Study On Antibacterial Activity And Mechanism Of Chitosan-Montmorillonite Composite

The side effect of the improper application of antibiotics into feed is more and more concerned in recent years, so the harmless substitute, which has similar effect but with tiny side effect, becomes a hot spot in research. Chitosan and montmorillonite are the most potential alternatives. The former has strong antibacterial activity, good absorption ability and biocompatibility, the latter has a strong ion exchange capacity. In our study, different chitosan-montmorillonite composite (CMC) were made by changing the proportion of chitosan and montmorillonite using intercalation hybrids method. The property of chitosan and montmorillonite is changed by expanding or narrowing the spacing in the layers of the materials. We selected the optimal proportion of chitosan and montmorillonite which is 15:1, named CMC15:1, to study the Minimum Inhibitory Concentration (MIC) and antibacterial curve and so on. The antibacterial activity of CMC 15:1 is studied by MIC, inhibition zones on Escherichia coli ATCC25922 and Staphylococcus aureus ATCC25923. The action site and mechanism of CMC 15:1 is discussed by studying the leakage of a variety of substances in Escherichia coli and Staphylococcus aureus such as phosphorus, ultraviolet light absorption material, protein, reducing sugar, P-galactosidase and TTC-dehydrogenase and by scanning electron microscopy (SEM) as well.We have also used X-ray diffraction (XRD) to study the property of different proportion of CMC, chitosan and montmorillonite. The results are as follows:The first part is mainly about the preparation and selection of CMC, as well as the antibacterial activity of CMC15:1. Firstly, We get CMC with different proportion by intercalation hybrids method, then select CMC whith optimal antibacterial property and suitable proportion by testing their MIC and antibacterial curve, which is CMC 15:1. The results also show MIC of Escherichia coli is 250 mg/L, MIC of Staphylococcus aureus is 125 mg/L. The maximum inhibiton zones of Escherichia coli is 7.87±0.50mm, at the concentration of 10 g/L. The maximum inhibiton zones of Staphylococcus aureus is 9.57+0.21mm, at the concentration of 1.25 g/L. The two bacteria have the integral of the cell morphology after treatment with CMC for 1 h, but the cell integrity is destroved after treatment with CMC for 2 h.The second part mainly studies on the antibacterial mechanism of CMC 15:1, which contains the location and strength of action by testing the leakage of various intracellular substances of bacteria, and observing the cell morphology via the scanning electron microscopy (SEM). In the study, the leakage of the intracellular substances such as inorganic phosphorus, protein, ultraviolet light absorption material, P-galactosidase and TTC-dehydrogenase in Escherichia coli is slower than the cell death, while the leakage of intracellular substances in Staphylococcus aureus is synchronized with the cell death. We also find the small molecules such as phosphorus in Escherichia coli leak by destroying the osmotic pressure of cell, while the macromolecules such as ultraviolet light absorption material leak through destroying the integrity of cell. But the leakage of phosphorus and ultraviolet light absorption material in Staphylococcus aureus is by disrupting the cellular integrity. The leakage of reducing sugar in the twobacteria may destroy the energy system of cell. Via SEM, we find the antibacterial mechanism of CMC15:1 on Escherichia coli and Staphylococcus aureus is not similar. For Escherichia coli, CMC 15:1 plays a major role in adsorption, flocculation and so on, an auxiliary role of destroying cell wall and cell membrane, even CMC 15:1 can enter into the cell and gather the cell contents. But for Staphylococcus aureus, CMC 15:1 mainly affects and destroys the cell wall and cell membrane, and causes the leakage of cell contents, then CMC 15:1 gathers the cell contents after entering into the cell and CMC 15:1 plays certain role in flocculating Staphylococcus aureus. Also CMC15:1 can play a destructive effect in energy system of the two bacteria. However, Whether the CMC 15:1 plays an role in DNA or RNA is unknown, which need further research.The third part is XRD of the different proportion of CMC, chitosan and montmorillonite. By the XRD, we can understand the reason that CMC 15:1 is better than the other materials, is the spacing in the layers of montmorillonite which is larger than the others after chitosan inserts into it.