| Cordyceps militaris,as an edible and medicinal fungus,contains a variety of biologically active substances and has a wide range of applications in the fields of food,medicine and health care.It was approved as a new food raw material in China in 2014.Cordycepin is one of the main active components produced by C.militaris.At present,there are serious problems that the subculture of Cordyceps strains is severely degraded,the liquid fermentation conditions still need to be optimized,the separation and purification methods are time-consuming and complicated,and the antibacterial spectrum and anti-cancer activity are not clear.In this study,C.militaris was used as the experimental material to explore the extraction method of cordycepin in fermentation broth,and the corresponding biological activity was analyzed.The main research contents and results are as follows:（1）After activating and cultivating 6 Cordyceps strains collected and preserved,the ITS-r DNA sequence determination and Gen Bank sequence comparison analysis showed that the 6 strains were all Cordyceps militaris.Through liquid culture and worm culture,taking the mycelium dry weight,superoxide dismutase（SOD）activity,cordycepin content,fruit body dry weight and morphology as indicators,the liquid fermentation YCC-W strain was used to produce cordycepin.（2）The liquid culture of YCC-W strain was performed by single factor experiment and orthogonal experiment.We obtained the optimal liquid culture conditions of the strain as:inoculation amount was4%,filling volume was 100 m L per 250 m L Erlenmeyer flask,culture temperature was 24°C,p H was5.5,culture days were 25 d（submerged culture for 3 d,rotation speed was 150 rpm,and then surface culture to 25 d）;the optimal medium composition was 20 g/L glucose,20 g/L fish peptone,1 g/L potassium dihydrogen phosphate,0.5 g/L magnesium sulfate,and 0.5 g/L manganese sulfate.The content of cordycepin in the fermentation broth could reach 874.13μg/m L.（3）A low-cost and simple method for the separation and purification of cordycepin was established:After the lyophilized fermentation broth powder was dissolved by methanol,it was centrifuged and concentrated under reduced pressure.Distilled water was added（the added amount was based on the standard that cordycepin can be naturally precipitated in the liquid after removing methanol）for further concentration,and after filtration,it was re-crystallized until the precipitation was white.After freeze-drying the precipitate,the purified product was qualitatively and quantitatively analyzed by HPLC、MS and IR,and the purity of the cordycepin was 94.26%.（4）Cordycepin’s antibacterial study on six common Gram-positive（3G+）and negative（3G-）bacteria showed that it could significantly inhibit the growth of three common G+,and the MIC of Bacillus subtilis was 2.5 mg/m L,the MIC of Bacillus thuringiensis and Staphylococcus aureus were both 5 mg/m L.Further analysis revealed that cordycepin caused bacterial nucleic acid leakage by affecting the morphology of the cell wall and membrane,which eventually led to bacterial death.However,cordycepin in the range of 0-10 mg/m L has no obvious inhibitory effect on three common G-bacteria such as Escherichia coli,Salmonella enterica and Pseudomonas aeruginosa.It was speculated that cordycepin had a certain selectivity to the composition of cell wall and membranes of different bacteria（G+/G-）.（5）Through in vitro cancer suppression experiments,cordycepin was found to significantly inhibit the proliferation of human colon cancer Caco-2 cells with IC50of 107.2μg/m L.With the aid of confocal laser scanning microscope to observe of cell live and death,we confirmed that the presence of cordycepin caused cell death and its morphology also changed.By flow cytometry,it was found that cordycepin could induce early apoptosis of Caco-2 and lead to the arrest of Caco-2 in G2 phase of cell cycle.In a certain range,apoptosis and cell cycle arrest of Caco-2 cells were both dose dependent.In this study,the methods of C.militaris fermentation,extraction,isolation and purification of cordycepin were obtained,and the antibacterial spectrum and mechanism of cordycepin against 6common G+and G-bacteria were analyzed.In vitro experiments proved that cordycepin could inhibit the proliferation and damage of human colon cancer Caco-2 cells.The results can provide reference for the further development and utilization of cordycepin. |
