壳寡糖对大肠杆菌的抑制作用

Antibacterial activity of chitosan oligosaccharide on Escherichia coli

背景:已有实验证明,抗菌抑菌作用是壳寡糖的最主要活性之一。目的:研究壳寡糖对大肠杆菌的抑菌效果。设计、时间及地点:生物工程实验,于2007-06/2008-10在河南师范大学微生物实验室完成。材料:壳聚糖为郑州世纪百川食品添加剂有限公司产品,脱乙酰度93%,相对分子质量290000。方法:高产壳聚糖酶菌株接种至5L的发酵罐中发酵培养,所得壳聚糖酶粗酶液经乙醇沉淀获得壳聚糖混合酶。壳聚糖混合酶经过HiTrapSP阳离子交换柱分离纯化得壳聚糖外切酶和壳聚糖内切酶,分别用壳聚糖混合酶,壳聚糖外切酶和壳聚糖内切酶降解质量浓度10.0g/L壳聚糖,最终制备5种不同相对分子质量壳寡糖S1,S2,S3,S4,S5,相对分子质量分别为500,2100,3700,5200,9000,在不同浓度不同pH值下对大肠杆菌抑菌活性进行实验分析。主要观察指标:不同相对分子质量的壳寡糖对大肠杆菌生长的影响;不同质量浓度壳寡糖对大肠杆菌抑菌活性;pH值对壳寡糖抑菌活性的影响。结果:在8h之内,5种壳寡糖对大肠杆菌均有明显的抑菌效果。培养8h之后:S1,S4和S5的抑菌效果逐渐减弱。培养16h之后:S1培养基中大肠杆菌的数量超过正常状态。相对分子质量2100的壳寡糖对大肠杆菌抗菌作用最强。其抑菌作用随壳寡糖质量浓度的增加而增强。测得pH值5.0,5.5,6.0,6.5和7.0壳寡糖S2溶液对大肠杆菌抑菌活性分别为0.097,0.086,0.061,0.081,0.091,在pH值6.0附近,壳寡糖S2对大肠杆菌抑制作用最好。结论:壳寡糖的相对分子质量大小是抑制大肠杆菌生长的主要影响因素。

BACKGROUND： Previous research has proved that one of the most roles of chitosan oligosaccharide is antibacterial and bacteriostatic activity. OBJECTIVE： To observe the antibacterial activity of chitosan oligosaccharide on Escherichia co/i. DESIGN, TIME AND SETTING： A bioengineering study was performed at Microbiology Laboratory of Henan Normal University between June 2007 and October 2008. MATERIALS： Chitosan oligosacchadde was yielded by Shiji Baichuang Food Additives Co., Ltd., Zhengzhou Province. Deacetylation degree was 93% and relative molecular mass was 290 000. METHODS： The chitosan strains were incubated in a 5-L fermentor, and the obtained raw solution of chitosan was precipitated to prepare mixed enzyme. Finally exonucleases and endonucleases were purified by HiTrap SP positive ion exchange column. The mixed enzyme, exonucleases and endonucleases with the chitosan concentration of 10.0 g/L were used to produce chitosan oligosaccharide of five different relative molecular masses （S1： 500, S2：2 100, S3：3 700, S4：5 200, and S5：9 000）. Thereafter, antibacterial activity on Escherichia coil was performed under the environments of different concentrations and different pH values. MAIN OUTCOME MEASURES： Effect of chitosan oligosaccharide with different relative molecular masses on growth of Escherichia coil; antibacterial activity of chitosan oligosaccharide with different concentrations on Escherichia coil; effect of different pH values on antibacterial activity of chitosan oligosaccharide. RESULTS： Within 8 hours, all concentrations of chitosan oligosaccharide had effectively antibacterial activity on escherichia coll. After 8 hours, antibacterial activity of S1, S2, and S5 decreased; in addition, after 16 hours, numbers of Escherichia coil in S1 culture medium were more than normal one. In particular, the antibacterial activity of chitosan oligosaccharide of the relative molecular mass 2100 was the strongest. Furthermore, the chitosan oligosacchadde was enhanced with the concentration increasing. Antibacterial activity of S2 was respectively 0.097, 0.086, 0.061, 0.081, and 0.091 at different pH values of 5.0, 5.5, 6.0, 6.5, and 7.0. Especially, the antibacterial activity of S2 was the strongest at pH=6.0. CONCLUSION： Relative molecular mass of chitosan oligosaccharide is the major factor of antibacterial activity.