Structural Characterization And Intestinal Probiotic Fuction Of Exopolysaccharides From Lactobacillus.Delbruecckii Ssp.Bulgaricus SRFM-1

Lactic acid bacteria(LAB)that produce exopolysaccharides(EPSs)play an important role in the dairy industry because of their contribution to the consistency and rheology of fermented milk products.LAB,like many other bacteria,are able to produce several types of polysaccharides that are classified according with their location relative to the cell.Those that are excreted outside the cell wall are called exocellular polysaccharides or EPSs.These can form an adherent cohesive layer and are called capsular polysaccharides.The EPS polymers can be considered as natural biothickeners because they are produced in situ by the LAB-starters that have General Recognised as Safe status(GRAS).Moreover,a lot of LAB EPS have been shown to it can regulate the bacterium's balance.In this paper,the research object is L.delbruecckii ssp.bulgaricus SRFM-1 which isolated from Xinjiang Sayram yogurt,our objectives are to study the isolate and purify,structure characteristics and intestinal probiotic fuction.1.Chose the milk as the medium of L.delbruecckii ssp.bulgaricus SRFM-1(culture temperature 40 ?,inoculum concerntration 4%,culture time of 28 h).The fermentation liquor was centrifuged to remove cells and collected the supernate,then removing proteins by a final concentration of 4%(w/v)TCA,centrifuging the supernatant and to be concentrated under reduced pressure.The yield of EPS was 141 mg/mL which were extracted from the supernatant with threefold volumes of pure alcohol.2.The obtained crude EPS was further purified by anionic chromatography of DEAE-Cellulose 52 to acquire three polysaccharide fractions,named with EPS-1,EPS-2 and EPS-3.The receiving rate of them based on the amount of crude EPS respectively were 34.4%,17.7%and 8.8%,the total recovery was 60.9%.EPS-1 and EPS-2 all showed just a single symmetrical peak on HPLC with the molecular weights respectively estimated to be 3.97×105 Da and 3.86×105Da.As to crude EPS,FEPS-1,EPS-2 and EPS-3,protein content which measured by applying the method of Bradford was 6.98%,not detected,not detected and 0.87%,respectively.Polysaccharide content which measured by using the method of sulfuric acid-phenol coloration was 72.45%,96.75%,93.54%and 70.92%,respectively.Uronic acid content,employing the method of cartazole-sulfuric acid assay,was 2.65%,0.49%,1.47%and 2.48%,respectively.Sulfuric radical content,measured by using the method of gelatin-barium chloride assay,was 1.84%,0.39%,0.51%and 0.88%.3.The structural characterizations of EPS-1,EPS-2 and EPS-3 were detemined by various methods.UV-wavelength spectra scanning showed no maximum absorbance in the range of 190?500 nm.The monosaccharide composition of EPS-1,EPS-2 and EPS-3 were glucose and galactose in a molar ratio of 1:1.23,1:1.33 and 1.34:1.The results of FT-IR spectra of EPS-1,EPS-2 and EPS-3 showed that they all had the obvious absorption of polysaccharide,and contained pyranose ring.Moreover,EPS-2 and EPS-3 had the similar FT-IR spectra,but their molar ratios of monosaccharide were different.EPS-3 had an N-H bending vibration that showed it had a high content of protein relatively.Methylation reaction,GC-MS and NMR analasis indicated the backbone of EPS-1 were composed of?6-p-D-Galp-(1 ?4)-?-D-Glcp-(1?4)?-D(1?4)-?-D-Galp-(1 ?6)-?-D-Galp-(1?4)-?-D-Glcp-(1 ?4)-a-D-Galp-(1 ?4)-?-D-Galp-(1 ?4)-?-D-Glcp-(1?,and had three branching points which existed in terminal Glcp residue;the EPS-2 was composed of?6-?-D-Galp-(1?4)-?-D-Glcp-(1?6)-?-D-Galp-(1?as the backbone chains with a branching point which existed in terminal Glcp residue,we could speculate the structure of EPS-1 and EPS-2.EPS-1:(?)EPS-2:(?)SEM analysis indicated that the results of EPS-1 are different from EPS-2 and EPS-3,EPS-2 and EPS-3 were aggregated in the form of filaments,and the accumulation was polluted and incomplete.There were spherical aggregates.EPS-2 and EPS-3 were aggregated irregular size of the empty.Congo red reaction indicated that EPS-2 and EPS-3 had triple helical structure.4.The antioxidant activities of EPS in vitro were evaluated and the results showed that they have strong superoxide radical scavenging activity,DPPH radical scavenging activity,hydroxyl radical scavenging activity and ferrous ion chelating activity.Their antioxidant activities enhanced in the order of crude EPS>EPS-1>EPS-2>EPS-3.5.Fermentations with ten pure strains of LAB were carried out using the purified EPSs as the sole carbon source,and bacteria growth was evaluated at 600 nm by means of a microplate reader during 48 h.Maximum growth rates(?max)and lag phase were calculated.In general,all the strains tested were able to utilize all the EPS when they were used as sole carbon source.Nonetheless,different structure and content of EPS affected the individual strains lag phase,cell densities and growth rates.Some strains showed higher cell densities and speeds of growth on these EPS than on commercial inulin.6.The different pure EPS was studied by anaerobe faecal fermentation in vitro.Total bacterial population,beneficial bacterium and harmful bacterium were enumerated by FISH.Prebiotic properties were evaluated by SI.In addition,their effects on production of SCFA were also investigated aiming to understanding of specific structure-prebiotic properties relationship of these EPS.EPS-1 and EPS-2 all had obvious promote the proliferation of probiotics activity and inhibited the activity of harmful bacteria,got higher SI,and produced a lot of acetic acid and lactic acid in the fermentation process,and showed good prebiotic properties.EPS-3 only obtained a lower SI,and the impact on the producing SCFA was not very obvious.