Fermentation And Regulation Of Erucamide Produced By Bacillus Megaterium

Erucamide,as an important derivative of erucic acid,is a long-chain unsaturated fatty acid amide with biological activity.It has broad application prospects in plastics,engineering plastics,cosmetics,medicine,environmental protection coatings,papermaking,food packaging and other fields.At present,erucamide is mainly derived from plant extraction or chemical synthesis,which still faces the problems of insufficient raw materials(high erucic acid rapeseed oil)and low yield.However,in the field of microbial fermentation,regulating cell metabolism by changing cell membrane permeability and promoting the accumulation of microbial metabolites has become one of the current research hotspots.Based on the previous work,this study used GC-MS to establish a detection method for erucamide in Bacillus megaterium L2,which can provide a basis for the determination of microbial erucamide.In addition,the production of microbial metabolites is usually affected by culture conditions.Response surface methodology is used to optimize the fermentation medium and fermentation conditions for erucamide production by B.megaterium L2to obtain the best fermentation conditions.On the basis of optimizing the medium,the effects of various accelerators on the production of erucamide by B.megaterium L2were studied,including surfactants(Tween 80,CTAB,SDS,PEG-6000,Triton X-100),organic solvents(n-butanol,methanol,absolute ethanol,chloroform,dichloromethane,n-hexane,dimethyl sulfoxide),metal ions(Mg²⁺,Ca²⁺,Mn²⁺,Fe²⁺,Fe³⁺,Zn²⁺)and rapeseed oil.Determine the accelerator with the best effect,optimize the concentration and time of its addition,design an orthogonal experiment with the content of extracellular erucamide as an indicator,achieve the purpose of further promoting the accumulation of erucamide,and explore its promotion mechanism.The results were shown as follows:(1)GC-MS was used to determine the content of erucamide in B.megaterium L2.This method uses dichloromethane as the solvent.The results show that the mass concentration of erucamide has a good linear relationship with the peak area(R²=0.9968)in the range of 5?60?g/m L,and the RSD of precision,repeatability and stability are all lower than 10%,the recovery rate was 82.78%?98.14%,indicating that GC-MS can meet the test conditions of erucamide.(2)Using single factor experiment and response surface method to study the medium composition and fermentation conditions of erucamide production by B.megaterium L2,the optimal fermentation conditions were obtained:5 g/L glucose concentration,16.40 g/L peptone addition amount,3 g/L beef extract,3 g/L NaCl addition,2.0%inoculum,34.8°C culture temperature,150 r/min,and initial pH 5.80.The results show that under these conditions,the yield of erucamide(1.325 mg/L)than before optimization by 39.03%.(3)The effects of various promoters on the production of erucamide by B.megaterium L2 were investigated.The results showed that 0.2%Tween 80 and 0.05%chloroform were added to the optimized medium before fermentation(0 h),and the yield of erucamide were 1.714 mg/L and 1.560 mg/L,which were 29.78%and17.37%higher than the blank group,respectively.In addition,0.006%Ca²⁺and 0.5%rapeseed oil were added to the optimized medium at 6 hours of fermentation.The yields of erucamide were 1.665 mg/L and 1.707 mg/L,which were 25.57%and27.67%higher than the blank group,respectively.(4)Taking Tween 80,chloroform,rapeseed oil,and Ca²⁺concentration as four factors,three levels were investigated,and the L₉(3⁴)orthogonal experiment was designed with the yield of erucamide as an indicator.The experimental results show that the order of the major and minor factors affecting the production of erucamide is:Tween 80>Ca²⁺>rapeseed oil>chloroform.The optimal regulation condition for the production of erucamide by B.megaterium L2 is 0.3%Tween 80.0.004%Ca²⁺,0.04%rapeseed oil,0.02%chloroform.Under these conditions,the yield of erucamide reached 1.778 mg/L,which was 32.59%higher than the blank group and60.26%higher than the initial culture conditions,indicating that strain L2 It has the potential for industrial production of erucamide.(5)By measuring the conductivity,soluble protein,reducing sugar content,intracellular nucleic acid and protein of the bacterial suspension,the effect of promoters on the cell membrane permeability of L2 strain was investigated.The results showed that the electrical conductivity of the bacterial solution was significantly increased,and the concentration of intracellular nucleic acid,protein,soluble protein,and?-galactosidase activity were all higher than those of the control group,while reducing sugar had no difference with the control group.It shows that without affecting the sugar metabolism,the promoters change the permeability and selectivity of the cell membrane of the L2 strain,so that the intracellular material is released from the cell without causing damage or death to the bacteria.(6)Fatty acid composition analysis showed that the ratio of unsaturated fatty acid components to saturated fatty acid components in membrane lipids increased from0.893 to 1.856 in the control,which improved the fluidity of the cell membrane,weakened the rigidity of the cell membrane,and increased the cell membrane.The permeability further shows that the additive increases the permeability of the cell membrane of the L2 strain to achieve the purpose of releasing more erucamide to the outside of the cell.