Production Of Rhamnolipids By Pseudomonas Aeruginosa 1.10452 And Its Efficient Purification And Antibacterial Activity

Rhamnolipids are a kind of biosurfactant that produced by microorganism,which have the functions of emulsifying,foaming,wetting,dispersing hydrophobic substances.Rhamnolipids display good biocompatibility,degradability and low toxicity,and show stable activity at extreme temperature,p H value and salinity.Thus,they hold great potential for bioremediation,agriculture,medical,daily chemical industry,and food application,become an ideal substitute for synthetic surfactants.Rhamnolipids are generally mixtures that compose of many homologues.The composition and properties of rhamnolipids are affected by fermentation strains,medium composition and fermentation conditions,the separation and purification of rhamnolipids have the problems of low purity and high cost,at present,there are relatively few studies on the antibacterial mechanism of rhamnolipids.In this study,Pseudomonas aeruginosa1.10452 was used as the fermentation strain,corn oil was used as the carbon source to produce rhamnolipids.Rhamnolipids were isolated and purified from the fermentation broth,and the component analysis and properties of rhamnolipids were studied,in order to provide theoretical support for the industrial application of rhamnolipids.First of all,the fermentation medium was optimized.Through single factor experiment,the optimal addition amount of corn oil was 60 g/L,sodium nitrate was 6.8 g/L,phosphorus source was 8 g/L,magnesium sulfate was 0.4 g/L,the optimal fermentation time was 96 h,and the optimal inoculation amount was 5%.Based on the experimental results of single factor experiment,five factors were selected for Plackett-Burman(PB)experiment,and the three main factors affecting rhamnolipids yield were the amount of carbon source,the amount of nitrogen source and the amount of inoculation.The steepest climb experiment was designed according to the results of PB experiment,and the central point of response surface optimization experiment was obtained.Rhamnolipids yield was taken as the response value.The amount of carbon source,nitrogen source and inoculation were independent variables.Response surface optimization(RSM)was used to optimize the composition of fermentation medium for rhamnolipid,and the optimal process parameters were as follows: 65.45 g/L of carbon source,6.47 g/L of nitrogen source and 4.12% of inoculation.Under these conditions,rhamnolipids yield was 25.23 g/L.The components of rhamnolipids which were isolated and purified by extraction and reverse extraction were analyzed by liquid chromatography-mass spectrometry(LC-MS)/MS,and the critical micelle concentration(CMC),emulsification,foaming and stability of rhamnolipids were investigated.The CMC of the rhamnolipids is 80 mg/L.the emulsification index of the rhamnolipids to soybean oil,liquid paraffin and n-hexane is 63.75%,65.69% and52.54% respectively.The rhamnolipids have good foaming ability and foam stability in pure water and hard water,and have good tolerance to extreme p H,temperature and salinity.As a surfactant,emulsifier and foaming agent,rhamnolipids have broad application prospects in the fields of environment,daily chemicals,food and so on.Taking rhamnolipids as the research object,the antibacterial activity of rhamnolipids against Gram-positive bacteria Staphylococcus aureus,Gram-negative bacteria Escherichia coli and fungi Candida albicans was investigated by inhibition zone test and minimum inhibitory concentration(MIC)test,and the changes of cell membrane integrity,permeability and extracellular reactive oxygen species(ROS)of tested bacteria were studied to discuss the antibacterial mechanism of rhamnolipids.The results showed that rhamnolipids had significant antibacterial activity against S.aureus,E.coli and C.albicans.The diameter of inhibition zone of 10 mg/m L rhamnolipids against the three tested bacteria were 17.52 mm,18.23 mm and15.73 mm,and the MIC were 0.313 mg/m L,0.157 mg/m L and 0.313 mg/m L,respectively.On the one hand,rhamnolipids changed the permeability of cell membrane,caused the leakage of nucleic acid,protein and other macromolecules,released nucleic acid,protein and other macromolecules,and increased the transmittance of crystal violet;on the other hand,rhamnolipids induced the production of reactive oxygen species(ROS)in bacteria,and accelerated the apoptosis of tested bacteria.