| Lactic acid bacteria exopolysaccharides (LAB EPS) have been generally researched by many workers because of its excellent physical properties and biological activities. LAB EPS are the LAB's metabolic products during growth, they include capsular and mucus polysaccharides which respectively combined with cell wall or secreted into extracellular. According to the reaearch reported, EPS are not merely have effects of mouthfeel, texture and rheology of fermentation dairy, but also the substitute for food additives such as stabilizer, emulgator and thickener. Moreover, a lot of LAB EPS have been shown to enhance immunity, antitumor, anti-inflammatory and cholesterol-lowering ructions.In this paper, the research object is Lactobacilus helveticus MB2-1 which isolated from Xinjiang Sayram yogurt, our objectives are to study the isolate and purify, structure characteristics and physiological function.1. Chose the whey powder as the medium of Lactobacilus helveticus MB2-1 in 50 L fermentation tank (culture temperature 42?, inoculum concerntration 4%, culture time of 32 h). The fermentation liquor was centrifuged to remove cells and collected the supemate, 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 789 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 and Sephadex G-100 to acquire three polysaccharide fractions, named with LHEPS-1, LHEPS-2 and LHEPS-3. The receiving rate of them based on the amount of crude EPS respectively were 10.47,46.96 and 2.81%, the total recovery was 60.24%. LHEPS-1, LHEPS-2 and LHEPS-3 all showed just a single symmetrical peak on HPSEC with the molecular weights respectively estimated to be 2.08×105,2.04×105and 2.01×105 Da. As to crude EPS, LHEPS-1, LHEPS-2 and LHEPS-3, protein content which measured by applying the method of Bradford was 4.08, not detected, not detected and 0.29%, respectively. Polysaccharide content which measured by using the method of sulfuric acid-phenol coloration was 71.68%,97.85%,94.54% and 67.62%, respectively. Uronic acid content, employing the method of cartazole-sulfuric acid assay, was 2.98,0.53,1.96 and 2.53%, respectively. Sulfuric radical content, measured by using the method of gelatin-barium chloride assay, was 1.43,0.27,0.42 and 0.87%.3. The structural characterizations of LHEPS-1, LHEPS-2 and LHEPS-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 LHEPS-1, LHEPS-2 and LHEPS-3 were mannose, glucose and galactose in a molar ratio of 1:2.75: 1.33,9.34:1.43:1 and 2.96:1:1.17. The results of FT-IR spectra of LHEPS-1, LHEPS-2 and LHEPS-3 showed that they all had the obvioys absorption of polysaccharide, and contained pyranose ring. LHEPS-1 had the P-pyranose form of the glucosyl residue and ?-pyranose form of the mannose residue. Moreover, LHEPS-2 and LHEPS-3 had the similar FT-IR spectra, but their molar ratios of monosaccharide were different. LHEPS-3 had an N-H bending vibration that showed it had a high content of protein relatively. Methylation and GC-MS analysis showed that LHEPS-1 was composed of ?4,6-D-Man/(-(1? and?3,6-D-Glcp-(1? as the backbone chain, and had two branching points which existed in Manp chain and Glup chain. The two terminal residues were D-Glcp and D-Galp. The backbone of LHEPS-2 was composed of ?4-D-Manp-(1? and ?6-D-Manp-(1?, the?6-D-Glcp-(1? and ?4-D-Galp-(1? could exist in the side chains or backbone chains. LHEPS-3 was composed of ?4,6-D-Manp-(1? and ?3,6-D-Manp-(1? as the backbone chain, and had two branching points which existed in Manp chain. The two terminal residues were D-Glcp and D-Galp. Combining the results of 1D NMR,2D NMR and above methods, we could speculate the structure of LHEPS-1, LHEPS-2 and LHEPS-3.Observing the results of SEM, we could find that EPS has a glaze surface and present a fragment state of aggregation. LHEPS-2 and LHEPS-3 had a siphonate state of aggregation, piled on one another and presented a state of launching. The images of AFM showed that EPS contain spike shaped lumps which have various heights ranging from 2 to 380 nm.4. The antioxidant activities of EPS in vitro were evaluated and the results showed that they have strong superoxide radical scavenging activity, moderate hydroxyl radical scavenging activity and ferrous ion chelating activity, weaker DPPH radical scavenging activity. Their antioxidant activities enhanced in the order of crude EPS> LHEPS-3> LHEPS-2> LHEPS-1, excepte the order of superoxide radical scavenging activity was crude EPS> LHEPS-2> LHEPS-3> LHEPS-1.5. Using the tumor cell model in vitro, we could find that the EPS of Lactobacilus helveticus MB2-1 has proliferation inhibition to human hepatoma cell, gastric cancer cell and colon cancer cell. Among these cells, the function of proliferation inhibition to HepG-2 was strongest, and then was BGC-823, the last was Caco-2, so we chosed HepG-2 cell to research in depth. The subsequent experiment indicated that the LDH of cell increase with the increase of EPS's concentration and make the HepG-2 cell shrinkage. Moreover, the study also found that the ROS of intracellular could be increased by EPS, keeping HepG-2 cell cycle from growing in the G2/M phase, and having a higher induction of apoptosis with the rate of 26.19% when the concentration of 600 ?g/mL. Finally, using the kit assay we test that EPS can make the Caspase-3/9 activities increase. |
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