降解烟碱微生物的筛选、鉴定及降解条件优化
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摘要
烟碱又名尼古丁(nicotine)属于吡啶族生物碱,在烟草生物碱中烟碱是最主要生物碱,烟碱是一种中枢神经兴奋剂,吸食后使人产生生理和心理上的依赖性,是让人成瘾的物质。利用微生物处理来降解烟碱这种方法不会造成烟香的损失还可以改进烟气品质,可以提高烟叶资源的利用率,有研究开发价值,更可以有效降低尼古丁对人的健康的损害,减少烟草工业产生的大量的废弃物对生态环境的污染。因此其开发应用将具有巨大的发展潜力和应用前景。
本文以湖南省浏阳县烟草土壤为试验材料,从中筛选提取一株有效降解烟碱的微生物,并且进行鉴定、确定其最佳生长条件及其降解烟碱最大的发酵条件,从而为微生物降烟碱的研究和应用提供了依据。
(1)以浏阳县烟草土壤为试验材料,进行筛选,得到20株对烟碱有降解能力的菌株,并且通过紫外分光光度法测所得菌株在烟碱培养基的培养下2、4、6天的降解烟碱能力,从中筛选出一株有效的降解烟碱能力最大的菌株z1-3-3-1,其48h内降解率为72.48%。
(2)对所筛选的到的有效降解烟碱能力最大的菌株z1-3-3-1先进行形态学鉴定,从菌落形态和个体形态进行分析；再进行分子水平鉴定,从提取DNA、电泳测试、PCR扩增、电泳验证、同源性基因比对鉴定菌株的属性,结果显示z1-3-3-1菌株为黄曲霉。
(3)对此菌株进行不同碳源、氮源的烟碱培养基培养,确定菌株最佳适应条件；还进行不同PH值、温度、烟碱浓度及不同接种量的测试进行烟碱培养基的培养,寻找菌株最大降解条件,并且最后进行交叉正交试验,确定菌株z1-3-3-1的最佳生长和降解烟碱的条件为：烟碱含量0.1%、温度28℃、pH7.0、转速120 r/m、接种量8%、柠檬酸三钠含量2.0%、蛋白胨1.0%。为菌株以后的研究和应用提供依据。
Alkaloid of tobacco is also known as nicotine, belonging to pyridine alkaloids. Nicotine is the main alkaloid in tobacco, which is a central nervous stimulator, and addictive substance after taking, causing physical and psychological dependence. Microbiology are used to degrade nicotine in tobacco, this approach will not cause the loss of incense smoke and can also improve gas quality、increase resource utilization value of tobacco, it can also reduce the damage of nicotine to human health, and lessen large amounts of waste generated in the tobacco industry on the ecological environment. Therefore, this study will have great development and application potential. Tobacco soil from liuyang of Hunan province was used as an experimental material. One microbiological strain was screened and selected to see the effectivity of the degrading nicotine. Then identification of the optimum growth and degrading of nicotine could be studied to provide a basis for the microbiology in practice.
(1) Tobacco soil from liuyang was used as experimental material to screen strains degrading nicotine, their rate of degrading nicotine wre determined by UV spectrophotometer after the stains were cultured 2、4、6 days. The best one which can degrade nicotine effectively is z1-3-3-1, its degration is 72.48% in 48h.
(2) Firstly the strain z1-3-3-1 was identificated from its Morphology, for example its colony morphology and individual morphology, then it was identificated from the molecular level, extracting its DNA.electrophoresis check, PCR amplification.electrophoresis check and homologous gene comparison to Clearly identified that strain's Properties.
(3) The strain was cultured in different carbon source、Nitrogen source、pH、temperature、nicotine concentration and inoculum concentration to determine its best growth conditions, the result is that nicotine concentration 0.1%, temperature 28℃, pH7.0, rotate speed 120 r/m, inoculum concentration 8%, Sodium citrate concentration 2.0%, peptone concentration 1.0%.
本文以湖南省浏阳县烟草土壤为试验材料,从中筛选提取一株有效降解烟碱的微生物,并且进行鉴定、确定其最佳生长条件及其降解烟碱最大的发酵条件,从而为微生物降烟碱的研究和应用提供了依据。
(1)以浏阳县烟草土壤为试验材料,进行筛选,得到20株对烟碱有降解能力的菌株,并且通过紫外分光光度法测所得菌株在烟碱培养基的培养下2、4、6天的降解烟碱能力,从中筛选出一株有效的降解烟碱能力最大的菌株z1-3-3-1,其48h内降解率为72.48%。
(2)对所筛选的到的有效降解烟碱能力最大的菌株z1-3-3-1先进行形态学鉴定,从菌落形态和个体形态进行分析；再进行分子水平鉴定,从提取DNA、电泳测试、PCR扩增、电泳验证、同源性基因比对鉴定菌株的属性,结果显示z1-3-3-1菌株为黄曲霉。
(3)对此菌株进行不同碳源、氮源的烟碱培养基培养,确定菌株最佳适应条件；还进行不同PH值、温度、烟碱浓度及不同接种量的测试进行烟碱培养基的培养,寻找菌株最大降解条件,并且最后进行交叉正交试验,确定菌株z1-3-3-1的最佳生长和降解烟碱的条件为：烟碱含量0.1%、温度28℃、pH7.0、转速120 r/m、接种量8%、柠檬酸三钠含量2.0%、蛋白胨1.0%。为菌株以后的研究和应用提供依据。
Alkaloid of tobacco is also known as nicotine, belonging to pyridine alkaloids. Nicotine is the main alkaloid in tobacco, which is a central nervous stimulator, and addictive substance after taking, causing physical and psychological dependence. Microbiology are used to degrade nicotine in tobacco, this approach will not cause the loss of incense smoke and can also improve gas quality、increase resource utilization value of tobacco, it can also reduce the damage of nicotine to human health, and lessen large amounts of waste generated in the tobacco industry on the ecological environment. Therefore, this study will have great development and application potential. Tobacco soil from liuyang of Hunan province was used as an experimental material. One microbiological strain was screened and selected to see the effectivity of the degrading nicotine. Then identification of the optimum growth and degrading of nicotine could be studied to provide a basis for the microbiology in practice.
(1) Tobacco soil from liuyang was used as experimental material to screen strains degrading nicotine, their rate of degrading nicotine wre determined by UV spectrophotometer after the stains were cultured 2、4、6 days. The best one which can degrade nicotine effectively is z1-3-3-1, its degration is 72.48% in 48h.
(2) Firstly the strain z1-3-3-1 was identificated from its Morphology, for example its colony morphology and individual morphology, then it was identificated from the molecular level, extracting its DNA.electrophoresis check, PCR amplification.electrophoresis check and homologous gene comparison to Clearly identified that strain's Properties.
(3) The strain was cultured in different carbon source、Nitrogen source、pH、temperature、nicotine concentration and inoculum concentration to determine its best growth conditions, the result is that nicotine concentration 0.1%, temperature 28℃, pH7.0, rotate speed 120 r/m, inoculum concentration 8%, Sodium citrate concentration 2.0%, peptone concentration 1.0%.
引文
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