蚯蚓蛋白酶解工艺及其产物分析

采用Asl.398枯草蛋白酶制备蚯蚓肽,通过单因素与正交试验设计(L9(34)),以氨基氮数,多肽浓度为衡量指标,对最佳酶条件进行筛选研究,并分析了酶解液氨基酸含量和相对分子量的分布。结果表明:最佳酶解条件为pH 6.5、酶浓度为1%、温度50℃反应、时间8 h,在此条件下,酶解蚯蚓蛋白的水解液氨基氮数可以达到16.55 mmol/100 mL,水解液多肽浓度达9.22 mg/mL,酶解液分子量大部分是在5 000以下的多肽、小肽及氨基酸的混合物,其中分子量在220以下的占了72.09%,氨基酸组成平衡,含量丰富,可用来制取新型氨基酸微量元素及小肽添加剂。     

蚯蚓对改良土壤和改善农业生态环境的作用

蚯蚓对土壤的耕耘比犁耕更有效,加强土壤通气;提供有益细菌,将土壤中的氨转化成为便于农作物吸收利用的形态;有助于将土壤中各种复合分子分解成为植物根系能够利用的成分。蚯蚓及时将地面残留的落叶枯草裂解转化为对土壤和植物的营养物质,可处理动物粪便以及城市垃圾,对有机农业和生态农业都有重要意义。     

污灌土壤的生态毒性研究

采用污灌区土壤现场采样及室内分析、培养试验方法,进行了污染物(重金属和矿物油)含量分析和植物毒性试验、蚯蚓毒性试验。结果表明,污灌区土壤中矿物油含量为145～1121mg·kg-1,重金属Cd为0.34～1.81mg·kg-1。土壤对植物和蚯蚓显示不同程度的毒性效应。种子发芽根伸长抑制率为2.0%～35.1%,蚯蚓死亡率为0%～40%。体重增长抑制率由14d的-2.3%～-19.4%增加到28d的-2.1%～10.7%。表明土壤中的污染物积累较低,但具有明显的生态毒性。     

蚯蚓脂肪及其在抗菌物理屏障中的作用

以赤子爱胜蚓(Eiseniafetida)为实验动物,采用室内培养方法,研究了细菌浸浴诱导对蚯蚓体内脂肪含量和脂肪酸组成的影响。结果表明:(1)索氏抽提法不能从蚯蚓体腔液中获得脂肪,但是可从蚯蚓的组织中获得脂肪。(2)细菌诱导后,蚯蚓体内的脂肪含量增加0.81%(干物质基础),总脂中的脂肪酸含量均有所增加,其中硬脂酸、油酸、亚油酸增加的幅度较大,分别增加了2.62%,1.58%和1.62%。(3)蚯蚓脂肪的抑菌作用具有选择性,对大肠杆菌的抑菌作用最强,抑菌圈直径为1.32cm;其次为绿脓杆菌,抑菌圈直径为0.97cm,但是对金黄色葡萄球菌没有抑菌作用;大肠杆菌诱导后,蚯蚓脂肪对绿脓杆菌和金黄色葡萄球菌的抑菌作用增强,但是对大肠杆菌的抑制作用减弱。(4)蚯蚓脂肪对大肠杆菌、绿脓杆菌的最低抑菌浓度(MIC)分别为:0.05、0.1g·mL-1;对金黄色葡萄球菌没有抑菌作用。但在细菌诱导后,蚯蚓脂肪对大肠杆菌、绿脓杆菌及金黄色葡萄球菌的最低抑菌浓度(MIC)分别为:0.05、0.1、0.1g·mL-1;(5)蚯蚓的抗菌物理屏障主要是体壁和消化道,而具有抑菌作用的蚯蚓脂肪也主要分布在这些组织中,因此蚯蚓的脂肪代谢在蚯蚓的生态免疫过程中发挥着重要作用。     

兽药添加剂喹乙醇的生态毒理学效应研究

采用水生生物斑马鱼(Daniorerio)、鲤鱼(Cyprinuscarpio)和土壤生物赤子爱胜蚓(Eiseniafoetida)作为生物材料,通过斑马鱼急性毒性试验、斑马鱼胚胎发育试验、鲤鱼肾细胞单细胞凝胶电泳试验、蚯蚓急性毒性试验、蚯蚓生化毒性试验以及蚯蚓体腔细胞单细胞凝胶电泳试验,对兽药添加剂喹乙醇分别从生物个体、细胞和分子水平进行了生态毒理学效应研究。急性毒性试验结果表明,喹乙醇实验浓度范围内对斑马鱼和蚯蚓的急性毒性均不大。胚胎发育试验结果表明,喹乙醇对斑马鱼胚胎的72hEC50为221.20mg·L-1,具有明显致畸效应。蚯蚓生化毒性试验表明,喹乙醇能促进蚯蚓体内SOD酶活性,并呈现一定剂量-效应关系,但对纤维素酶影响不明显,单细胞凝胶电泳试验结果表明,喹乙醇具有一定遗传毒性,能引起鲤鱼肾脏细胞和蚯蚓体腔细胞DNA损伤,与空白对照相比有显著差异,并呈现出一定剂量-效应关系。     

蚯蚓蛋白酶解工艺及其产物分析

采用Asl.398枯草蛋白酶制备蚯蚓肽,通过单因素与正交试验设计(L9(34)),以氨基氮数,多肽浓度为衡量指标,对最佳酶条件进行筛选研究,并分析了酶解液氨基酸含量和相对分子量的分布。结果表明:最佳酶解条件为pH 6.5、酶浓度为1%、温度50℃反应、时间8 h,在此条件下,酶解蚯蚓蛋白的水解液氨基氮数可以达到16.55 mmol/100 mL,水解液多肽浓度达9.22 mg/mL,酶解液分子量大部分是在5 000以下的多肽、小肽及氨基酸的混合物,其中分子量在220以下的占了72.09%,氨基酸组成平衡,含量丰富,可用来制取新型氨基酸微量元素及小肽添加剂。     

蚯蚓对土壤-植物系统中菲、芘降解的强化效应研究

采用盆栽试验方法,研究了蚯蚓（Pheretima sp.）对土壤-植物系统中菲、芘的去除效果的强化作用及对土壤酶活性的影响。结果表明,在试验浓度范围（0~322 mg.kg-1）内,蚯蚓对供试植物苏丹草（Sorghum vulgare L.）生长促进作用显著。接种蚯蚓的土壤中,苏丹草在菲、芘各浓度下总生物量较未接种蚯蚓土壤分别提高14.41%~25.91%、14.39%~23.9%;蚯蚓活动可促进土壤-植物系统中菲、芘的降解,接种蚯蚓处理中菲、芘各浓度下降解率较未接种蚯蚓处理分别高4.20%~9.76%、3.69%~9.38%。土壤酶活性测定结果也显示蚯蚓活动可增加土壤酶的活性。     

土壤中添加蚯蚓及秸秆对油菜积累镉的影响

采用盆栽试验,在不同镉(Cd)污染浓度的灰漠土中分别添加蚯蚓和新鲜秸秆,研究了添加蚯蚓和秸秆还田条件下油菜(Brassica rapa L.spp.Chinenesis)-土壤体系中Cd的分布情况.结果发现:(1)添蚯蚓可有效增加土壤中Cd向油菜体内转移;蚯蚓可以在Cd污染的土壤中生存,蚯蚓体内Cd浓度随土壤环境中Cd...     

Short- and long-term effects of the endogeic earthworm Millsonia anomala (Omodeo) (Megascolecidæ, Oligochæta) of tropical savannas,on soil organic matter

Summary This study aimed to establish the effects of Millsonia anomala, a tropical geophagous earthworm common in the humid savannas of Lamto (Ivory Coast), on soil organic matter dynamics over different time scales under laboratory conditions. The texture of casts produced by the worms fed on a shrub savanna soil was not significantly different from that of the soil, which showed that M. anomala ingested soil particles without selection. Physical fractionation of soil organic matter showed that the coarse organic fraction (250–2000 m) was depleted by 25–30% in fresh casts compared to the control noningested soil; this was mainly due to a fragmentation of coarse organic debris. Incubation of casts and a 2-mm sieved control soil under laboratory conditions for more than 1 year showed that the C mineralisation rate was almost four times lower in the casts (3% year^-1) than in the control soil (11% year^-1). We therefore concluded that on a long time scale M. anomala populations may significantly reduce the decomposition rate of soil organic matter in Lamto savannas.     

Development of biomarkers inAporrectodea caliginosa andLumbricus rubellus for detecting exposure to benzo[a]pyrene

Objectives and Methods. The sensitivity of two biomarkers, the neutral red retention assay (NRRA) and cytochrome P450 were evaluated in the earthwormAporrectodea caliginosa for their potential to detect exposure to benzo[a]pyrene (BaP). Cytochrome P450 was also evaluated in the earthwormLumbricus rubellus, and measured using the substrate ethoxycoumarin. Optimal assay conditions (pH, and temperature) were determined, followed by exposure of earthworms to 20 mg/kg BaP (a typical concentration at contaminated sites in New Zealand). Ethoxycoumarin-O-dealkylase (ECOD) activity was measured after 7, 14, and 21 days exposure. The NRRA was evaluated in earthworms exposed to 0.2, 20, and 100 mg BaP/kg, and biomarker responses were compared with effects on body weight. Results and Discussion Benzo[a]pyrene failed to induce ECOD activity in either earthworm species, and therefore it is not useful as a biomarker of BaP exposure and was not evaluated further. In all cases, the NRRA was significantly affected in the absence of any effects on earthworm body weight, indicating that this assay can detect exposure to BaP at a range of concentrations comparable to those found at contaminated sites. The NRRA should be linked to reproductive endpoints, then it can be used as an early warning indicator of adverse effects. Establishing biomarker stability under environmental conditions is an important step in biomarker development. Therefore, the effects of soil type (sandy soil, silt loam, and a clay soil), moisture content (15–30%), and temperature (5–20°) on the NRRA were determined. In all cases, there was no effect on the NRRA, indicating that this assay is very stable under varying environmental conditions. Conclusion and Outlook In conclusion, cytochrome P450 activity does not appear to be a useful indicator of PAH exposure in eitherA. caliginosa or L.rubellus, and due to the inherently low activity, it is not suitable as a routine biomarker for detecting environmental contamination by these compounds. In contrast, the NRRA in the earthworm A.caliginosa is a promising indicator of PAH exposure at the concentrations likely to be found in contaminated sites in New Zealand, and therefore has potential for evaluating these contaminated sites. If the NRRA can be linked to ecologically important life-cycle endpoints, such as reproduction, then it could be used as an early warning indicator of adverse effects at contaminated sites, i.e., by measuring biomarker responses in earthworms from a ‘contaminated area’ and comparing these with earthworms from a matched control area.