Study On Preparation, Function And Structure Of Exopolysaccharides From Lactobacillus Casei LC2W

The exopolysaccharides (EPS) produced by the food-grade lactic acid bacteria (LAB) have been extensively studied during the last decades. These LAB EPS are considered to not only play an important role in the rheology, mouthfeel and texture of fermentation products, but also provide beneficial physiological effects on human health, such as antimutagenic, antitumour, immunomodulating and cholesterol-lowering activity. The objectives of the current work are to study the fermenting conditions of EPS produced by Lactobacillus casei LC2W, to isolate and purify EPS, and to study the bioactivity, physicochemical properties and structure characterization of EPS.The effects of different culture media on EPS produced by Lactobacillus casei LC2W were studied. Skim milk in which the yield of EPS was 83 mg/mL was chosen as a basic medium of Lactobacillus casei LC2W. Addition of soybean peptone as nitrogen, glucose as carbon and K2HPO4 to skim milk could remarkably increase the yield of EPS. The culture media of Lactobacillus casei LC2W were optimized by the orthogonal experiment design as follows: skim milk powder 12%, soybean peptone1.0%, glucose1.5% and K2HPO4 0.1%.The effects of inoculum concentration, incubation temperature and time on EPS produced by Lactobacillus casei LC2W were studied by single factor analysis. Optimal parameters of the fermenting procedure of EPS by RSM were obtained: inoculum concentration 4.0%, incubation temperature 32.5°C and incubation time of 26 h. The yield of EPS was 137 mg/L under the optimum culture medium and fermenting conditions without controlling pH, but if controlling pH 6.0 the largest yield 160 mg/L was obtained and increased by 17% compared with the yield of no controlling pH.The fermentation broth was pretreated by heating to inactivate enzymes and then centrifuging to remove cells and coagulated proteins, and trichloroacetic acid was added into the supernatant to a final concentration of 4.0% (w/v) to remove proteins. The EPS of which the yield was 153.4 mg/L were precipitated from the supernatant with three volumes of cold ethanol.LCP were fractionated by anion exchange chromatography of DEAE-Sepharose FF column, LCP1 and LCP2 were first obtained by the elution of 0.05 mol/L Tris-HCl buffer (pH 7.6) and LCP3 was followed by a linear gradient of NaCl concentration (0-1.2 mol/L). The total recovery of LCP was 81.7%, and the contents of LCP1, LCP2 and LCP3 were 35.74%, 12.61% and 33.34% based on LCP, respectively. Three polysaccharides identified by gel filtration chromatography on Sepharose CL-6B column and HPLC were homogeneous. Combined with UV-spectroscopy, FT-IR and monosaccharide composition analysis, LCP1 and LCP2 were thought to be neutral polysaccharides, and LCP3 was a complex of protein-polysaccharide which protein content was 60%.SHR were continuously fed respectively with the test samples (LCP1, LCP2 and LCP3) at the daily dosage of 15 mg kg–1 and the control sample (normal saline) for seven days. The results showed that LCP1 produced a significant decrease in the systolic blood pressure (SBP) of SHR (P<0.01), the maximal decreases (20 mmHg) were noticed from 2 h to 4 h after the oral administration, and no significant changes were found in SBP of SHR fed with LCP2 and LCP3. Meanwhile, LCP1, LCP2 and LCP3 showed no effect on heart rate of SHR, indicating they had no disadvantage effect on the circulatory system of SHR.The study on the blood-pressure-lowering mechanism showed that LCP1 reduced obviously ACE activity in aorta while no effects on ACE activity in the lung, kidney and blood serum, and that the ET-1 and CO contents in SHR blood serum made no difference between the control and LCP1 groups.Lymphocyte culture test in vitro showed that LCP1, LCP2 and LCP3 had no cytotoxicity. Immunocompetence test in vitro indicated these polysaccharides all inhibit the proliferation of T- and B-lymphocytes and mixed lymphocytes and had the obvious immunosuppressive bioactivity.The physicochemical properties of LCP1 were as follows: the purity of LCP1 was further confirmed by capillary electrophoresis (CE) analysis; the specific rotation measured by the polarimeter was [α]52859 = +62.0°(H2O, c 1.12); the intrinsic viscosity in aqueous solution determined by Ubbelohde viscometer was 1.930 dL/g; the refractive index increment (dn/dc) in 0.1 mol/L NaNO3 solution tested by Differential Refractometer was 1.20; and the molecular weight, intrinsic viscosity, polydispersity and Mark-Houwink exponent of LCP1 determined using HPSEC were 1.236×106 Da,1.875 dL/g, 1.202, and 0.654, respectively. Mark-Houwink exponent 0.654 indicated LCP1 molecule in NaNO3 solution was a random coil conformation.LCP1 molecules in aqueous solution formed the aggregates of which the aggregation rate was 45% while in NaNO3 solution there was only one population without aggregation. Therefore, 0.1 mol/L NaNO3 solution was a good solvent for studying the molecule information of LCP1. The Zimm plot from static light scattering measurement for LCP1 in 0.1 mol/L NaNO3 solution was obtained, and weight average molecular weight (Mw), radius of gyration (Rg) and the second virial coefficient (A2) measured were 1.276×106 Da, 52.9 nm, 2.44×10-4 cm3 mol/g2, respectively. Hydrodynamic radius (Rh) from dynamic light scattering obtained was 34.8 nm by extrapolating to angle zero. LCP1 molecules in NaNO₃ solution was found to be a random coil conformation by analyzingρ=Rg/Rh (1.61).Steady-shear rheological measurements of LCP1 showed a non-Newtonian shear-thinning flow behavior at high concentrations and a Newtonain flow behavior at low concentrations. The apparent viscosity decreased with the increase of temperature, and pH had a little effects. Salt might reduce the viscosity of LCP1 but the decrease was relative to salt concentration. LCP1 was found not to form a gel by viscoelastic tests.LCP1 was composed of rhamnose, glucose and galactose in mol rate of Rha:Glc:Gal=3:5:2. The structure of LCP1 was carried out by combining periodate oxidation, Smith degradation, methylation analysis and partial acid hydrolysis with GC-MS, GC, HPAEC-PAD and 1D 2D NMR. The possible structure was as follows:...