. aureus have been explored in this paper.
Experiment
Materials
CS (provided by special new fast food Industrial Co., Ltd. Chaozhou City Guangdong Province, China); lead nitrate; cupric sulfate; potassium dichromate; sodium hydroxide, were analytical reagent; nutrient broth; nutrient agar.
Tested bacteria: S. aureus (from microbiology laboratories of food college of South China University of Technology). CTS24 Hall effect of strong magnetic figures; culture chest; Vernier caliper.
Preparation of CS metal chelates
Metal salts used for the batch adsorption experiment were of analytical reagent grade: Pb(NO3)2, CuSO4, KCr2O7. CS weighed accurately was reacted with 10 mL ion solution in 15 mL centrifuge tube, and according to our previous researches, the optimal magnetic field intensity of magnetic field device used the experiments was 400 kA/m (9). After 4 hours, it was centrifuged and filtered, then dried under vacuum until a constant weight. So CS metal chelates (CS-Cu, CS-Pb, CS-Cr) were prepared under magnetic field. Similarly, they were prepared without magnetic field.
Antibacterial assay
The antibacterial activities of CS metal chelates, CS against S. aureus were carefully evaluated by agar diffusion method. The bacterial growth curve and the antibacterial curves were measured by turbidimetric method.
Preparation of cell suspensions: S. aureus was incubated overnight in nutrient broth (1% peptone, 0.3% beef extract and 0.5% sodium chloride, pH 7.4) at 37°C. The culture mediums obtained were diluted with autoclaved distilled water to obtain cell suspensions with optical absorbance at 580 nm of 0.3 ± 0.02.
Agar diffusion method
Filter papers of 6 mm were put into the different concentration solutions of CS metal chelates and CS, respectively. Then the solutions and distilled water were autoclaved at 121°C for 15 min. Cell suspension of 100 mL were evenly coated in the surface of plate of nutrient agar (1% peptone, 0.3% beef extract and 0.5% sodium chloride, 1.5% agar, pH 7.3). After that, one filter paper from autoclaved distilled water were affixed in the middle of the plate around with four filter papers from the solution of CS metal chelate or CS, incubated 24 h at 37°C. The diameter of each inhibition zone was measured by Vernier caliper. The concentrations of solutions were 50, 25, 12.5, 6.25 mg/mL, respectively. Every experiment was repeated four times.
Minimum inhibitory concentration (MIC): different concentrations of solutions were dropped into melting agar of 15 mL to make each tube contents of 50, 25, 12.5, 6.25, 3.125, 1.5625 mg/mL, respectively. After sterilization and cooling, 107-108 FU/mL cell suspension of 100 mL were evenly coated in the surface of the plate and incubated for 24 h at 37°C. Observing the growth of S. aureus, the MICs of CS metal chelates in whose plate bacteria did not grow were considered as the MICs.
Growth curve analyses and the time-kill study
Cell suspension of 100 mL was put into two shares nutrient broth and compared the cultivations of CS metal chelates and CS. Samples were withdrawn at selected time points, and absorbency was estimated at 600 nm.
Results and discussion
Antibacterial activity of CS metal chelates
Agar proliferation of paper was applied to measure diameters of the inhibition zones. The results were shown in Table I. From Table I, we can see that the inhibition of CS on S. aureus increased with the CS concentration.
Diameters of the inhibition zones of CS metal chelates on S. aureus
Chitosan metal chelates treated by 400 kA/m magnetic field.
The diameters of the inhibition zones of all kinds of CS metal chelates were sharply different. The inhibitory effect of CS-Cr was most significant in the experiment without assistance by magnetic field and the diameter of inhibition zone was 9.8 mm when the concentration of CS-Cr was 50 mg/mL.
Table I shows diameters of the inhibition zones of different concentrations of CS metal chelates synthesized by assistance with 400 kA/m magnetic field. It was known that the inhibition effect of all kinds of CS-Cu, CS-Pb, CS-Cr treated by magnetic field on S. aureus had strengthened. Especially, the most marked increase belonged to CS-Cr. So it proved that the inhibition effect of CS metal chelates on S. aureus could be strengthened after chelates treated by magnetic field because of increase of preparation yield.
The MIC of all kinds of CS metal chelates prepared in magnetic field of 400 kA/m to S. aureus is shown in Table II.
MIC of chitosan metal chelates before and after treated by 400 kA/m magnetic field to S. aureus
Antibacterial activity was poor and the MIC was not measured.
From Table II, it can be seen that after being treated by the magnetic field of 400 kA/m, the MIC of all kinds of CS metal chelates to S. aureus was CS-Cu:12.5 mg/mL, CS-Pb: 6.25 mg/mL, CS-Cr: 3.125 mg/mL.
The time-kill curves
Time kill curves are shown in Figure 1. The experimental concentrations of all CS metal chelates were 50 mg/mL. It can be seen from Figure 1, the antibacterial activities of all CS metal chelates treated by the magnetic field were enhanced and different chelates treated by the same magnetic field were different (9). The strength of the antibacterial activities of the CS metal chelates dealt with magnetic field of 400 kA/m was CS-Cr >CS-Pb >CS-Cu.
The Time-kill curves of chitosan metal chelates.
Discussion
From the results of the experiment, the inhibition of CS on S. aureus was increased with the CS concentration. Antibacterial activity of CS due to positively charged substituents -NH3+ on the molecular chain. S. aureus cell wall contains a lot of teichoic acid with negative charge which will combined with -NH3+ of CS to change the cell membrane permeability, disrupting the cell's normal physiological function (10). Therefore, the higher the concentration of CS containing more -NH3+ in the solution, the higher the antibacterial activity of CS on S. aureus.
Research shows that metal ions can weaken the antibacterial activity of CS to some extent. This may be because CS is able to chelate the metal ions in the environment, forming salt – CS complexes so as to reduce the CS activated charge, and then reduce the CS antibacterial activity caused by the ion concentrations in the environment change (11).
In addition, antibacterial activity of CS metal chelates on S. aureus were strengthened by magnetic field of 400 kA/m. The mechanisms of magnetic field on the antibacterial activity of biological systems are mainly the following: cytoplasmic membrane science ion theory, quantum state interference theory, free radical theory and theory of Lorentz force. The theory of this article needs further research.
Conclusion
Experimental results show that the extent of antibacterial effect of various CS metal complex on S. aureus was different and the inhibitory effect of CS-Cr was the most notable; antibacterial effects on S. aureus were strengthened by magnetic field of 400 kA/m.
The studies of this article show that chelates prepared after the recovery of metal ions can be used to inhibit bacteria as a secondary energy; CS metal chelates have a certain relevance in food and water treatment processing.
Footnotes
Financial support: The authors are thankful to the financial support from the Science and Technology Foundation of Guangdong (No. 2014A020221004 and 2014A020215004), Technology Research Foundation of Basic Research Project of Shenzhen (No. JCYJ20150401171152771 and JCYJ20150330102401102), Postdoctoral Science Foundation of China (No. 2015M580759).
Conflict of interest: None of the authors has financial interest related to this study to disclose.