Study On Extraction Of Polysaccharides From The Fresh Sarcotesta Of Ginkgo Biloba And Inhibition Of Its Against Staphylococcus Aureus Biofilm

ObjectiveThis paper directly carry out a series of experiments,with the raw material of the fresh sarcotesta of Ginkgo biloba,to protect environment and provide a theoretical basis to further clarify the mechanism of antibacterial activity and antibiofilm activity of polysaccharide from the fresh sarcotesta of Ginkgo biloba(GBSP)against Staphylococcus aureus(S.aureus).MethodsThrough the single factor experiment and response surface methodology,the optimum extraction process of GBSP was determined,the crude polysaccharides were obtained,and the purified polysaccharides by decolorization with activated carbon were obtained from the fresh sarcotesta of Ginkgo biloba.The supernatant obtained by extraction of the PMP derivative was added TFA and determination of monosaccharide composition by high performance liquid chromatography(HPLC).The minimum inhibitory concentration(MIC)of S.aureus was determined by the method of broth double dilution.Congo red was used to determine whether S.aureus could produce biofilm.The formation of biofilm of S.aureus was successfully established,which observed by crystal violet staining and confocal laser scanning microscope(CLSM)in vitro.The crystal violet was detected quantitatively to determine the time point of the biofilm formation of S.aureus.The minimum biofilm inhibitory concentration(MBIC)and minimum biofilm bactericidal concentration(MBBC)of GBSP on biofilm of S.aureus at early and mature stage were detected by the method of micropore plate.The inhibitory effect of GBSP on biofilms of S.aureus was observed by crystal violet staining and CLSM.Finally,the effect of GBSP with sub-bacteriostatic concentration on the expression of major regulatory genes in the process of its formation was detected by fluorescence quantitative PCR,and the inhibition effect of GBSP was explained from the gene level.Results1.The optimal conditions included a liquid-solid ratio of 30:1(volume/mass),an extraction time of 5.3 h,the extraction times of 4 times and an extraction temperature of 100 ?.Under these conditions,the extraction rate of polysaccharide was(6.657±0.3018)%.The decolorization rate by the method of activated carbon was 98.39%.2.According to the results,GBSP was composed of Man,Rha,Glu,glucuronic acid,galacturonic acid,and two unknown monosaccharides.The content of polysaccharide by the method of anthrone sulfuric acid was 52.21%.3.The MIC of GBSP on S.aureus was 1.563 mg/m L.The congo red plate identified from red to black,indicating that the S.aureus produce polysaccharide intercellular adhesin(PIA)that S.aureus can form a biofilm in vitro.Crystal violet staining showed that S.aureus aggregated gradually with the passage of time and varied in thickness after 24 h to form lumps.The observation by CLSM showed that as the culture time increased,the thickness of the membrane increased gradually.By compared with blank,it was showed that the maturation of S.aureus biofilm gradually.Quantitative determination of crystal violet showed that S.aureus biofilm matured gradually with incubation time,adhered to the surface of the carrier at 12 h and reached maturity after 24 h.The MBIC and MBBC of GBSP against the early stage of S.aureus biofilm were 100 mg/m L,however the inhibition of GBSP against the mature biofilm was not obvious.4.GBSP,with sub-antibacterial concentration,has a certain inhibitory effect on the expression of regulatory genes of the formation of PIA.And when the concentration was decreased,the inhibition was weakened in a dose-dependent manner.Especially GBSP,which the concentration was 0.78 mg/m L,inhibited the expression of ica A,ica B,and ica D genes significantly(P<0.05).The level of expression of ica A,ica B,and ica D genes were decreased by more than 90%,which compared with the blank group,and the expression of ica C gene was decreased by 55%.ConclusionsThe extraction process and monosaccharide composition of GBSP were determined for the first time with raw material of the fresh sarcotesta of Ginkgo biloba.In conclusion,GBSP is a new polysaccharide,which can inhibit the expression of regulatory gene of PIA in order to inhibit the formation of biofilm of early stage of S.aureus.