Study On Bifidobacterium Biofilm Formation Mechanism And Fermentation Regulation

Bifidobacterium species are the most commonly used probiotics in foods for human consumption,however,it’s easy to die during industrial processing.Biofilm is a self-protection model for bacteria to improve environmental resistance.How to promote Bifidobacterium biofilm formation has important industrial application value.However,the current biofilm study mainly focused on how to prevent and inactivate the biofilm formation of harmful bacteria,and the biofilm mechanism of bifidobacteria is still unclear.In this study,the bifidobacterial biofilm fermentation system was first established,and then the bifidobacterial biofilm formation mechanism was studied,and finally,different fermentation conditions were used to control the biofilm fermentation.The main findings are as follows:The biofilm formation in the insoluble dietary fiber system was evaluated by the carrier particle size,the count of viable cells,and the scanning electron microscope,which found that the bifidobacterial biofilm formation stages can be divided into several stages including cells attached to the carrier surface,extracellular substances secretion and microcolony formation,biofilm growth and maturation,and biofilm dispersion,biofilm maturation and disperse.There are differences in the biofilm formation ability of different bifidobacteria,among which Bifidobacterium pseudocatenulatum can form biofilm particles with a diameter of 2 mm on a100μm carrier.The cell resistance of bifidobacteria was enhanced after biofilm formation,and there were almost no viable cells after 60 h of carrier-free culture,while the number of biofilm cells on the carrier still exceeded 10~6 CFU/g.These results indicated that the insoluble dietary fiber was beneficial for bifidobacteria biofilm formation.The biofilm formation ability of Bifidobacterium species was different,all Bifidobacterium bifidum strains can form strong biofilms,B.pseudocatenulatum has strong,weak,and non-biofilm-forming strains,while Bifidobacterium longum has only weak and non-biofilm-forming strains.Therefore,we speculate that there may be differences in the biofilm formation mechanisms of these three Bifidobacterium species.To study the biofilm formation mechanism of Bifidobacterium bifidum,B.pseudocatenulatum and Bifidobacterium longum,we first used comparative genes to find their biofilm functional genes;then exogenously added signal molecules and inhibitors to verify related genes,key samples were selected for transcriptome and targeted metabolome to analysis biofilm formation related pathways;finally,a time series transcriptome and untargeted metabolome analysis of both biofilm and planktonic cells of bifidobacteria in the insoluble dietary fiber fermentation system was performed to complete the biofilm formation pathways.It was found that there were differences in the biofilm formation mechanisms of these Bifidobacterium species.B.bifidum mainly regulates biofilm formation through Qse C/Qse B two-component system(TCS),AI-3/quorum sensing system(QS),and c AMP second messenger system by regulating Tad IV pilin and extracellular substances secretion,among which Qse C/Qse B is the main regulator.c AMP can promote cells adsorbed to the carrier by regulating Tad IV pilin and increase cell proliferation to form microcolonies in the early biofilm stage of B.bifidum,and the main metabolites at this stage include threonine.Threonine can be further metabolized to form AI-3 and activate Qse C/Qse B TCS,which can promote biofilm growth by enhancing stress response and extracellular substances secretion.With the increase in the number of biofilm cells,the expression of the QS-related gene tam was up-regulated,enhancing the intercellular communication and acidic substances secretion that caused p H decreases,and then biofilm entered to disperse stage.B.pseudocatenulatum mainly regulates biofilm formation through c-di-GMP,c AMP,AI-2/QS system by regulating Tad IV pilin and exopolysaccharide secretion,among which c-di-GMP is the main regulator.In the initial biofilm stage of B.pseudocatenulatum,c-di-GMP and c AMP can reduce the motility of Tad IV pilin and increase the synthesis of exopolysaccharide to promote cell adsorption to the carrier.At this stage,galactose synthesis was inhibited(galactose can inhibit AI-2/QS).As the number of cells increases,AI-2/QS promotes biofilm growth by enhancing cation transport,glyoxylic acid and dicarboxylic acid metabolism,and peptidoglycan biosynthesis.With the secretion of acidic substances increased,the cell’s stress response was decreased.The SOS response gene lex A was inhibited and eventually aggravated cell damage,and the biofilm cells begin to fall off.B.longum mainly regulates biofilm formation through AI-2/QS,c AMP,Lux C/Lux E TCS system by regulating Tad IV pilin and extracellular substances secretion,among which AI-2/QS is the main regulator.In the initial biofilm stage of B.longum,c AMP reduced cell motility through Tad IV pilin to promote biofilm state transition and enhanced cell repair through Lux C/Lux E TCS.With increasing cell numbers,AI-2/QS promoted biofilm growth by enhancing the transport of extracellular matrix by the Tat and Sec secretion systems.During the biofilm disperse stage,AI-2/QS enhanced the stress response to external stimuli through grp E and promoted the secretion of propionic acid,glyoxylic acid,dicarboxylic acid,butyric acid,and other substances.Bifidobacteria growth and biofilm formation under different glucose,p H,and threonine fermentation conditions were analyzed,and biofilm-promoting samples were selected for transcriptome and targeted metabolome analysis,and the mechanism of these factors regulating biofilms was analyzed.The results showed that 1.0%glucose can regulate the synthesis of lipids and polypeptides in B.bifidum to promote biofilm formation through c AMP;the synthesis of aromatic amino acids and peptidoglycan in B.pseudocatenulatum to promote biofilm formation through c-di-GMP;cation transport,pyruvate,aspartate,histidine metabolism in B.longum to promote biofilm formation through c AMP.p H 6.0 can regulate alanine,aspartate and glutamate,and pyruvate metabolism in B.bifidum to promote biofilm formation through c AMP and Qse C/Qse B;sulfur-containing amino acid metabolism,glyoxylic acid and dicarboxylic acid metabolism in B.pseudocatenulatum to promote biofilm formation through c-di-GMP;amino sugar and nucleotide sugar metabolism in B.longum to promote biofilm formation through Lux C/Lux E.2 m M threonine can regulate response to extracellular stimuli and cell communication,amino sugar and nucleotide sugar metabolism in B.bifidum to promote biofilm formation through AI-3/QS.Finally,to find the regulatory targets of Bifidobacterium biofilm fermentation,we performed collinearity analysis on the genomes of B.bifidum,B.pseudocatenulatum and B.longum,and weighted gene co-expression network analysis(WGCNA)of transcriptome samples under different biofilm fermentation conditions.It was found that the Turquoise module containing 414 genes was positively correlated with the bifidobacteria biofilm formation ability.There were 59 genes involved in 11 types of metabolism-related pathways in the Turquoise module,including peptidoglycan biosynthesis;glyoxylate and dicarboxylate metabolism;histidine metabolism;nicotinate and nicotinamide metabolism;propanoate metabolism;galactose metabolism;phenylalanine,tyrosine and tryptophan biosynthesis;arginine biosynthesis;lysine biosynthesis;alanine,aspartate and glutamate metabolism;pyruvate metabolism.These results provide targeted substances and genes references for precise regulation of Bifidobacterium biofilm fermentation.