Effect Of Cryopreservation On Species Specificity And Activity Of Cordyceps Militaris

Cordyceps militaris,as a new resource food for both food and medicine,has attracted public attention.However,due to the unstable quality of the strains in the production,the yield of the strains often decreases sharply,which brings serious hidden dangers for the factory production.Therefore,the application of excellent and stable strains in the production has become the core problem in the production of Cordyceps militaris.In this experiment,the effects of storage temperature and storage time on the species specificity and vitality of Cordyceps militaris were studied through different storage methods of the same species,and the mechanism of low temperature storage on the species specificity and vitality preservation of Cordyceps militaris and the preliminary effect of antioxidants in the storage process of Cordyceps militaris were explored,so as to realize the medium and long-term storage of excellent and stable strains.The test results are as follows:1.Within the range of the test,the hyphae and mushroom appearance stored at-80℃were significantly better than those stored at 4℃.In terms of the hyphae appearance,the hyphae color stored at-80℃ was bright,orange yellow,without aerial hyphae,and the rhythm ring was clear.In the early stage of mycelium growth,the average daily growth rate was 4.84mm/d,which was significantly higher than that of the 4℃ preserved strains.The strains had strong activity,early primordium differentiation,and high biological transformation efficiency.2.Under the experimental conditions,the strains stored at-80℃ for 12 months were double mating type,MAT1-1 and MAT1-2 were detected as single mating type,only MAT1-1 was detected,and MAT1-2 was lost;In the decolorization of bromothymol blue(BTB),the decolorization ability of the preserved strains at-80℃ was significantly higher than that of the preserved strains at 4℃.3.Ergosterol,an important component of cell membrane,plays an important role in cell viability,membrane fluidity,integrity maintenance and cell material transportation.-80 ℃ultra-low temperature preservation cordyceps militaris hyphae is significantly higher than 4℃storage ergosterol content in mycelia and mycelial biomass was significantly higher than4℃storage strains,combined with the subcellular structure of membrane structure under different preservation temperature changes can be seen that-80 ℃ cryogenic storage cell membrane integrity,full content,cell membrane structure is rich,and the membrane structure independent gathered more,However,the cell membrane of the strains stored at 4℃ changed significantly,the loss of contents occurred,the abundance of membrane structure decreased,and the formation of large membrane complexes,and the phenomenon of cell autophagy was found.4.Accumulation of reactive oxygen species has an important impact on the quality and degradation of strains.The activity level of relevant antioxidant enzymes in the ASA-GSH cycle can reflect the activity and quality of strains in a certain sense.The activities of GR and MDHAR of strains stored at-80℃ were significantly higher than those stored at 4℃,and with the prolongation of storage time,the activities of GR and MDHAR decreased significantly.5.With the extension of storage time,the expressions of ERG6-1,ERG6-2 and ERG9 in ergosterol biosynthetic genes showed a significant downward trend,except ERG4 and ERG7,which had no significant change.The expression of ERG6-1,ERG6-2,ERG7 and ERG9 of the ergosterol biosynthesis related genes were significantly up-regulated,and the expression of ERG6-1,ERG6-2,ERG7 and ERG9 were significantly down-regulated with the prolonging of storage time.6.In the preservation process of strains,exogenous antioxidants were screened and added in this study to verify the effect of antioxidant GR in the preservation,which is a more appropriate antioxidant applied in the cryogenic preservation of Cordyceps militaris.In conclusion,strain degradation is a common biological phenomenon,which not only affects the development of fruiting body,but also affects the content of metabolites.The occurrence or deterioration of this phenomenon can be avoided by adopting appropriate preservation methods.Through the identification of biological characteristics,species and activity of the strains,and the verification of the expression levels of several genes related to the synthesis of the cell component(ergosterol),it was found that the ultra-low temperature of-80℃ was the suitable method for medium and long-term storage of Cordyceps militaris,and 4℃ was more suitable for short-term storage.Among the methods for identification of strain preservation effect,identification of biological characteristics,determination of antioxidant enzyme activity(GR,MDHAR)and determination of ergosterol content are all effective methods for identification of strain preservation effect.