Study On The Inhibition And Removal Of Dihydromyricetin On Staphylococcus Aureus Biofilm

Staphylococcus aureus widely exist in the natural environment.It not only causes infections in various tissues of the human body,but also endangers life.It may also cause various degrees of biological contamination and harmful food spoilage in food production,processing and packaging.S.aureus has a strong ability to form biofilms,which can cause long-term repeated infections,which can make it difficult to completely cure many diseases.In addition,the resistance mechanism of biofilms is also one of the reasons to the ineffectiveness of antibiotics.Therefore,it is of great significance to find an environmentally friendly and non-drug resistant biofilm control method.Dihydromyricetin(DMY)is an important flavonoid in vine tea,with very active chemical properties and physiological activities.DMY and its formulations have good antibacterial properties against planktonic S.aureus,but the inhibitory effect and the emulsion of DMY on S.aureus biofilm control are still poorly understood.This paper discusses the effect and preliminary mechanism of DMY and its emulsion on the control of S.aureus biofilm.Firstly,the S.aureus biofilm model in vitro was established,and the influence of external factors on the total formation of S.aureus biofilm at different stages was discussed.Secondly,the minimum inhibitory concentration(MIC)of DMY was determined by the growth curve and 96-well plate micromethod.The effect of DMY on the formation of S.aureus biofilm was further investigated.At last,DMY emulsion was fabricated to improve the removal of mature S.aureus biofilm by emulsion synthesis,the effect of the biofilm removal efficiency was verified by laser confocal scanning microscope.The enzymatic hydrolysis experiment confirmed the main components of the biofilm extracellular polymer formed by S.aureus and chemical methods were used to quantitatively determine the changes in extracellular protein,extracellular polysaccharide and eDNA secretion under the intervention of DMY emulsion.The main findings are as follows:(1)External factors,such as medium TSB concentration,bacterial concentration,glucose concentration,and salt concentration affect the formation of S.aureus biofilm.High-concentration(100%)TSB medium can promote the formation of S.aureus biofilm,and has a greater impact on the initial adhesion and mid-term development of S.aureus biofilm.The effect of bacterial concentration on the growth and development of S.aureus biofilm t is not obvious.Low concentration(0.5%)glucose promotes more obviously in the growth and development stage of biofilm.The addition of Na Cl in the early stage of biofilm formation can promote the formation of S.aureus biofilm,and the effect is more obvious when the concentration is 3%,but when the biofilm is mature,the addition of Na Cl will inhibit the growth of the biofilm.The in vitro culture conditions of the S.aureus biofilm model were 100%TSB medium,10~6cfu/m L bacterial concentration,3%Na Cl and 0.5%glucose.(2)The minimum inhibitory concentration(MIC)of DMY was determined to be512?g/m L by the growth curve and the 96-well plate micromethod DMY with different concentrations can effectively inhibit the formation of biofilms.DMY mainly inhibits the growth of biofilms by intervening in the initial adhesion and proliferation stages duiring the formation of S.aureus biofilms,but has no obvious effect on removal of mature S.aureus biofilms.(3)Crystal violet staining and laser confocal microscopy confirmed that the synthesized DMY emulsion had an obvious effect on the removal of mature biofilm of S.aureus,and most of the bacteria in the biofilm lost their activity.Enzymatic hydrolysis experiments proved that in the EPS formed by S.aureus,the main component was extracellular protein,followed by eDNA and extracellular polysaccharide.DMY emulsion can inhibit the synthesis of extracellular polysaccharide and extracellular protein to a certain extent and also has an inhibitory effect on eDNA autolysis releaseThe results of this thesis show that DMY has a certain inhibitory effect on the biofilm of S.aureus,but it has no obvious effect on the mature biofilm removal.The synthesis of DMY emulsion improves its removal efficiency of S.aureus biofilm,and influenced the secretion of extracellular matrix of S.aureus biofilm.This study provides a theoretical basis for the application of natural flavonoids in the antimicrobial field and provide a theoretical and scientific basis for the further establishment of biofilm control strategy.