Construction Of Liquid Ferme Ntation System For High Carotenoid Production By Cordyceps Militaris And Its Extraction Technology

Cordyceps militaris is a valuable medicinalfungus that may have antioxidant,anti-inflammatory,anti-tumor,anti-cancer effects.It contains a variety of bioactive ingredients such as cordycepin,cordycepic acid,cordyceps polysaccharides,adenosine and carotenoid.Carotenoid is one kind of natural pigments that has beendiscovered in Cordyceps militaris in recent years.Compared to those from other resources,carotenoid from Cordyceps militarisismore hydrophilic,leading to a higher bioavailability and exploitation value.Several factors,including culture,medium and enrichment condition would affect the production of carotenoid.In this study,a liquid fermentation system was constructed by selecting appropriate Cordyceps militaris strains and optimizing the fermentation media and condition.Besides,a preliminary discussion of different kinds carotenoids from Cordyceps militariswas conducted.Several results were achieved as listed below:?1?Carotenoid high-yield strains were selected.Thirteen Cordyceps militarisstrains werecompared for the production of carotenoid.The results showed thatCM-03,CM-04and CM-11 strainsproduced high amount ofcarotenoid,which wereselected for further liquid fermentation.?2?Fermentation mediawere optimized.According to the nutritional characteristics of Cordyceps militaris,fermentation media for each strain were prepared with rice,wheat,corn,bran and bean at different ratios.The highest carotenoid production was achieved at1.4 g/L of wheat,2.8 g/L of corn,0.3 g/L of bean for CM-03;1.2 g/L of rice,0.6 g/L of corn,0.3 g/L of bean for CM-04;1.7 g/L of wheat,2.6 g/L of corn,0.3 g/L of bean for CM-11.These results indicated that the medium for high carotenoid production is strain-dependent.?3?Fermented conditions of two liquid culturefor Cordyceps militariswere optimized.Taking the yield of carotenoid as index,fermented conditions of CM-04 were optimized through single factor experiment and response surface methodology.The results showed that for one-step fermentation,highest level of carotenoid?662.9±48.4?g/g?was obtained after dark shaking for 5d,followed by light shaking-flask culture for 6d.For two-step fermentation,highest level of carotenoid?1217.5±115.9?g/g?was obtained after dark shaking for 4d,followed by light shaking-flask culture for 10d with the flat working volumeof 100mL.The later scale-up experiment of two-step fermentation achieved a high amount of 1839.2±145.9?g/g carotenoid.?4?Based on shaking-flask fermentation,abiotic elicitors?mental ion?and biotic elicitors?tomato juice,peanut oil,chitosan and Rhodotorula glutinis elicitor?were added to the media to analyze the correlation between elicitors and carotenoid production.The results showed that Na⁺?tomato juice and Rhodotorula glutinis have no significant effect on carotenoid production,whilefour mental ions(Ca²⁺,Fe²⁺,Mn²⁺and Zn²⁺)and peanut oil could inhibit carotenoid synthesis.The increase?by 24.2%compared with control?of carotenoid production was only observed when 2ml of chitosan?4 mg/mL?was added.?5?The kinetics models for mycelial growth and carotenoid synthesis from shaking-flask fermentation were established.The results showed that the kinetics model for dx/dt=0.09947(1-X/?7.602?X mycelial growth and carotenoid synthesis were:dp/dt=0.5857X,respectively.?6?Safe extraction methodsof carotenoid from Cordyceps militariswereestablished.For ethanol extraction method,cell walls were ground by using quartz sand,followed by submergingin 60%ethanol with solid-liquid ratio of 1:40,maintaining in a water bath at80?and extraction 2 times.For water extraction method,fresh fruit bodies were added with solid-liquid ratio of 1:20,followed by boiling in a water bath at 100?and extraction 2times.?7?The varieties of carotenoids of Cordyceps militariswere analyzed through HPLC-MS.The results revealed that there are ten substances in Cordyceps militaris pigments with relative molecular massof512,526,496,498,510,524,494,508,522 and 536.The reported lutein and zeaxanthin were not found out in carotenoid of Cordyceps militaris.Relative molecular mass of 4 substances could match the cordyxanthin,but with unclear structure.Meanwhile,the other 6 substances were first found out in this study,which requires further exploration.