Study On Biological Characteristics Of Initial Degradation Of Cordyceps Militaris During Successive Transfer Culture

Cordyceps militaris is a famous edible and medicinal fungus with important pharmacological effects.However,in the process of subculture,strain degradation led to a sharp decline in its yield,which brought serious hidden trouble to factory production.Detection of strain degradation early became the core technical problem of factory production of Cordyceps militaris.This experiment from the angle of the accumulation of reactive oxygen species quality influence,To address Cordyceps militaris transgenerational morphological changes in the process of cultivation,accumulation of reactive oxygen species and strains of correlation between degradation,explore the iconic index of strains in the early degeneration,Which are for the accumulation of reactive oxygen species by means of successive transfer culture Cordyceps militaris strains,microstructure characteristics of apparent traits,and antioxidant enzyme activities of active oxygen eliminating system are studied.The main results are as follows:1.The effect of subculture of Cordyceps militaris on its species characteristics was studied.However,the color of hyphae became lighter and the number of aerial hyphae increased.Compared with the first generation,the average daily growth of the fourth generation hyphae decreased by 10.3%,and the colony angle of the fifth generation hyphae became obvious;The results of transmission electron microscope showed that the membrane was twisted,the cell wall was loose to collapse,and large vacuoles were gradually formed in the cells;Under the scanning electron microscope,the hyphae were distorted,uneven in thickness,and the diameter of hyphae decreased significantly,which was 12.89% lower than that of the first generation;The conidiophore is deformed.Compared with the first generation,the number of conidia decreased by 14.02%.The hyphae of the fifth generation were seriously constricted.These can be used as physiological indexes for the early degradation of Cordyceps militaris.2.The relationship between the degradation of mycelium and the change of subcellular structure during the subculture of Cordyceps militaris was found.Under the transmission electron microscope,with the subculture,the cell wall of mycelium gradually thinned,and the cell contents changed.After the subculture to the third generation,the cytoplasm decreased significantly,the mitochondria swelled and the cristae deformed,and a lot of autophagy phenomena were observed.When subcultured to the 4th-5th passage,the cells were deformed,some of them formed large cavities,some of them extravasated cell fluid,some of them contracted protoplasts,and the nuclear area increased significantly.In the fifth passage,the cells were severely stressed,some of them formed a large number of cavities and went to apoptosis,some of them enlarged the nuclear area,formed a very thick secondary wall inside the cell wall,and divided into chlamydospores.It was found that wolunin could protect itself from the stress of Cordyceps militaris cells.Wolunin formed at the top of mycelium.With the increase of stress,wolunin blocked the septum of diaphragm to prevent the loss of protoplasm and protect the top growth point cells.The diameter of vorunin in Cordyceps militaris is about150-200 nm,which is significantly larger than that of other filamentous fungi.3.NBT staining observation and determination of reactive oxygen species(ROS)content of the subcultured strains showed that the strain was stressed by ROS in the subculture process.The accumulation of H2O2 and MDA reached the maximum in the third generation,increased by 46.15% and 16.67% respectively compared with the first generation,and decreased slightly after the fourth generation.The verification test of exogenous active oxygen also proved that the accumulation of active oxygen caused the initial degradation of the strain.The accumulation of reactive oxygen species was consistent with the change of malondialdehyde content,so malondialdehyde content could be used as an early and rapid detection index for the degradation of Cordyceps militaris.Combined with transmission electron microscope and laser confocal microscope,it was observed that the accumulation of reactive oxygen species at the tip of hyphae increased at first and then decreased during subculture.The staining of the tip of hyphae in the third generation was significantly deepened,and the reactive oxygen species at the tip was the most.With the increase of subculture times,the reactive oxygen species at the tip decreased,that is,the ability of eliminating reactive oxygen species at the tip of hyphae became weaker with the increase of subculture times.4.In the antioxidant enzyme system,the response speed of superoxide dismutase(SOD)and glutathione reductase(GR)to stress was faster.In the second generation hyphae,the enzyme activities increased by 55.87% and 17.14%,respectively,and decreased with the second generation.At the beginning of subculture,the reactive oxygen species increased,and the strain accelerated the scavenging reaction of reactive oxygen species.With the increase of subculture times,the scavenging effect of reactive oxygen species was weaker than that of reactive oxygen species.