Separation And Preparation Of Sophorolipids And Studies On Its Anti-biofilm Properties

Surfactants have become one of the most widely used chemicals in a wide variety of industrial sectors,such as food,cosmetics,petroleum,environmental protection,personal healthcare and pharmaceutical areas.However,most chemically synthesized surfactants derived from petroleum products,and massive petroleum consumption has led to resource exhaustion and environmental contamination.Therefore,it is necessary to develope eco-friendly,biodegradable and renewable surfactant alternatives to chemical surfactants.In recent years,biosurfactants have gained more and more attention owing to their higher biodegradability,lower toxicity,thermostability,and tolerance in extreme conditions.Sophorolipids(SLs)are one of the most representative glycolipid biosurfactants.Because of the highest yield of SLs among all the surfactants,they are the most likely substitutes for chemical surfactants.SLs can not only be used in many industrial fields instead of chemical surfactants,but also can be used in medicine field as anti-bacterial,anti-fungal,antiviral and anti-tumor medcines due to its unique biological activity.The application of SLs in environmental protection,food,medicine,healthcare and otherindustrial fields has been paid more and more attention.Biofilm is a form of microbial population,which is one of the important reasons that lead to greater resistance to antibiotics than planktonic bacteria and difficult-to-treat microbial infection.It has been reported that SLs have the potential to disrupt microbial biofilms.In addition to the advantages of high safety,wide drug spectrum and rapid effect over other natural antimicrobial agents,SLs also have good environmental compatibility.It can be fully developed to be used as novel natural antimicrobial products to inhibit the formation of biofilm in pharmaceutical industry.The inhibitory effect of SLs in microbial biofilm and how they affect the physiological state of biofilm,and how to develop SLs into the specific medicines for anti-biofilm according to the function characteristics of SLs on biofilm,all need to be further studied.Thus,to develop effective materials and methods to control the formation of biofilm,it is necessary to study the inhibitory activities of SLs on the main types of biofilms formed by bacteria.The main research contents and results of this thesis are as follows:1.Fermentation strategy of lactonic SLs using glucose as the sole carbon and purification and preparation of C18:1DLSLNaturally synthesized sophorolipids are composed of more than 20 different structures of sophorolipid molecules,the proportion of each sophorolipid molecule in SLs mixture and different molecules will affect its biological activity,it is necessary to prepare lactonic SLs even pure sophorolipid molecule with better biological activities to meet the requirements in medicine field.In this work,a strain with high-yield lactonic SLs was genetically constructed and used to produce di-acetylated lactonic SL with a C18:1 fatty acid(C18:1 DLSL)with the best biological activity.Using glucose as a sole carbon source for sophorolipids production by fermentation of Starmerella bombicola,a mixture with a higher lactonic SLs ratio was obtained,and the C18:1 DLSL with a purity of more than 95%was prepared by preparative HPLC.Using glucose as the sole carbon source for SLs production.When using the glucose-feeding strategy by feeding 1%of glucose per day from the third day of fermentation,the production of lactonic SLs was nearly twice as high as that of non-feeding fermentation.The production of lactonic SLs was increased 1.3 times from 1.79 g/L of the wild-type strain to 4.16 g/L of Peno::sble strain.The increase of lactonic SLs yield can reduce the cost of separation and purification of C18:1 DLSL,and is beneficial to study the application potential of C18:1 DLSL.2.Research on the antibacterial and anti-biofilm activities of sophorolipidsAt present,there are few studies on the antimicrobial activity of a single pure sophorolipid molecule.In view of the excellent biological activity of C18:1 DLSL,two common pathogenic bacteria Staphylococcus aureus and Pseudomonas aeruginosa,were selected as the research objects,and the antibacterial and anti-biofilm activities of high-purity C18:1 DLSL and crude lactonic SLs were investigated.The concentrations of C18:1 DLSL and lactonic SLs completely inhibited the planktonic gram-positive bacterium S.aureus were 15 mg/L and 1500 mg/L,respectively.However,neither C18:1 DLSL nor lactonic SLs showed significant inhibitory effect on planktonic gram-negative bacterium P.aeruginosa.In addition,it was the first time to report the inhibition of C18:1 DLSL on the formation of S.aureus and P.aeruginosa biofilms and the dispersion of their mature biofilms,and it was also found that C18:1 DLSL had a lower minimum inhibitory concentration(MIC)to P.aeruginosa in the biofilm state than in the planktonic state.When the concentration of C18:1 DLSL was 20 mg/L,the inhibition ratios of C18:1 DLSL to S.aureus and P.aeruginosa biofilms were 89%and 70%,respectively.The antibacterial and anti-biofilm activities of lactonic SLs were weaker than that of C18:1 DLSL.Observed by scanning electron microscope(SEM),after treatment with C18:1 DLSL and lactonic SLs,the number of bacteria was significantly reduced and the cells became smaller,the morphology of the bacteria was seriously damaged and the cells were broken.The adhesion of amorphous substrates was reduced.3.Preliminary study on the anti-biofilm activity in medical catheters by the combined action of sophorolipid and zinc ionsSo far,there have been no reports on the use of sophorolipid biosurfactant produced by microorganisms in combination with zinc ions to develop anti-biofilm activity in medical catheters.The purpose of this work was to investigate the synergistic antibacterial and anti-biofilm activity of C18:1 DLSL/lactonic SLs and zinc ions,and to develop anti-biofilm coatings of sophorolipids and zinc ions binding materials in medical catheters.It was found that when low concentrations of C18:1 DLSL(12 mg/L)and zinc ions(40 mg/L)were used together,more than 80%of the S.aureus biofilm were inhibited,compared with 20%inhibition of C18:1 DLSL and 30%inhibition of zinc ions at their individual same concentration,which indicated that the effect was obviously enhanced in the case of combinational use of C18:1 DLSL and zinc ions.The inhibitory effect of lactonic SLs combined with zinc ions on S.aureus was also significantly greater than that of the same concentration alone.It showed that the combination of C18:1 DLSL/lactonic SLs and zinc ions had synergistic inhibition on S.aureus in both the planktonic state and the biofilm state.For the mature biofilm of P.aeruginosa,when the concentration of C18:1 DLSL and zinc ions was both 100 mg/L,the dispersion ratio reached 70%,and compared with 40%of C18:1 DLSL and 2%of zinc ions at the same concentration,the dispersion ratio was distinctly increased.Similarly,the dispersion effect of lactonic SLs combined with zinc ions was significantly stronger than their inhibition at the same concentration alone.The results showed that the composition of C18:1 DLSL/lactonic SLs and zinc ions had a synergistic dispersion effect on the mature biofilm of P.aeruginosa.Observed by SEM,it was found that after treatment with the composition of C18:1 DLSL/lactonic SLs and zinc ions,the cells of both S.aureus and P.aeruginosa were seriously damaged and the biofilm was severely damaged.The combination of sophorolipid and zinc ions will be a potential medicine against microbial biofilms.