| Lactic acid bacteria (LAB) exopolysaccharides (EPS) are long-chain polysaccharides secreted by LAB into the extracellular environment during growth. Due to their unique physical and rheological properties, EPS are widely used in the food industry as thickeners, emulsifiers, gelling agents and stabilizers. In addition, these polysaccharides have been demonstrated to possess important biological properties, such as antitumor, immunomodulatory, immunostimulatory, antinociceptive and antiinflammatory, antiviral, antimutagenicity and antioxidant activities. EPS produced by LAB have gained considerable attention mainly because LAB are food-grade bacteria known as GRAS and their EPS could be easily utilized in foods in a juridical point of view.The isolation and identification of this EPS-producing Lb. plantarum strain70810from Chinese Paocai and the analysis of its EPS were carried out systemically. The main results are as follows:1. LAB isolated from various traditional Chinese fermented foods were screened for the production of EPS. The rod-shaped strain70810from Chinese Paocai, which was identified as Lb. plantarum (HQ259238) by morphological, physiological, biochemical and16S rDNA tests, was selected due to its highest EPS production capability for further study. Effects of various culture conditions (media, carbon, nitrogen, inoculation volume, initial pH, temperature, and culture time) on EPS production were investigated firstly by single factor method. For EPS production with, the preferable culture conditions were30℃and pH6for24h with sucrose30g/L, soybean peptone10g/L, as the carbon, nitrogen of MRS meadium, respectively. The fermentation conditions of exopolysaccharides produced by Lb. plantarum70810were optimized. Three main factors were screened by Plackett-Burman (PB) design from fourteen nutritional and cultural factors affecting Lb. plantarum70810EPS yield. The screened significant factors were then optimized by response surface methodology (RSM). The PB experimental results showed that sucrose, inoculation volume and temperature were significant factors for the production of EPS. The optimal conditions were confirmed by RSM as follows:sucrose34g/L, inoculation volume5%(V/V) and temperature31℃. Under these optimal conditions, the70810EPS yield reached425.16mg/L. Conclusion:It is feasible to apply PB design and RSM to optimize the fermentation conditions of exopolysaccharides from Lb. plantarum70810.2. To remove proteins, two methods were tested:Sevage method and trichloroacetic acid (TCA) method. The results indicated that TCA method is better. TCA final concentration4%was selected and the deproteinization rate was86.4%. The optimal conditions of ethanol precipitation of EPS produced by Lb. plantarum70810were investigated by single-factor experiments and response surface methodology. A mathematical model was then developed to show the significant effect of each factor and their interactions on the yield of EPS. The optimal parameters for ethanol precipitation of Lb. plantarum70810EPS were confirmed as follows:precipitation time12.56h, volume of ethanol3.9times, concentration multiple of original fermentation supernatant3.54times. Under the optimal condition, the model-predicted and experimental values of EPS yield were323.11mg/L and320.17mg/L, respectively, revealing0.91%relative error between them. The optimal decolorization conditions of EPS from Lb. plantarum70810were studied with macroporous adsorptive resin by single-factor experiments. The results showed that resin S-8demonstrated the highest decolorization ratio among various resins tested. The optimized decolorization conditions were determined as follows:resin S-8of4%(W/V), pH value of EPS solution6, concentration of EPS solution2mg/mL, for4h at25℃in static state. Under these conditions, the decolorization ratio and retention ratio of EPS were72.58%and69.15%, respectively. The decolorized and deproteinized EPS obtained from S-8resin was further purified by chromatography of DEAE-52and Sephadex G-100to afford two polysaccharide fractions (EPS-1and EPS-2). EPS-1and EPS-2both showed only one symmetrical peak on HPLC with the molecular weights estimated to be1.75×104and1.20×105Da, respectively. As to crude EPS, EPS-1, and EPS-2, polysaccharides content, measured by employing the method of sulfuric acid-phenol coloration, was76.75%、94.45%and90.55%, respectively. Uronic acid content, determined by using the method of cartazole-sulfuric acid assay, was3.19%、2.09%and1.71%, respectively. Protein content, measured by applying the method of coomassie brilliant blue coloration, was0.32%、0.82%and1.58%, respectively. Sulfuric radical content, determined by employing the method of gelatin-barium chloride assay, was not detected, respectively.3. The structural characterizations of EPS-1and EPS-2were investigated by various methods. The analysis of monosaccharide compositions of EPS-1and EPS-2were carried out by GC. EPS-1was composed of glucose, mannose and galactose with a molar ratio of53.24:10.41:1.00. EPS-2was composed of glucose, mannose and fucose with a molar ratio of3.48:9.61:1.00. Spectra of FTIR implied that there were a-glycosidic bond and pyranose rings in EPS-1and EPS-2. On the base of the results of monosaccharide compositions, FTIR, methylation analysis, GC/MS,1H and13C NMR, it was possible to conclude that the repeated unit of EPS-1contained a backbone composed of1'4-linked-glucose and mannose, which attached to galactose. EPS-2contained a backbone composed of1'4-linked-glucose and branched by1'2,6-linked-mannose, which attached to fucose.4. Biosorption of Pb(II) from aqueous solutions by70810EPS was studied with parameters of initial pH, contact time, initial Pb(II) concentration, adsorbent dosage, and temperature, respectively. Maximum adsorption of Pb (II) was observed at pH5,30℃, and contact time6h, respectively. The adsorption capacity was also found to be dependent on initial Pb(II) concentration and adsorbent dosage. The adsorption capacity increased when initial Pb(II) concentration increased from0.1to1000mg/L. The metals uptake increased from25.44to160.62mg/g with70810EPS dosage between0.5and1mg in5mL volume, but decreased as70810EPS dosage further increased. Fourier transform infrared spectroscopy (FT-IR) spectra analysis indicated that some functional groups (e.g.,-OH, COO-, C=O and-NH) of70810EPS were involved in Pb(II) biosorption process.5. The inhibition effects of crude EPS, EPS-1and EPS-2on the growth of PC-3cells, HCT-116cells, Hep G2cells and HT-29cells were evaluated in vitro by using Cell Counting Kit-8(CCK-8) method. The results showed that, crude EPS, EPS-1and EPS-2all presented significantly higher antitumor activity against the PC-3cells, HCT-116cells, Hep G2cells and HT-29cells in vitro than blank control groups, and the inhibition ability was dose-dependent and time-dependent. At the concentration of400μg/ml for72h, the inhibition rates of crude EPS, EPS-1and EPS-2against the PC-3cells were93.74%,38.54%and39.40%, respectively. The inhibition rates of crude EPS, EPS-1and EPS-2against the HCT-116cells were57.35%,55.01%and57.48%, respectively. The inhibition rates of crude EPS, EPS-1and EPS-2against the Hep G2cells were96.76%,52.73%and59.76%, respectively. The inhibition rates of crude EPS, EPS-1and EPS-2against the HT-29cells were46.10%,39.78%and57.45%, respectively. |
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