微生物膜诱导刺参幼体附着变态的活性物质研究

Chemical cues derived from bacteria and benthic diatoms of biofilms induce larval settlement and metamorphosis of many marine invertebrates, whereas few have been fully characterized. Biofilms are commonly formed as the larval substrata in the breeding of the sea cucumber Apostichopus japonicus, but their inductive mechanism remains unclear. This leads to the low and unstable percentages of larval settlement and metamorphosis, and high mortality of larvae and juveniles prior to and after this process. Therefore, it is crucial to elucidating the inductive activities of bacteria and benthic diatoms of biofilms and their derivative chemical cues..In this research project, culture conditions of the inductive bacterium and benthic diatom, which were isolated and screened for activity by our preliminary study, will be optimized for the large-scale fermentation and culture. The active ingredients will be crudely extracted from the bacterial and diatom metabolites, respectively, by monitoring the inductive activity of larval settlement and metamorphosis. Chemical cues will be subsequently isolated and purified through chromatographic procedures and further characterized by spectrographic techniques. Furthermore, the structure-activity relationship of the chemical cues from the two different organisms will be analyzed by comparing their structures and thereafter combing the examination of inductive activities of their analogues. The last is to in situ determine the presence and concentrations of the active compounds in biofilms and biofilm conditioned-seawater, which are either characterized or deduced by the structure-activity relationship, and to further propose the theoretical basis for the optimization of biofilm inductive activities..Our research achievements will further the understanding of the larval development of echinoderms from the viewpoint of marine chemical ecology and also contribute to the sustainable development of sea cucumber aquaculture.

微生物膜中的细菌与底栖硅藻产生的活性物质诱导多种海洋无脊椎动物幼体的附着变态，但被完全鉴定的仍然较少。刺参苗种生产中常采用微生物膜形成幼体的附着基，而由于微生物膜诱导机理不明，幼体附着变态率低且不稳定、附着变态前后幼体与稚参大量死亡。因此，阐明膜中细菌与底栖硅藻的活性以及其产生的活性物质是解决问题的关键。本项目拟针对前期工作已筛选的诱导活性细菌和底栖硅藻株，优化其培养条件进行规模化发酵和扩大培养，以幼体附着变态活性为导向分别从其代谢产物中粗提取诱导活性组分，通过色谱手段和现代光谱技术分离、纯化并鉴定活性物质。进而比对两种来源的活性物质结构并结合结构类似物的诱导活性进行构效关系解析。最后，原位检测微生物膜及其浸提海水中已鉴定的以及构效关系推导的活性物质的存在与浓度，提出微生物膜诱导活性的优化依据。研究成果将从海洋化学生态学角度加深我们对棘皮动物幼体发育的理解，并有利于刺参养殖业的可持续发展。

刺参养殖业是我国海水养殖的特色产业，总产值超过300亿元；其发展对苗种数量和质量提出了更高的要求。制约苗种生产方面主要存在三个问题。1、幼体的附着变态是发育的关键环节，存在附着变态率低与不稳定、幼体和稚参“滑板”大量死亡等问题。2、幼参保苗的周期一般为一年，期间每隔5-10天换水，如何提高幼参存活率以及劳动效率是关键。3、育苗、保苗中药物的无序使用引发了食品安全问题。此外，海参目前主要以粗放式加工为主，在加工过程中营养成分都会有的流失、初加工产品的消化吸收率也低。如何进行精深加工，充分发挥其药膳同源的功效，进行产业升级成为行业面临的瓶颈。. （一）刺参幼体无公害化培育理论研究与应用：①幼体饵料的明确。设置植物性饵料组（新鲜的大叶藻液），动物性饵料组（海洋红酵母为主），混合饵料组设（植物性与动物性饵料各一半），研究了不同饵料对幼体生长、生理、附着以及育苗水体水质的影响效果。结果表明，植物性饵料以及混合饵料更适合于幼体的发育、附着以及消化与免疫酶的活力，且利于维持相对优良的水质。②底栖硅藻的规模化培养。底栖硅藻是幼体附着变态的诱导物，也可作为后期生长的饵料。但其附着特性以及大量培养所需时间与技术等条件限制了大规模利用。利用充气与添加抗絮凝剂的方法研发了“浮游化”大规模培养技术。硅藻的附着性能与诱导活性不受影响，只用了6天时间，密度可高达800万cells/ml，显著优于传统的2-3个周。③附着变态信号通路的初步研究：三种不同性质的活性因子（离子、神经递质、天然微生物膜）作用下，Ca2+通道呈现了同样的参与性，表明幼体附着变态过程中信号通道与传导方面具有共性。④生物絮团调控下的绿色保苗理论研究与应用。添加糖蜜与芽孢杆菌生成生物絮团，絮团可有效净化水质，换水由10天一次延长了一个周期，有益菌数量显著高。将以上技术进行集合，实现了育苗、保苗完全不用任何药物，SGS检测的46项指标全部达标。. （二）海参低聚肽的研发及其对小鼠抗疲劳功效的研究：采用特异性酶解技术，设计出包含膜分离、去腥、去色、杀菌、干燥等全程物理性工艺，开发出了海参低聚肽产品。质谱法和色谱法测定结果表明99.58%以上肽的分子量小于1000 Da。海参低聚肽能显著延长小鼠游泳时间，且运动后小鼠的血清尿素氮含量，肝糖原含量指标相对大豆低聚肽效果更为明显，显示出很强的抗疲劳功效。