A Method For Extracting Inorganic Selenium And Organic Selenium And Effect Of Exogenous Selenium On Biological Indices In Sea Cucumber(apostichopus Japonicas)

In the study, method of extracting inorganic selenium and organic selenium was optimized, determined procedures of inorganic selenium and organic selenium were established and selenium of different forms in Sea cucumber from different regions were invested. Physiological and biological indices of Sea cucumber feed with added selenium were studied, accumulation of selenium was also analyzed at the same time. The research would provide a theoretical basis for exploring a new green aquatic immune feed and achieving safe organic selenium-rich sea cucumber. The contents and results in detail are stated as follows:1. Optimizing and Establishing Method for Extracting Inorganic Selenium and Organic Selenium.Selenium is a necessary trace element for the health of people and animals. In recent years, many investigations show that many diseases are related to intaking level of selenium. It is not fit to judge the nutrition and toxicity of selenium by the content of total selenium, owing to the significant relevance between its availability and speciations. So it isnecessary to establish and extend the method for speciation analysis of selenium.Among the four extractions including HCl-boiling water extraction, Tris-HCl-Glycerin extraction, dialysis extraction, water extraction, water extraction is the best. The conditions including sonication time, bath time, bath temperature, extraction times of ultra-pure water extracting method were optimized. Under optimum operating conditions, there is a good linear relationship between selenium concentration and the fluorescence intensity as long as the selenium concentration is in 0~30 μg / L, the linear equations is I = 133.06 * C + 21.76 and the correlation coefficient r is 0.999, meanwhile the minimum detection limit of selenium is 0.0005 ng/m L. The spiked recoveries were 95.43%~99.74% for total selenium and 92.25%~95.36% for inorganic selenium, and the RSD of the analytical methods were in the range of 2.48%~5.26% and 2.73%~6.10% respectively. This method has a wide linear dynamic range and good precision and accuracy, so it is suitable for the determination of selenium in different forms in aquatic products.2. Analysis of Total Selenium and Inorganic Selenium in Apostichopus japonicus of Different Regions by Hydride Generation-Atomic Fluorescence Spectrometry.Selenium that humans absorb mainly comes from daily food, and is converted to the form that the body can use through metabolism. Theselenium content especially the organic selenium that would be used in Sea cucumber is higher than many aquatic products, while the study of selenium in sea cucumber is still relatively weak. How about the proportion, the biological effect of organic and inorganic selenium in different tissues, and the difference between different organizations and different regions, such all are still unclear. This study aims to ascertain the impact of the environment on the sea cucumber selenium and selenium metabolism in different forms, and then provides a scientific reference for further exploring the different form of selenium metabolism and molecular biology research.In order to explore selenium status in Sea cucumber of different regions, selenium level was studied in four populations of Sea cucumber from Dalian, Dongying, Yantai, Qingdao, Rizhao by hydride generation-atomic fluorescence spectrometry(HG-AFS). The level of organic selenium(14.17±3.41 mg/kg) and inorganic selenium(6.19±3.83 mg/kg) in respiratory tree are both the highest of all the tissues, and the amount of total selenium and inorganic selenium concentrations in tissues followed the decreased order: respiratory tree >intestinal>muscle >inner body wall>outer body wall. The proportion of organic selenium in body wall and muscle(the edible part) is between 75.63% ~ 89.41%, except in Dongying’(the proportion of organic selenium in muscle is the lowest that arrived at 53.64%). There is a significant difference in contentof total selenium and inorganic selenium in the same tissues between different regions, inorganic selenium in respiratory tree, intestinal and muscle of Dongying population was higher than other regions.3. Research on Physiological and Biological Indices and accumulation of exogenous selenium in Sea cucumberSelenium also has a variety of important physiological functions for aquatic animals. The use of antibiotics and chemotherapeutics to combat sea cucumber factious diseases has the risk of generating resistant pathogens, bioaccumulation and environmental pollution. Studies show that adding the right amount of exogenous selenium can improve aquatic animals resistance to disease by enhancing immune function. This study is carried out to analysis the biological effects of selenium on the sea cucumber by feeding sea cucumber different concentrations of selenium enriched yeast, measuring the survival rates and related enzymes of sea cucumber, which can enrich nutritional knowledge; In addition, we investigate the distribution and transformation of selenium and find content variation of sea cucumber by measuring the selenium content of different tissues in sea cucumber, and meanwhile, obtain high activity organic selenium alleviating the lack of selenium status.In this study, Apostichopus japonicas were cultured with different exogenous selenium concentrations(0.00 mg/kg、0.20 mg/kg、0.40 mg/kg、0.80 mg/kg、1.60 mg/kg、3.20 mg/kg、6.40 mg/kg), and after 4 W,biological indicators and selenium contents were detected. The results showed that adding appropriate concentration of selenium can increase the survival rate of sea cucumber(up to 100%) and digestive enzymes(The value of protease and amylase activity is 35.13 U/mg prot and 0.51 U/mg prot, and that’s 2.45 and 2.07 times than the control group respectively). Selenium plays a significant role in promoting immune activity(The value of SOD, GSH-Px, AKP, ACP, was respectively highest while the selenium concentration is 0.80~1.60 mg/kg and meanwhile were significantly higher than the other groups’); Further, selenium contents in the sea cucumber tissues increased with different levels, and the organic selenium multiples larger than inorganic selenium, which indicating enrichment of organic selenium into the body is the main way. In conclusion, the selenium concentration between 0.80 mg/kg and 1.60 mg/kg can have best effects in sea cucumber cultivation.