Production Of Extracellular Saponins From Cordyceps Militaris By Liquid Fermentation

Cordyceps militaris is a traditional medicinal and edible fungus.Its mycelium contains a variety of bioactive components,such as cordyceps polysaccharides,saponins,adenosine,and cordycepins,which can be used as a substitute for wild Cordyceps.The cultivation of edible fungi has defects such as long cycle and difficult control of cultivation conditions,and it is not easy to achieve large-scale production of Cordyceps militaris.In order to meet the market demand,the production of Cordyceps militaris by liquid fermentation technology has attracted much attention.During the liquid fermentation of Cordyceps militaris,secondary metabolite saponins will be produced.Saponins have various biological activities such as antioxidant and antibacterial,and have broad application prospects.Therefore,this paper mainly studied the production of saponins by liquid fermentation of Cordyceps militaris,including the technological conditions for the production of extracellular saponins by liquid fermentation of Cordyceps militaris,the metabolic mechanism of the synthesis of extracellular saponins by different nitrogen sources,and the separation,purification and antioxidant activity of extracellular saponins from Cordyceps militaris.The main conclusions are as follows:（1）The medium components and ratio of extracellular saponins produced by liquid fermentation of Cordyceps militaris were optimized by single factor experiment and response surface methodology,and the optimal medium was obtained as xylose 2.35%,peptone 1.18%,Cu SO₄ 0.009%,VB₁ 0.002%;Single factor experiment was used to study the culture conditions,and the optimal culture conditions were obtained as follows:culture temperature 25℃,rotation speed 150 r/min,inoculation amount 10%,liquid volume 100 m L/（250 m L conical flask）;Under these conditions,the extracellular saponin content of Cordyceps militaris reached 2.22 g/L,which was 53.10%higher than that of the basic medium.Using the strain growth kinetic model to fit the biomass curve of Cordyceps militaris in the rapid growth period,it is found that the growth of the strain is closely related to the fermentation process conditions;Logistic equation was used to fit the extracellular saponins production curve,and it was found that the growth type of extracellular saponins of Cordyceps militaris was growth-related.（2）Metabolomics was used to study the effects of different nitrogen sources on the synthesis of extracellular saponins in Cordyceps militaris.Principal component analysis（PCA）showed that the metabolomes of the urea group and the peptone group were significantly different.Orthogonal projection to latent structures-discriminant analysis（OPLS-DA）)identified the differential metabolites of the two groups of mycelia,157 differential metabolites were found in the positive ion model,of which 98 were down-regulated and 59 were up-regulated,and 105 differential metabolites were found in the negative ion model,of which 61down-regulated,44 up-regulated;The differential metabolites between the urea group and the peptone group can be mainly divided into the following seven types:carbohydrates,amino acids,lipids,nucleotides,vitamins,hormones and other secondary metabolites;The metabolic pathways of seven types of differential metabolites were analyzed,and it was found that peptone as a nitrogen source in cells could increase the synthesis of NADH and ATP by enhancing the tricarboxylic acid cycle（TCA）,thereby increasing the biomass of Cordyceps militaris;The up-regulation of differential metabolites such as amino acids（Proline,Arginine,Glutamine,Glutamate）,nucleotides（Adenine,Guanine,Thymine,Uracil,Aminopurine,Xanthine,Deoxyadenosine,5-Methylcytosine）and lipids（Adenine,Guanine,Thymine,Uracil,Aminopurine,Xanthine,Deoxyadenosine,5-Methylcytosine）were indicating that using peptone as nitrogen source is beneficial to increase the synthesis of protein,nucleic acid and fatty acid,thereby increasing the biomass of Cordyceps militaris and the content of extracellular saponins;The down-regulation of abscisic acid content and the up-regulation of pantothenic acid,nicotinamide,pyridoxine,tetrahydrofolic acid and retinoic acid indicated that peptone as a nitrogen source could help increase the biomass of Cordyceps militaris;The up-regulation of the level of caffeosterol and the down-regulation of the level of periplogenin indicated that peptone as a nitrogen source could increase the content of extracellular ergosterol.（3）AB-8 macroporous resin,C18 column and DEAE-52 cellulose column were used to separate and purify extracellular saponins in Cordyceps militaris fermentation broth,the highest concentration of saponins was respectively obtained from 60%ethanol eluent,20%ethanol eluent and 0.3 M Na Cl solution,and a relatively pure saponin S was obtained.The antioxidant activity of saponin S was studied from four aspects:inhibition of DPPH·,·OH,O₂-·and total antioxidant activity,which showed that saponin S had strong antioxidant activity.The antioxidant kinetic model of saponin S was established,and it was found that saponin could scavenge DPPH·,O₂-·by affinity with free radicals.In the reaction of hydroxyl radical scavenging,saponins can achieve the scavenging effect of·OH through competitive inhibition.Saponins,as antioxidants in the total antioxidant test,improve the reaction rate with Fe³⁺-TPTZ,which is a non-competitive inhibition.