| Food safety has always been a hot issue concerned by the whole society,and bacterial infection has become the main factor resulting in food poisoning events.Therefore,it is meaningful to exploit novel efficient antimicrobial agents applied to the food industry.Mannosylerythritol lipid-A(MEL-A)is a kind of natural glycolipid biosurfactants with plenty of physicochemical properties and biological activities.However,there are few reports on their antimicrobial activities against food-borne pathogens,such as gram-positive bacteria,which greatly restricts their application in the field of food microbial safety.In this dissertation,the food-borne gram-positive pathogens were taken as the research object,and an in-depth study was conducted on the antimicrobial effect of MEL-A on planktonic bacteria and biofilm and its application in the food system.The antimicrobial mechanism of MEL-A on planktonic bacteria and biofilm was systematically explained from the cellular level to the molecular level,providing the theoretical basis for the application of MEL-A in food safety control and industrial application.The main results of this study are as follows:(1)MEL-A had significant inhibitory effects on the planktonic cells of Staphylococcus aureus and Bacillus cereus with minimum inhibitory concentration(MIC)of 0.625 mg/m L and 1.25 mg/m L,respectively.The survival rate of both bacteria was lower than 20%,and the growth curve showed a reduction in proliferation rate and stagnation of growth cycle under the MIC level.The treatment of MEL-A has caused the loss of cell membrane integrity and promoted the late apoptosis of bacteria through PI staining and flow cytometry analysis.The morphology observed by scanning electron microscopy and transmission electron microscopy proved MEL-A changed the cell surface and internal structure,leading to cell membrane damage,uneven distribution of cytoplasm and cell lysis.Besides,MEL-A inhibited the gemination of B.cereus spores with a decreasing geminating rate.(2)MEL-A had a significant inhibitory effect on the biofilm formation of S.aureus,and the minimum biofilm eradication concentration(MBEC)was 32 μg/m L.Crystal violet staining was used to quantitatively analyze the effects of MEL-A on biofilm formation in different stages,and it was the initial adhesion stage when MEL-A caused the highest removal efficiency of biofilm.The persistent bacteria and extracellular polymeric substances(EPS)in the biofilm structure of S.aureus were influenced by MEL-A with reduced bacterial quantity and decreasing content of protein and lipid,thus the entire structure lacked the necessary integrity.Also,MEL-A inhibited the biofilm formation of S.aureus on the surfaces of different materials(polystyrene,glass,stainless steel)demonstrated by confocal laser scanning microscopy.(3)The killing effect of MEL-A combined with other physical approaches on methicillin-resistant Staphylococcus aureus(MRSA)was studied.The synergistic treatment of MEL-A and ultrasound could inactivate 1 Log planktonic cells of MRSA within 5 min,whose bactericidal effect was much better than the single treatment(ultrasound or MEL-A treatment only).Ultrasound,as an external physical field,can improve the permeability of bacterial cell membrane and promote MEL-A to enter cells to exert an antibacterial effect.The combined treatment of MEL-A and ultrasound could effectively eradicate MRSA biofilm with an eradicating rate of up to90%.(4)The molecular mechanism of MEL-A against planktonic S.aureus was studied by transcriptomics and RT-q PCR.Transcriptomics analysis revealed the changes in intracellular transcription level induced by MEL-A in planktonic S.aureus and exposed the key metabolic pathways.Firstly,the transmembrane transportation of ABC transporters was dysfunctional,resulting in the decrease or increase of the intake of essential substrates such as metal ions,sugar and phosphate.Multiple pathways of amino acid biosynthesis and metabolism were up-regulated,leading to the loss of the dynamic balance of intracellular amino acid levels.Metabolic pathways such as glycolysis and TCA cycle were significantly down-regulated,which blocked the production and transformation of energy,causing insufficient energy supply for normal life activities.In addition,MEL-A has significantly regulated the signal system in S.aureus.The Sae RS two-component regulation system is significantly down-regulated,leading to a decrease in the expression of several downstream virulence genes,which effectively weakens the virulence of S.aureus and its defense ability against host cells.Agr quorum sensing system was up-regulated,which could induce the detachment of mature S.aureus biofilm.(5)Aiming agr-related genes as the targets,the interaction mechanism between agr quorum sensing system and MEL-A was further explored.A mutant strain with the gene agr A knockout was successfully constructed,and the relationship between the action mechanism of MEL-A on S.aureus biofilm and agr quorum sensing system was explored.The biofilm eradication rate of the wild-type strain was significantly higher than that of the mutant Δagr A at the same concentration of MEL-A.The number of persistent bacteria and content of EPS protein in wide type biofilm was much lower than that of the mutant Δagr A biofilm.Due to the significant difference in the results of the biofilm formation between the S.aureus wild type and the mutantΔagr A after treated by MEL-A,it is believed that the detachment of S.aureus biofilm through up-regulating agr quorum sensing system accounts for the excellent biofilm scavenging effect.(6)MEL-A was used as a dough improver to significantly improve the rheological properties of frozen dough and reduce the content of freezable water by1.88%,effectively preventing the formation of ice crystals from physical damage to the structure of the gluten protein network.Besides,MEI-A reduced the content of free sulfhydryl groups in frozen dough and decreased the disulfide bond breaking between gluten proteins,thus strengthening the structure of the gluten protein network.The microstructure of dough and texture characteristics of bread indicated that MEL-A can effectively improve the air retention capacity and react in evenly-distributed pores inside the bread,rendering enlarged the volume and better texture of the bread.Importantly,MEL-A has a selective antimicrobial effect on the dough microorganisms.In the simulated fermentation process,the bacteriostatic rate of MEL-A on vegetative cells of Bacillus cereus was higher than 90%,and the bacteriostatic rate on spores was up to 75.54%,while there was no significant effect on the cell activity of yeast. |
