| Pathogenic bacteria and fungi have always been one of the problems that perplex the healthy life of human beings.They are the pathogens of many diseases.Common pathogens can lead to various kinds of inflammation,infection and other diseases.At present,the treatment of common pathogens is mainly drug therapy,but long-term use of drugs will have side effects and drug resistance.Sophorolipids(SLs),a natural product,have been widely concerned for their excellent properties.Generally speaking,SLS can be roughly divided into two types,namely acid sophorolipid(ASL)and lactonic sophorolipid(LSL).Among them,lactonic sophorolipid has been studied by many scholars because of its significant antibacterial,antiviral and antitumor effects.However,due to the high production cost of sophorolipid from microbial fermentation,many researches on sophorolipid are still in the laboratory stage.Atmospheric and room temperature plasma(ARTP)is a compact and powerful physical microbial mutagenesis tool,which can be used for mutation propagation of a variety of microorganisms.By changing the dose or time of ion treatment,we can obtain mutants with multiple random breaks in DNA and oligonucleotides.Therefore,this study first explored and investigated the possibility and practical significance of using ARTP to obtain high-yield sophorolipid strains,and then extracted lactonic sophorolipid from the mixed products,and investigated the effects of lactonic sophorolipid on common pathogenic bacteria(Staphylococcus aureus,Escherichia coli,Pseudomonas aeruginosa,Lactobacillus)and fungi(Candida albicans,Trichophyton rubrum,Microsporum caninum,Pseudomonas aeruginosa,Lactobacillus).Then,representative strains were selected to investigate the antibacterial mechanism of lactonic sophorolipid on them.Through the above research,the potential of sophorolipid in industrial production and as a common pathogen inhibitor was explored,and its application scope was expanded.The research contents and results are as follows:1.ARTP mutagenesis of sophorolipid producing strain: Candida bombicola CGMCC 1576.Using this method,a mutant library composed of 106 mutants was constructed,and stable mutants with high yield of Sophorae were screened.After 5generations of subculture,8,7 and 4 mutants could increase the yield of lactonic,acid and total sophorolipids by more than 30%.Among them,A6-9 and A2-8 mutants were fed batch culture.The lactone sophorolipid produced by A6-9 and acid sophorolipid produced by A2-8 were 74.3% and 52.8% higher than those produced by wild strain,respectively;In addition,the total yield of sophorolipid of A2-8 was 34.17% higher than that of the original strain.In addition,the surface activity and HLB of sophorolipid from wild and mutant strains were determined.It was found that sophorolipid from different strains had different characteristics.2.The inhibition effect of lactonic sophorolipids on pathogenic bacteria was investigated.The results showed that lactonic sophorolipids had inhibitory effect on Staphylococcus aureus and Pseudomonas aeruginosa,but not Escherichia coli and Lactobacillus,indicating that lactonic sophorolipids had no absolute preference for gram-positive and negative bacteria.The minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of Staphylococcus aureus and Pseudomonas aeruginosa were 0.05g/L and 0.20g/L?4.00g/L and 6.00g/L,respectively.3.The inhibition mechanism of lactonic sophorolipids on pathogenic bacteria.The results of SEM showed that lactonic sophorolipids formed folds,depressions,cell wall rupture and incomplete cell morphology.The permeability analysis of cell membrane,the change of intracellular ATP and UV absorption showed that under lactonic sophorolipids treatment,the permeability of bacteria membrane increased,the contents were leaked out and the macromolecular matter was lost.The results showed that lactonic sophorolipids destroyed the cell membrane and cell wall of bacteria,and then inhibited the growth of bacteria.In addition,the loss rate of Staphylococcus aureus content was significantly higher than that of Pseudomonas aeruginosa under mic concentration.Therefore,it was boldly speculated that lactonic sophorolipids had stronger inhibition on Gram-positive bacteria than gram-negative bacteria.4.The inhibition effect of lactonic sophorolipids on pathogenic fungi was investigated.The results showed that lactonic sophorolipids had inhibitory effect on Candida albicans,Trichophyton rubrum and Microsporum canis,but not Malassezia.The minimum inhibitory concentration and minimum fungicidal concentration(MFC)of Candida albicans,Trichophyton rubrum and Microsporum canis were 6.00g/L and18.00g/L?10.00g/L and 17.00g/L?8.00g/L and 18.00g/L,respectively.5.The inhibition mechanism of lactonic sophorolipids on pathogenic fungi was investigated.The results of SEM showed that lactonic sophorolipids could fold and collapse the surface of Candida albicans cells,and the cell wall was destroyed and the cells were incomplete.The permeability analysis of cell membrane,the changes of intracellular ATP and UV absorption content indicated that lactonic sophorolipids enhanced the permeability of Candida albicans cell membrane and lost the content flow to the outside of cell.The results showed that the mycelium growth of Trichophyton rubrum was inhibited under lactonic sophorolipids treatment,the morphology of mycelium was obviously changed,the content of mycelium was lost,and the cell wall was discontinuous or damaged.The changes of electrical conductivity and ergosterol content indicated that the synthesis of cell membrane of ringworm rubrum was disturbed and the membrane function was abnormal,and the growth was inhibited.The results showed that lactonic sophorolipids destroyed the cell membrane and cell wall of fungi,and then played an antibacterial role.But at the concentration of MIC,LSL had a stronger damage to yeast fungi than tinea fungi,and the content logistics rate of yeast fungi was higher. |
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