| Polysaccharide, a crucial component of organisms, has special biological activities. However, as a kind of second metabolite, polysaccharide usually has too low content in organisms to meet the requirement of mass production, which is the main problem during the research and development of polysaccharide industry. Much evidence shows that the polysaccharide in Spirulina, a kind of photoautotrophic blue-green microalga, has special molecular structure and various biological activities, such as antitumor, antivirus and antiradiation. Because of its superior physiological functions, the Spirulina polysaccharide has become a focus of pharmaceutical industry and clinic application. However, due to its low and inconstant content (approximately 2~6% in dry powder), apparent influence of cultivation conditions and difficulty in separation, the extraction ratio of Spirulina polysaccharide is very low and the preparation cost is rather high, which have restricted its industrialization a lot. Mass studies are conducted on the purification, structure and function of polysaccharide while few on high-yield techniques.In this dissertation, using nuclear and its relative biological techniques, a new Spirulina platensis strain with high-yield polysaccharide was bred, and the factors of cultivation conditions were studied, then the conditions of polysaccharide synthesis was optimized. Moreover, the molecular mechanism was preliminary investigated in order to finally elucidate the mechanism of its high-yield polysaccharide. 1. Establishment and optimization of the methods for Spirulina polysaccharide quantitationThe phenol-sulfuric method used for Spirulina polysaccharide quantitation has some disadvantages, such as instability and poor repetition. In order to solve the problems, we modified the method as follow: (1) Glucose is usually used as the standard in polysaccharide quantitation. The maximal absorption (Amax) of the products of glucose reacted with phenol and sulfuric acid is at 490 nm, but Amax of the products of Spirulina extract reacted with phenol and sulfuric acid is at 484 nm. Thehigh purity (95%) Spirulina polysaccharide was used as standard in this dissertation and named Sp-Std, because Amax of the products of Sp-Std, reacted with phenol and sulfuric acid, was at 484 nm. (2) The sulfuric acid was added into the reaction system at the ratio of 5.5:8.5 (V/V), followed by incubating for 30 minutes at 25癈. (3) The products of Spirulina extract reacted with phenol and sulfuric acid was stable in 60 minutes. The results showed that using this modified method, the linear range of polysaccharide content was 0-320 u g, which is nearly twice of that of other methods, and the correlation coefficient was 0.999. Besides, this method used for Spirulina polysaccharide quntitation has been proved stable, precise, sensitive and reproducible.2. Studies on polysaccharide synthesis characteristics of different genotypic Spirulina strains and determination of parent strain for high-yield polysaccharide mutant breedingDetermined by the modified method, the polysaccharide contents of forty Spirulina strains were 2-6%, and their average value was 3.21%. Among these strains, the polysaccharide contents of Sp-D, Sp-J, Sp-B, Sp-S and Sp-E were higher than those of others. Moreover, an apparent difference was showed among the total DNA of five Spirulina strains, that is, a 1100 bp DNA existed in Sp-B, Sp-S and Sp-E, while not in Sp-D and Sp-J.The growth and polysaccharide synthesis of microagla were usually affected by some factors such as Mg2+, K+, SO42", NO3", C/N, N/P and C/P. Therefore, the effects of the seven factors on growth and polysaccharide synthesis of two different genotypic Spirulina strains Sp-E and Sp-D were investigated. According to the results, the difference of polysaccharide content and yield between Sp-E and Sp-D was due to their different molecular genetics backgrounds. The growth ratio and the polysaccharide yield ratio of Sp-E were 6.06% and 20.9% higher than those of Sp-D, respectively. Ther...
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