Study On Fermentation Technology Of Candida Tropicalis With High Level Ribonucleic Acid

Ribonucleic acid(RNA)is a kind of biological macromolecule that can participate in physiological functions such as protein synthesis and immune regulation.With the deepening of research,the application of RNA has become more widespread.Microorganisms are rich in RNA,especially yeast strains.Therefore,the production of RNA by yeast fermentation is an effective way for industrial production.This paper starts from a strain of Candida tropicalis AY91009.The research was carried out from three aspects:medium optimization,fermentation condition optimization and fermentation regulation,and the following conclusions were drawn:1.Using Candida tropicalis AY91009 as the experimental strain and molasses as the carbon source of the medium,the fermentation medium was optimized by response surface method,and a quadratic regression model with yeast extract,ammonium chloride and ZnSO₄·7H₂O were established.The fermentation medium was determined as following:molasses 14%,yeast extract 2.90%,ammonium chloride 1.37%,NaH₂PO4·2H₂O 0.10%,MgSO₄·7H₂O 0.20%,FeSO₄·7H₂O 0.05%,and ZnSO₄·7H₂O0.10%.The RNA content in this medium was 11.58%,which was 35.60%higher than before optimization.2.In the 20 L ventilated stirred fermenter,during the ribonucleic acid fermentation of Candida tropicalis,the changes in RNA content were clearly divided into two stages:pre-accumulation(stage I)and late degradation(stage?).The results of optimization of fermentation conditions showed that the pre-accumulation and late degradation of RNA were affected by different fermentation conditions.The optimal fermentation conditions for RNA accumulation(C_max=87.22 mg/g DCW)were pH 5.5,speed of 600 r/min,aeration of 1.2 vvm.The fermentation conditions for minimum RNA degradation rate(r_ave=1.867 mg/(g·h))were pH 4.5,rotation speed 400 r/min,and aeration rate 0.8vvm.Based on the fermentation conditions,the initial inoculum size(wet weight)was optimized.The results showed that the lower the inoculum,the maximum RNA value and final RNA content are higher.When the initial inoculum was 40 g/L,the highest RNA content was 113.30 mg/g DCW,which was 24.71%higher than the control.The final intracellular RNA was 66.93 mg/g DCW and the dry weight was 32.38 g/L.3.In order to improve the RNA content of Candida tropicalis,the effects of different feeding strategies and different fermentation methods on ribonucleic acid fermentation were studied.The results showed that:(1)The index feeding strategy 4-1:Adding molasses at the beginning of the 4th h,can increase the RNA_unitnit content per unit cell,but because the feed volume is too large,the biomass is low,so the total RNA concentration is not high.The index feeding strategy 4-2:0 h begins to index supplemental carbon source,is more favorable for cell growth.(2)The strategy of intermittently adding carbon and nitrogen sources(C:N=10:1)has improved both biomass and RNA.The maximum RNA_unitnit value was 123.75 mg/g DCW,which was 9.22%higher than the control(113.30 mg/g DCW).The fermentation end point RNA_unit content was 72.62 mg/g DCW increased by 8.50%,the biomass was39.88 g/L,and the RNA_total was 2.90 g/L,which was 33.64%higher than the initial control.On the basis of intermittent addition of carbon and nitrogen sources,the effects of supplemental amino acids,bases and nucleosides on the intracellular RNA_unit content were compared,and the effect of supplemental base was the most obvious.The final RNA_unit content was 83.44 mg/g DCW.The batch fermentation was initially increased by 44.24%,so supplemental bases are the best exogenous nutrients.(3)The split fermentation is divided into 4 h as the node.When the split volume is small,the effect on biomass is small,but the degradation tendency of RNA_unit is relieved.When the volume is 1/2 volume,the bacterial cells almost stop growing,while the trend of RNA_unit decline has changed,and there is still a certain rebound.With a high initial wet weight and two 1/2 division conditions(strategy 5-5),the final RNA_unitnit content reached 86.61 mg/g DCW,which was 29.40%higher than the batch fermentation,it is indicated that moderate cell density and medium renewal rate are beneficial to the accumulation of yeast RNA.(4)In the quasi-continuous fermentation,When the dilution rates are set to 0.5 h^-1and 0.6 h^-1(both have exceeded the critical dilution rate),although the biomass is decreasing,the RNA_unit content is significantly increased,up to 134.07 mg/g DCW,and the trend of RNA_unit decline almost disappeared.When the dilution rate is set to 0.5 h^-1while controlling the temperature and adding bases,the intracellular RNA_unit content is up to 150.94 mg/g DCW,and the trend of RNA decline is completely changed.