| With such a long coastline, our country has rich resources of shrimp and crab which are widely found in crustaceans. The annual yield of chitosan is 10 billion tons which is slightly lower than cellulose. Compared with the general inhibitor, chitosan has its own characters such as wide antibacterium, high antibacterial rate, non-toxic and no side effect. Therefore, it is necessary to study the degradation of chitosan. In this paper, four factors that affected chitosan degradation in acetic acid homogeneous system were investigated. Then the inhibitory effect of chitosan with different molecular weight on four kinds of bacteria was studied. Finally, the dynamics and morphology of different bacteria acted by chitosan were examined, and we preliminarily expored the antibacterial mechanism of chitosan. The results were showed as below:(1) High molecular weight chitosan was degraded in hydrogen peroxide-acetic homogeneous system. Terminal group method, viscosity method and titration method were used respectively to determine the molecular weight, intrinsic viscosity and deacetylation degree. The effect of degradation temperature (T), reaction time (t), concentration of H2O2 (C1) and acetic acid(C2) to molecular of chitosan were explored. The results showed that when degradation temperature was at 30-70℃, reaction time was 30-80 minutes, concentration of H2O2 was 3%-8% and concentration of acetic acid was 1%-2%, the best degradation effect was obtained.(2) Orthogonal experiment was designed to investigate the significant influence of T, t and C1 on the degree of deacetylation (D.D.), intrinsic viscosity (η) and molecular weight (M). The results showed that the significance of T, t and C on η, D.D. and M were T>C>t, T>t>C and T>C>t, respectively. The optimum condition of oxidative degradation of chitosan was 70℃,70 min,12% H2O2 and 1% HAC.(3) The antibacterial effect of different molecular weight chitosan, D.D. of chitosan, concentration of chitosan and solution pH value on 76-Ralstonia solanacearum, RS-Ralstonia solanacearum,199-Bacillus cereus and 302-Paenibacillus polymyxa were systematically explored. Then, minimum inhibitory concentration (MIC) values of chitosan to the strains above were determined. The results were as follows:the growth of four strains was significantly inhibited by seven different molecular weight chitosan; the better antibacterial effect of chitosan was obtained at low molecular weight, high degree of deacetylation and high concentration. The chitosan with molecular weight of 1500 and 40000 had the best inhibitory effect on 76-Ralstonia solanacearum and RS-Ralstonia solanacearum; but to 199-Bacillus cereus and 302-Paenibacillus polymyxa, inhibition of the chitosan above significantly weakened. When degree of deacetylation of chitosan was bettwen 88%-92%, the antibacterial rate was above 80%. When the pH was 6.0-6.5, the antibacterial rate was nearly 100%.(4) The results showed that the influence of divalent metal ion on antimicrobial activity of chitosan was greater than monovalent metal ion to some extent. The effect order of antibacterial activity on 76-Ralstonia solanacearum and RS-Ralstonia solanacearum was Ca2+>Na+>K+>Mg2+. For 199-Bacillus cereus and 302-Paenibacillus polymyxa the order was Ca+>Mg+>K+>Na+.(5)The dynamics and antibacterial curve of bacteria acted by chitosan with molecular weight of 1500 and 40000 were studied. The reaserch showed that:the antibacterial mechanism of chitosan with different molecular weight were distinct; chitosan with the same molecular weight on different bacteria also had different antibacterial mechanism.(6) The morphology of bacteria acted by chitosan were then studied. The results showed that:treated by chitosan of molecular weight 1500, the morphology of 76-Ralstonia solanacearum was completely induced from circular to filament, but the morphology of RS-Ralstonia solanacearum and 302-Paenibacillus polymyxa were only partially induced. The morphology of RS-Ralstonia solanacearum was completely induced to filament while treated by chitosan with molecular weight of 40000, while the morphological of 199-Bacillus cereus and 302-Paenibacillus polymyxa were partially induced from rod to circle. |
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