Synthesis And Characterization Of Prebiotics Produced From Acceptor Reaction Of Glucansucrase

Prebiotics are defined as selectively fermented ingredients that result in specific changes in the composition or activity of the gastrointestinal microbiota,thus conferring benefit upon host health.As the most important part of prebiotics,functional oligosaccharides are metabolized by intestinal bacteria instead of enzymes in upper gastrointestinal,which play a great role in altering the intestinal microbiota composition,producing the short chain fatty acids(SCFAs),enhancing the host immunity and preventing cancer.Recently,enzymatic synthesis has been a mainly effective way to develop functional oligosaccharides.Glucansucrases(GTF)are classified as glycoside hydrolase enzymes that synthesize a variety of α-glucan polysaccharides from sucrose while oligosaccharides are synthesized in presence of maltose as acceptor.This study focused on enzymatic synthesis of prebiotics from L Leuconostoceros citreum SK24.002 GTF acceptor reactions including purification,structural analysis,evaluation of prebiotic properties in vitro fermentation.Firstly,it was carried out to analyze products from maltose and maltitol acceptor reactions catalyzed by L.citreum SK24.002 GTF.It showed that three oligosaccharides were synthesized including one trisaccharide(DP3),one tetrasaccharide(DP4),one pentasaccharide(DP5)when maltose was the acceptor,and four oligosaccharide alcohols were synthesized including one trisaccharide alcohol(DP3-H),two trasaccharide alcohols(DP4-H),one pentasaccharide alcohol(DP5-H)when maltitol was the acceptor.The reaction conditions were then optimized on the ratio of donor/acceptor,enzyme amount and substrate concentration.The optimum reaction conditions for maltose were as follows: the ratio of donor/acceptor 1:1,enzyme amount 0.63 U/mL,substrate concentration 150 mg/mL,as a result at which the total production of oligosaccharides was 161.0 mg/mL,including DP3 78.7 mg/mL,DP4 65.0 mg/mL and DP5 17.3 mg/mL.The optimum reaction conditions for maltitol were as follows: the ratio of donor/acceptor 1:1,enzyme amount 0.80 U/mL,substrate concentration 100 mg/mL,as a result at which the total production of oligosaccharide alcohols was 89.6 mg/mL,including DP3-H 61.5 mg/mL,DP4-H 16.6 mg/mL and DP5-H 11.5 mg/mL.Secondly,oligosaccharides were isolated and purified via gel chromatography and high performance liquid chromatography,then whose structure was analyzed by mass spectrometry and nuclear magnetic reasonance.It showed that three oligosaccharides synthesized in maltose acceptor reaction were DP3,α-D-Glcp-(1→6)-α-D-Glcp-(1→4)-D-Glcp,also called panose,DP4,α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→4)-D-Glcp and DP5,α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→4)-D-Glcp;four oligosaccharide alcohols synthesized in maltitol acceptor reaction included DP3-H,α-D-Glcp-(1→6)-α-D-Glcp-(1→4)-D-Gol,DP4-H,α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→4)-D-Gol and α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→4)-D-Gol as well as DP5-H,α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→4)-D-Gol.Finally,all oligosaccharides were studied by anaerobe faecal fermentation in vitro as well as FOS,GOS,inulin.It was found that all pure oligosaccharides effectively promoted the growth of intestinal flora with the great increase of OD600 after 48 h fermentation.Besides,pH in vitro system of all pure oligosaccharides dropped rapdily by 2.6 to 3.0,which was attributed to the synthesis of SCFAs.Further,SCFAs were measured by gas chromatography(GS),which showed the yield of acetic acid,propionic acid and butyric acid of DP5,DP5-H,DP4 and DP4-H groups were higher than those of FOS,GOS and inulin groups,DP3 and DP3-H showed similar capabilities to the control groups.Total bacterial population,beneficial bacterium and harmful bacterium were enumerated by fluorescent in situ hybridization(FISH).It was found all pure oligosaccharides promoted the proliferation of Bifidobacterium and Lactobacillus,and DP3,DP5,DP4-H and DP5-H performed better on proliferation of Bifidobacterium than GOS and FOS groups.Only DP4 and DP4-H groups showed the inhibition on Bacteroides Levalliella.The prebiotic properties could also be expressed in the form of Prebiotic Index(PI).The PI values at 12 h of all products were higher than those at 24 h.The PI values of DP3,DP5,DP4-H and DP5-H at 12 h were higher than those of GOS,and the PI values of pure products except DP4 at 24 h were higher than those of FOS,GOS and inulin.