Improve Biosynthesis Of Carotene In Cordyceps Militaris By Fungal Elicitors

Cordyceps militaris has been proved to have values like regulating the immune system,anti-tumor,anti-oxidation,anti-aging,hypoglycemic,sedativation,hypnosis and regulating the endocrine system and other effects.Recent studys have proved that Cordyceps militaris contains rare carotenoids which is water-soluble and has unique physiological values.Thus,there are more and more studys focus on it,most of which are still in the stages of extraction and identification.But there is almost no article concerning about improving the carotenoid content.In this study,two mearsures are used to improve the carotenoid content including optimizing the fermentation conditions and adding elicitors.Meanwhile,a study of clarifying the mechanism of carotenoid biosynthesis is also tried.The main results are as follows:(1)Taking carotenoid contents as the only index,the study combined single factor experiment with orthogonal experiment to optimize the solid fermentation conditions.The optimum culture conditions include 30 g rice,1.5% peptone concentration in nutrient solution,6 days for dark culture and 8 days for illumination.It was verified that the carotenoid content was up to 309.54 mu g/g under this culture condition.(2)Taking five species of fungi as elicictors,including Rhodotorula glutinis ?Saccharomyces cerevisiae?Monascus rubber?Blakeslea trispora?Flammulina velutipes,to investigate their influence on the carotenoids in Cordyceps militaris.The results showed that except Rhodotorula glutinis that could improve the carotenoid significantly,the rest of them had no positive effects.(3)Taking the Rhodotorula glutinis fungal elicitor as the study subject,the cultivation time,adding concentration and adding time were studied.The results showed that the cultivation time and adding time of elicitors both have significant effects on carotenoids.But after narrow the concentration range,the effect of it is not that obvious.The result showed that 84 hours is the best fermentation time for Rhodotorula glutinis,1.5 g/L is the optimal elicitation concentration,and the fifth day is the most suitable adding time.The elicitor is separated into three spicies of main components,in order to figure out which of them is the active ingredients.The results showed that Part O is the active components,which included mainly small molecules by removed most proteins and polysaccharides.(4)By establishing the local blast genome database of Cordyceps militaris,the DNA sequence of car T and car D were gained,and then they were cloned and sequenced successfully.The results show that the car T in Cordyceps militaris and Beauveria bassiana are quite conservative and expression of the protein in a variety of fungi are highly homologous,showing that the evolution of frequency is not high.And car D genes in homologous evolution process is quite conservative too,the amino acid sequence reached 100% homologous with Beauveria bassiana,although some differences are exist in the gene sequence.But t the influence caused by the differences in the structure of a protein it is not clear.(5)Real-time quantitative PCR was used to monitor the effects of fungal elicitors on Cordyceps militaris carotenoid synthesis genes.The results showed that both car T gene and car D gene had been significant inhibited,while ggpps 191 genes proved to be less infected.