Zein Porous Materials Promoting Probiotics Biofilm Formation And Their Effects On Intestinal Flora

Biofilm culture is a way to learn from nature to protect microbes from environmental stresses.Compared with planktonic microbial cells,microbial cells immobilized on material surfaces in the form of biofilm have better stabilities and tolerances towards harsh evironments.Although biofilm formation is a natural property of microorganisms,the biofilm formation abilities of microorganisms on material surfaces vary.In this work,electrospun zein nanofiber membranes and zein/cellulose composite hydrogels were prepared and used as the scaffolds for biofilm formation,exploring the biofilm formation abilities of probiotics on these two scaffolds.Compared with planktonic Lactobacillus reuteri,the tolerances of L.reuteri biofilms in simulated digestive fluids in vitro were analyzed and the effects of L.reuteri biofilms on intestinal flora was evaluated in vitro.The detailed research results are as follows:（1）The unique 3D porous structures of electrospun zein nanofiber membranes provide plenty of adhesion sites and growth space for microorganisms,as well as perfect size matching degrees and hydrophilicity/hydrophobicity differences between electrospun zein nanofiber membranes and microorganisms,which cooperatively acting as strong driving forces to enhance biofilm formation of microorganisms with different morphologies and properties on the material surfaces.Five microorganisms all formed dense biofilms on electrospun zein nanofiber membranes in a rapid way.Alive cell densities of three probiotics and two yeasts on electrospun zein nanofiber membranes were about 0.9～1.4 Log cfu/cm²and 1.7～3.3 Log cfu/cm² higher than the values on polystyrene（PS）plates and zein films,respectively.Electrospun zein nanofiber membranes could be used as universal scaffolds for microbial biofilm formation.（2）The prepared zein/cellulose composite hydrogels had appropriate pore size,high porosity,and high specific surface area,which also enhanced L reuteri biofilm formation on the material surfaces.The pore walls and gullies of hydrogels were completely covered by L reuteri biofilms,and the maximum viable cell density reached 9.15 Log（cfu/mL）.Compared with planktonic L reuteri,L reuteri biofilms had better tolerances towards in vitro gastrointestinal fluids and bile salt.After 3 h digestion in simulated gastric fluids with pH 2.5and pH 3.5,the viable cell densities of L reuteri biofilms were about 0.35 Log（N/N₀）and 0.48Log（N/N₀）higher than the values of planktonic L reuteri,respectively.After 3 h digestion in simulated intestinal fluids,the viable cell densities of L reuteri biofilms were about 0.36Log（N/N₀）higher than the values of planktonic L reuteri.After 3 h digestion in 0.03%,0.1%,and 0.3%artificial bile salt solutions,the viable cell densities of L reuteri biofilms were about0.54 Log（N/N₀）,0.84 Log（N/N₀）,and 1.24 Log（N/N₀）higher than the values of planktonic L reuteri,respectively.（3）In fecal fermentation in vitro,zein/cellulose composite hydrogel integrated L.reuteri biofilms decreased the abundance of Acinetobacter and Klebsiella,while increased the abundance of Bifidobacterium as compared to planktonic L.reuteri;electrospun zein nanofiber membranes integrated L.reuteri biofilms increased the abundance of Lactobacillus and Bacteroidetes.L.reuteri biofilms increased the concentrations of acetic acid,propionic acid,butyric acid,and total short-chain fatty acids of fecal fermentation.Both planktonic and biofilmL.reuteri can promote the growth of probiotics,inhibit the increase of harmful bacteria,enhance the production of short-chain fatty acids,and regulate the balance of intestinal flora.L.reuteri in biofilm phenotype has a better effect on regulating intestinal flora than planktonic phenotype.