Study On Optimization Of Fermentation Parameters In Preparation Of Antimicrobial Peptide CC31 In Pichia Pastoris

Is this study,the effect of culture medium of submerged fermentation,solid-state fermentation and its culture condition on the fermentation of Pichia pastoris CC31 were evaluated.The optimum fermentation medium combination was obtained though optimization,the fermentation effect was verified,the influencing factors were explored,and the correlation between fermentation combination and fermentation effect was explored.Methods:(1)In the shake flask test,the best carbon source was selected from glycerol,glucose and sucrose by single factor test,the best nitrogen source was selected from ammonium sulfate,ammonium carbonate and ammonium oxalate,from PTM1 and yeast extract,the best trace elements were selected as the main components of the fermentation medium.On the basis of selecting the components of the fermentation medium,the orthogonal test design table design was used to determine the cell density by using different carbon source ratios,carbon source addition amount,nitrogen source addition amount and trace element addition amount as variables.OD600 nm is an indicator to obtain the relationship of the combination to the fermentation effect.Under the optimized medium conditions,the inoculum size,p H value and temperature were optimized by one-way.(2)The conditions of submerged fermentation culture using uniform design.The effects of fermentation was explored by using protein content and wet bacteria weight as indicators.The quadratic multiple stepwise regression analysis was used to fit the equations of protein concentration,wet bacterial weight,liquid loading and rotational speed,and optimized by DPS software to obtain the best combination of culture conditions.And predict the optimal equation solution.Protein concentration and wet bacterial weight were determined after the end of fermentation,and the correlation of the equation was verified.The one-way design of the feeding mode intermittent feeding and continuous feeding was optimized,after the end of verification test.(3)The selection of solid-state fermentation particle size was carried out by one-way design,which screening at 40~60 mesh,60~80 mesh and 80 mesh.Based on the uniformity of the particle size design,the DPS software was used to design the test,and the relationship between the fermentation conditions and the fermentation effect was explored by using the small molecule protein concentration as an indicator.The quadratic multiple stepwise regression analysis was used to fit the equations of protein concentration and inoculum size and water content,and optimized by DPS software to obtain the best combination of culture conditions and predict the optimal equation solution..Based on the optimized solid-state fermentation conditions,the optimal ratio was verified.The fermentation material was routinely measured to observe the effect of Pichia pastoris engineering bacteria on the composition of the fermented material.Results:(1)Carbon source glycerol,glucose,sucrose,nitrogen source ammonium sulfate,ammonium carbonate,ammonium oxalate,trace elements PTM1,and yeast extract can promote the growth of yeast cells.Considering the high price of glycerol,glycerin and glucose are selected as mixed carbon sources,the glycerin,ammonium sulfate and yeast extract have the best effect.The best combination obtained by orthogonal design is glycerin: glucose 3 : 1,carbon source addition amount 6%,nitrogen source addition amount 1.5%,and trace element addition amount 1%.The optimum fermentation BSM medium composition was 85 % H3PO4 26.7 m L/L,Ca SO4·2H2O 0.94 g/L,K2SO4 18.2 g/L,KOH 4.13 g/L,Mg SO4·2H2O 14.9 g/L,glycerol 30 g/L,glucose 30 g/L,(NH4)2SO4 15 g/L,and yeast extract 10g/L.The optimal ratio of the inoculum size was 10%,p H value was 6 and temperature were 30 ?.(2)The uniform design optimization showed that the optimal fermentation conditions was 6L and rotation speed was 150 r/min,the fermentation effect of the bacteria was obviously improved.The relative error of the test was 2.6 %.The one-way optimization results of different feeding modes,intermittent feeding and constant flow feeding showed that the wet weight of the bacteria can reach 17.5 g/L and the actual value was 273.35 mg/L,and improve effectively the fermentation effect and shorten the fermentation time.(3)The uniform design optimization showed that the optimal fermentation conditions of the particle size was 40~60 mesh,the inoculum size was 10 % and the water content was 50%,a small molecular protein of 156.23 mg/L would obtained.Through the solid fermentation of Pichia pastoris,not only the Pichia pastoris was successfully expanded,but also the protein content in the fermented material was improved.