Application Of The Response Surface Methodology(RSM) For Optimizing The Production Of Pleuromutilin By Pleurotus Mutilus

Pleuromutilin,a diterpenoid compound,was produced from Pleurotus mutilus and Pleurotus passeckerianus Pila.It selectively binds to Ribosomal 50S Subunit of Prokaryote,which inhibits peptide bond formation,and has no effect on the synthesis of proteins in eukaryotic organism and mammals.Pleuromutilin possesses good security in clinical applications and also has a better therapeutic effect against a variety of multi-resistant bacteria,its derivatives as antibiotics for veterinary or human have broad market prospects.With the ever-increasing of market demand,it is urgent to improve the fermentation level and the yield of pleuromutilin which serves as semi-synthetic precursors to meet the needs of medical and health undertakings.In this paper,Response Surface Methodology（RSM） was employed to optimize fermentation medium and culture condition for utmost improvement of the pleuromutilin production.The main research was constructed by the single factor experiment used for optimizing the culture condition,such as the age of second-grade seed,pH value and inoculum liquid volume;the Placken-Burmen design（PB） experiments used to screen factors which had a significant impact on the value of response;the Central Composite Design（CCD） experiments intended for optimizing the response surface methodology;and the 50L fermentor model enlarging experiment with the optimal ingredient obtained by flask fermentation tests.Through single-factor screening experiments,we identified that the cultivate time of second-grade seed liquid was 60h,the volume of inoculum was 10%and the pH value was 6.7. Useing PB experiments and experimental data statistical analysis,we could determine that the P value of glucose,corn steep liquor and soybean powder were less than 0.05,and three media had very significant impact on the production of pleuromutilin.And we got the first-order linear regression equation Y（g/L）=3.762+0.189X₁+0.413X₂-0.446X₃+0.248X₄+0.579X₅-0.002X₆+0.203X₇ which R² was 0.90.By employing Central Composite Design（CCD） experiments and the fitting of the data,we got the second-order linear regression equation Y（g/L） = 6.03+0.453X₂+0.293X₃+0.551X₅-0.16X₂×X₃-0.147X₂×X₅+0.106X₃×X₅-0.595X₂²-0.25X₃²-0.498 X₅² which R² was 0.91.Finally,the best medium composition was obtained as soybean oil,20 g/L;Soybean powder,25 g/L;corn steep liquor,5 g/L;soluble amylum,10 g/L;glucose,70 g/L; MgSO4·7H₂O,0.5 g/L;CaCO₃,0.9 g/L.Y1=6.121±0.312 g/L,Y2=6.098±0.286 g/L and Y3=6.106±0.021 g/L,the verification result of these three production models was close to that of the forecasted production model Y=6.054±0.371g/L.The verification experiments definited the stability of the model.On the basis of its stability,the craft parameters such as fermental termination and ventilatory capacity were determined and the yield of pleuromutilin was improved.Optimization of medium verification experiment was undertaken in 50L fermenter.Through controlling and regulating the progress parameters such as on line pH value,amino nitrogen, reducing sugar,dissolved oxygen,rotational speed and observing the process of the whole fermentation,the regularity of thallus vegetation and product composition Was found and the production of pleuromutilin increased.We found that the biomass was rapidly accumulated from 12%to 56%during the 12th hour to 48th hour.After 12th hour of fermentation there was a sharp decline in dissolved oxygen.According to preliminary experiments,15%was the critical dissolved oxygen volume.We adjusted the speed and air flow to meet the requirement of the dissolved oxygen in fermentation process.The content of amino nitrogen decreased from 165.2 mg/100mL to 61.6 mg/100mL in the initiate 36 hours and then maintained at about 117.6 mg/100mL from 60th hour to 168th hour.After that the content of amino nitrogen increased to126 mg/100mL dgradually till the end of the fermentation.The pH value was changing during the fermentation:dropping in the initiate 24 hours then rising until arriving at the maximum 7.07 in 96th hour,declining from 96th hour to 180th hour at last.Pleuromutilin began to accumulate substantially in this falling progress.Owing to the ascension of pH value and the descend of biomass from 180th hour to 192ed hour at the end of fermentation,the autolysis of mycelium appeared.Through Response Surface Methodology（RSM）,the unit production arrived to 6.06±0.04g/L in 180th hour comparing with the unit production of 3.29±0.33g/L by using original fermentation medium.Compared with the initial unit production in 180th hour,the unit production in 192ed hour reached 6.67±0.21 g/L and increased 102.64%via improving the fermentation termination and ventilatory capacity on the basis of optimal ingredient obtained by flask fermentation tests.50L fermentor fermentation was explored after the establishment andvalidation of fermentation flask models and then the fermentation unit production was upgraded to 10.06±1.09g/L in 192ed hour,which was 165.9%of the flasks’ production and 305.8%of the original unit production.During the flask fermentation tests and its enlarging process,the stability of the model was good,and we got the anticipated purpose of the experiment.