| Staphylococcus aureus(S.aureus)is an important food-borne pathogen that can form biofilms.Its coatings are extremely resistant to the population and difficult to remove completely,which has a great impact on the food industry.Lactobionic acid(LBA)is classified as a bionic acid rarely found in nature.It is composed of polyhydroxygluconic acid bound to galactose.It is now known for its antioxidant,antibacterial,chelating,stabilizing,acidifying and moisturizing properties,so it is a multifunctional food additive.In order to further study the biofilm inhibition characteristics of LBA,this topic takes S.aureus biofilm as the research object to study the effect and mechanism of LBA on S.aureus.The main results are as follows:(1)The adhesive ability and activity of the biofilm showed that the adhesive form of the biofilm in 24 h was the small bacterial clusters,and there were still individual bacteria scattered in some parts.With the incubation time reaching 3 d,the number of purple colonies on the surface of the cover glass began to increase,and the area of the biofilm also gradually increased,showing a compact and uniform sheet structure.The number of viable bacteria in the biofilm reached the maximum and the activity was good.After 7 d,it was found that when the biofilm accumulated to a certain thickness,it would fall off and disintegrate locally.(2)The MIC value of LBA to S.aureus is 8 mg/mL,the MBC value is 32 mg/mL,the MBIC value to S.aureus biofilm is 16 mg/mL,and MBBC >128 mg/mL which showed that LBA had good bacteriostatic and membrane inhibition effects on S.aureus.The silver staining method and crystal-violet adhesion test found that compared with the control group,the formation of S.aureus biofilm was reduced to 56% under the action of MIC of LBA.From the SEM and CLSM images,with the increase of the treatment concentration,the membrane-like adhesion substance in the observation field gradually decreased,which proves that the amount of biofilm was reduced.There was almost no adhesion substance on the surface of the microbial cells in the MIC treatment group,and almost aseptic bodies gathered and CLSM found dead cells appeared in the biofilm samples,which directly demonstrated the inhibitory effect of LBA on S.aureus biofilm.(3)After treatment with LBA,the contents of extracellular polysaccharides,extracellular proteins,and eDNA in the S.aureus biofilm decreased significantly.The extracellular membrane protein was quenched with the addition of LBA,and its conformation was affected.The surface hydrophobicity,metabolic activity,athletic ability,and ATPase enzyme activity of S.aureus were significantly inhibited,which inhibited the formation of S.aureus biofilm.In addition,a decrease in the amount of hemolysin secretion can indirectly prove the interference of LBA on quorum sensing and biofilm formation.(4)RT-PCR showed that lactose acid inhibited the expression of icaA,icaR,agrA,sigB and hla genes in S.aureus and up regulated the expression of luxS gene.The results showed that LBA could inhibit the production of extracellular polysaccharide by regulating the expression of ica operon,and further inhibit the release of hemolysin by affecting the quorum sensing system.In iTRAQ study,274 differentially expressed proteins were statistically significant,164 of which were up-regulated,and 110 were down-regulated after LBA treatment.In the early stage of biofilm formation,LBA affected the expression of autolysin,indirectly inhibited eDNA release and accelerated cell death.With the addition of LBA,the secretion of anchor protein was affected,and the adhesion and infection of S.aureus biofilm were inhibited.In addition,LBA significantly affected the expression of virulence factors of S.aureus and the formation and infection of biofilm. |
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