Study On The Production Of Helvolic Acid By Aspergillus Fumigatus

Helvolic acid is a typical tetracyclic triterpenoid and regarded as a promising bioactive substance against various pathogenic bacteria and cancer cells.But the further research and medicinal application of helvolic acid was restricted because of its low yield in fermentation.The initial HA-producing strain Aspergillus fumigatus used in this paper was a marine microorganism,which was provided by Prof.RX Tan?Nanjing University?.Single-factor experiments were conducted to improve the inoculum size,inoculation time,nitrogen source,carbon source,fermentation time and initial pH,finding that the accumulation of helvolic acid was affected by primary metabolism majorly,so the biomass in fermentation process was further studied later.After that,the statistic methods were used to optimize the fermentation medium.Firstly,Placket-Burman Design was conducted to screen the significent factors,which included mannitol,NaNO₃ and beef extract.Then,path of steepest ascent design was used to find vicinity of the optimum response.Response surface methodology was applied to optimize medium composition for helvolic acid production in shake flask on the basis of path of steepest ascent design.The optimal fermentation medium is listed as followed:39.88 g/L mannitol,2.25 g/L NaNO₃ and 3.54 g/L beef extract,8.4 g/L sodium succinate,0.3 g/L MgSO4-7H20,0.01 g/L FeSO4·H2O,0.67 g/L KH2PO4.Initial pH is adjusted to 4.2.The seed broth is inoculated into 250-mL shake flask containing 50 mL of seed medium and incubated at 28? with shaking at 160 rpm for 9 days.A maximum helvolic acid production of 68.5 mg/L was achieved under the optimized conditions,which was 4.1-fold compared to the initial medium.The effects of mycelial morphology and oxygen supply on biomass and helvolic acid production were further studied.The research found that Aspergillus fumigatus was shear-sensitive;appropriate cell aggregates had positive effect on helvolic acid production;low-frequecy ultrasonic was associated with the accumulation of biomass and helvolic acid.The loading liquids in earlier and later fermentation stage were changed,finding that adequate oxygen supply tended to increase biomass in earlier fermentation stage,while appropriate control of oxygen supply was beneficial to helvolic acid biosynthesis.On the basis of flask-shaking optimization,mycelial morphology and oxygen supply research,the fermentation medium,agitation rate and air flow rate were determined.The fermentation condition in 5-L bioreactor is listed as followed:inital pH is adjusted at 4.2,agitation speed is set as 300 rpm,initial air flow rate is 6 L/min.The helvolic acid production reached 54.81 mg/L in 5-L bioreactor.By combined use of the scale-up strategy,similar impeller linear velocity,similar kLa and similar pH,the fermentation condition in 500-L bioreactor was set as:inital pH 4.2,agitation speed 80 rpm,initial air flow rate 15 m3/h,and the helvolic acid production reached 41.00 mg/L.