Effect On Biofilm Formation Of Staphylococcus Aureus By Rabdosia Rubescens Extract

Staphylococcus aureus is a kind of common food-borne pathogens,which is caused to equipment corrosion and food pollution by adhere to the surface of food processing equipment and packaging materials easily.Rabdosia rubescens is Chinese characteristic herbal medicine.Oridonin and ponicidin are of the main active component of R.rubescens.Modern pharmacological studies indicate that Rabdosia rubescens has many functions,such as anti-cancer,antibacterial and diminishing inflammation,antioxidation,enhancing immune and so on.The research of inhibiting food spoilage microorganisms biofilm has not been reported up to now.Therefore,this study provides scientific basis for the control of Staphylococcus aureus biofilm formation in food industry and animal husbandry,first,GC-MS was used to analyze the antibacterial active constituents of Rabdosia rubescens extract.Secondly,the effect of Rabdosia rubescens extract on biofilm formation and removal of Staphylococcus aureus,as well as the effect of extracellular polysaccharide.The main results are as follows:(1)Isolation of Rabdosia rubescens by the ultrasonic assisted ethanol reflux extraction and liquid-liquid extraction.Screening out antibacterial fractions by bacteriostatic test of the extract fractions,as well as GC-MS used to analyze the extract fractions with antibacterial.The results show that extract fractions with antibacterial mainly concentrated in chloroform extract and ethyl acetate extract,and the chloroform extract was higher than ethyl acetate extract.Oridonin is the mainly antimicrobial component in the chloroform extract,and the content is 5.87%.High Performance Liquid Chromatography(HPLC)analysis was carried out for the determination of oridonin.The results show that the chloroform extract has 91mg/g of oridonin.(2)The ability of S.aureus ATCC6538 biofilm formation was identified by Congo red plate and crystal violet staining method.The results show that S.aureus ATCC6538 was biofilm positive strains and moderately strong bacteria.The biofilm formation was observed by silver staining.The results show that the biofilm began to form after 1 day of culture,which reached maximum at the 3rd days,then slightly peeled off.It significantly peeled off after 4 days.There was no significant difference between 6th and 7th days.And viable bacterial counts were determined by dilution plate method,drawing a growth curve of S.aureus biofilm formation.The results show that from 0h to 24 h,the bacteria began to multiply,which reached maximum at the 72 h,and then bacterium number began to decline.(3)Inhibitory and remove effects of R.rubescens extract on the biofilm formation of S.aureus ATCC6538 were analyzed by MTT and sliver staining methods.The results show that the MIC and MBC of R.rubescens crude extract to S.aureus were 125?g/mL and 250?g/mL separately.R.rubescens crude extract can significantly inhibit the biofilm formation of S.aureus ATCC6538.With the increase of the concentration of R.rubescens crude extract,the inhibitory effect was more obvious.The inhibiting rate was over 81% when the concentrations of R.rubescens crude extracts were 2MIC.R.rubescens crude extract can not significantly remove the biofilm formation of S.aureus ATCC6538 when the concentrations of R.rubescens crude extracts were 2MIC and the remove rate was only 63%.The changes of biofilm of S.aureus ATCC6538 using R.rubescens crude extracts were observed under scanning electron microscope(SEM)5000×.The results show that R.rubescens crude extracts have a good inhibitory effect on S.aureus ATCC6538 biofilm formation.With the increase of the concentration of R.rubescens crude extracts,the inhibitory effect was more obvious.(4)On the basis of the above experiment,the inhibition effects on S.aureus ATCC6538 and its biofilm synthesis were studied.The results show that When the concentration of R.rubescens crude extracts was more than 1MIC,S.aureus ATCC6538 does not grow and the cell membrane has cleavage.The conductivity increased with the electrolyte flow out of the cell.Using SDS-polyacrylamide gel electrophoresis analysis,R.rubescens crude extracts can inhibit the expression of bacterial protein and protein expression decreased or not seen.The inhibition of extracellular polysaccharide(EPS)synthesis was studied by using Fluorescence microscopy.The results show that an obvious decrease of extracellular polysaccharide(EPS)was observed after R.rubescens crude extracts at the initial time of biofilm formation when the concentration of R.rubescens crude extracts was more than 1MIC.However,the inhibition on extracellular polysaccharide(EPS)synthesis was weak at 24 h of biofilm formation after adding R.rubescens crude extracts.The inhibition of EPS synthesis was studied by using sulfuric acid anthracene method.The results show that an obvious decrease of EPS was observed after R.rubescens crude extracts at the initial time of biofilm formation.