Optimization Of Lactobacillus Plantarum RS66CD Biofilm Formation Conditions And Comparative Transcriptomic Analysis

This experiment explored the conditions and the expression of differential genes during the formation biofilm of L.plantarum RS66CD.The optimum conditions for the biofilm formation of L.plantarum RS66CD were determined.Analysis of differential genes with compared to planktonic cells by RNA-seq technology(Illumina sequencing)to reveal gene regulation during L.plantarum biofilm formation.The tolerance of form biofilm strains compared with planktonic strains to temperature,p H,salt concentration and bile salts.And the effect formation of biofilm on spray drying powder was studied.The results are as follows:(1)After adding Na⁺,Mn²⁺,Ca²⁺,Mg²⁺and Fe³⁺,biofilm formation was determined by the96-well method and SEM.Select the most significant metal ions that promote formation biofilm of L.plantarum RS66CD.The biofilm formation under different ion concentration,different growth temperature,and different growth time were also studied to identify the optimal condition for biofilm formation.The result show that Na Cl concentration of 53 g/L,the culture time of 24 h,and the culture condition of 40?are the optimal condition for RS66CD biofilm formation.(2)Comparative transcriptomic analysis of L.plantarum biofilm and planktonic cells,found that compared to planktonic cells 447 genes were up-regulated and 426 genes were down-regulated in the biofilm.KEGG pathway analysis showed that genes coding for D-Alanine metabolism,peptidoglycan biosynthesis,two-component system,carbon metabolism,bacterial secretion system,lysine biosynthesis and fatty acid metabolism were crucial for biofilm formation.L.plantarum form biofilm by sense the changes of external signals,enhance the synthesis of the cell wall,regulate protein secretion,enhance bacterial adhesion,and regulating the type and composition of fatty acids.As the q RT-PCR validation of differential expression was consistent with the RNA-Seq,the transcriptomics results were considered representative.(3)The tolerance was significantly increased after formation biofilm of L.plantarum by comparing the survival rate of the experimental group and the control group.In the temperature tolerance experiment that the average survival rate of the experimental group was5 times higher than the control group;the p H tolerance was 2.8 times higher;the salt tolerance was 5 times higher;the bile salt tolerance was 7 times higher.After spray drying to make bacterial powder,the survival rate of 67%in the experimental group was much greater than 1.7%in the control group.In summary,in our study that L.plantarum as common fermentation bacteria biofilm formation was promoted when grown at Na Cl.Transcriptome results show that L.plantarum can promote the formation of biofilm by enhancing cell wall synthesis,regulating protein secretion,enhancing cell adhesion,controlling substance entry and exit,affecting fatty acid synthesis and regulating cell membrane fluidity,which can improve their salt tolerance.The environmental tolerance of strains after biofilm formation was improved.Our results provide insights into the differential gene expression involved in biofilm formation,which can help reveal gene regulation during L.plantarum biofilm formation.