Comparative Study On The In Vitro Effects Of Pseudomonas Aeruginosa And Seaweed Alginates On Human Gut Microbiota

Microbial resources are variety,widely distributed,and easily to be modified with the advantages of growth very fast.Extracellular polysaccharide generated from bacteria has high security,unique physical and chemical properties.Alginate,which widely exists in kelp,pelvetia silquosa,fucus and sargasso cell walls,is a kind of water-soluble dietary fiber for human body.Alginate have been reported owns the functions on reducing the absorption of protein and fat,lowing blood fat,preventing obesity,hyperlipidemia,and other heart and head related diseases.Alginate also has functions on anti-tumor,prevention of colon cancer,protecting intestinal health,eliminating heavy metal ions.Alginate are widely used in biology,food,cosmetics,textiles,printing and dyeing and other fields due to its good physical and chemical properties.Alginates are extensively distributed in the cell walls of brown alga such as giant kelp,seaweed,silquosa,fucus and sargasso.However,alginates derived from brown algae are non-renewable.In addition,the composition of seaweed alginates varies with different breeding environments and harvest seasons.It is thus hard to maintain the consistent quality of alginates during production.Bacterial alginates may therefore be utilized as a substitute for the development of new products and other commercial applications.Two gram-negative bacterial genera,Azotobacter and Pseudomonas have been reported to have capacity of producing alginates.Because the growth conditions of Azotobacter are stringent,its alginate production is relatively low.Moreover,the genetic manipulation tools for Azotobacter have not been optimized to date.Current research studies are focused on ways to regulate alginate overproduction in Pseudomonas.The detailed mechanisms underlying the regulation of alginate overproduction in Pseudomonas have been extensively studied,and genetic tools for Pseudomonas manipulation have been developed,thereby enhancing alginate yield in overproducing Pseudomonas strains.In the present study,we obtained an alginate overproducing P.aeruginosa mutant by screening transposon mutagenesis libraries.We then comparatively studied the in vitro functions of human gut microbiota in degrading seaweed and mutant Pseudomonas alginates.The specific results are as follows:1.Conjugations were performed for transposon mutagenesis,using pFAC plasmid-carrying E.coli as the donor strain and PAO1 as the recipient strain.The transfer of plasmids from E.coli to PAO1 was performed via triparental conjugations using the helper plasmid pRK2013.To identify the insertion site of the transposon,inverse PCR and sequencing were performed,which showed that the site of transposon insertion was located upstream of the sigma factor gene algU,whose upregulation induced alginate overexpression2.A mucoid variant was isolated on a PIA plate,and its alginate production was determined to be substantially higher than that of PAO1 after growth at 37 °C.The composition of the bacterial alginate and seaweed alginate used in the present study was measured on a DIONEX ICS 5000 system.Compared to the standard alginate monomers M and G,both bacterial and seaweed alginates consisted of M and G.Its output about 32?g per OD600.3.Batch culture fermentations were conducted to comparative study the in vitro effects of Pseudomonas aeruginosa and seaweed alginates on human gut microbiota.Thin layer chromatography(TLC)analysis showed that the gut microbiota obtained from most volunteers degraded both bacterial and seaweed alginates after 24 h of fermentation,and all of them can degraded the alginate after 72h.SCFA production was determined by using Gas chromatography.At 72 h of fermentation,the propionic,butyric and total SCFA concentrations were higher in the alginates-added groups than that of the soluble starch-added group.However,SCFA production of acetic,propionic,butyric,and total SCFA was similar between the seaweed and bacterial alginate groups.To compare the effects of alginates on the composition of fecal microbiota,changes in the bacteria communities of 5 fecal samples were measured using PCR-DGGE and high through-put.The results demostrated that SCFA production of acetic,propionic,butyric,and total SCFA was similar between the seaweed and bacterial alginate groups,which were higher than 90%.4.Isolation and identification of alginate-degrading bacterium.Two Bacteroides were isolated which can degraded both bacterial and seaweed alginates.Sequence alignment showed that the alginate lyase genes of these BacteroidesA3 and P9 were 100%homologous.