The Research Of Genome Shuffling Breeding And In Vitro Antioxidant Activity Of Cordyceps Kyushuensis

Cordyceps genus is a large class of insect pathogenic fungi, many of which possess high medical walue, such as Cordyceps sinensis and Cordyceps sobolifero. In recent years, many Cordyceps fungi with many pharmaceutic effects have been found. Cordyceps kyushuensis is one of these promising Cordyceps fungi. In recently studies, many bioactive sustances with anti-tumor, antibacterial and other pharmacological activities have been found in this fungus. The active components in Cordyceps kyusheusis are similar to Cordyceps sinensis, which could be developed as the alternative products of Cordyceps sinensis. Focused on the development and utilization of Cordyceps kyushuensis, this dissertation investigated three aspects including the quanlity control method, quanlity optimization as well as antioxidant substances extraction prossess.First, high performance liquid chromatogram (HPLC) was used to detect the concentration of AMP, uridine, inosine, guanosine, adenosine, cordycepin in Cordyceps kyushuensis. The contents of six nucleosides were3959.17ug/g,2324.80ug/g,243.57μ/g,1257.81μg/g,1744.37μg/g,3659.79μg/g, respectively. The result indicated that the Cordyceps kyushuensis contained a wealth of AMP, uridine, guanosine, adenosine, cordycepin and trace amounts of inosine. The methodological study results showed that the method has a good linear relationship, precision, stability and recovery, which could be used for the constraction of Cordyceps kyushuensis fingerprint to apply for the identification and quality control of Cordyceps Kyushuensis.Secondly, genome shuffling was used to improve the strain of Cordyceps Kyushuensis and the contents of cordycepin, adenine, uracil, adenosine, hypoxanthine, guanosine and uridine in the genome shuffling strains were detected by high-performance capillary electrophoresis. Finally,10improved strains with good genetic stability were obtained. The content of cordycepin in strain R5was978.25μg/g, which was9.63times higher than the parent strain. Compared with the parent strain, the contents of cordycepin, adenosine, guanosine and uridine in R18and R19were also improved significantly. Based on the phenotypes of cordycepin, adenosine, guanosine and uridine, the relationships between genome shuffling strains and mutants were infered by Cluster analysis and principal component analysis.Finally, the antioxidant substances extraction prossess of cultured Cordyceps Kyushuensis of strain R9was optimized by Response Surface Methodolog (RSM) by measuring the scavenging activity of DPPH and the chelating rate of Fe2+. The optimal conditions of extraction for scavenging DPPH was the ratio of liquid to solid of500:1, ultrasonic power of161.95W, extraction time of30min and water as extract solution without any ethanol. The optimal conditions of extraction for chelating Fe+was the ratio of liquid to solid of499.96:1, ultrasonic power of78.91W, extraction time of30min and ethanol solution with a concentration of37.48%as extract solution. Under the optimal condition, the scavenging ratio of DPPH can reach to71.40%, and the chelation ratio of Fe2+could reach to85.67%, which indicated that cultured Cordyceps Kyushuensis possesses a high antioxidant activity. However, the bioactive components such as nucleosides, polyphenols. and polysaccharides showed no obvious correlation between the contents and the scavenging ratio of DPPH and chelation ratio of Fc2+. Thus, further study was needed to investigate the antioxidant mechanism of Cordyceps Kyushuensis.