Structure, Chain Conformation And Rheological Behavior Of The Exopolysaccharide From Bacillus Mucilaginosus SM-01

Bacterial exopolysaccharides are biopolymers which have diverse structures and unique characteristics. Because of their interesting physicochemical properties and biological activities, bacterial exopolysaccharides are widely used in food, pharmaceuticals and chemical technology. Although there are a vast number of bacterial exopolysaccharides have been reported over recent decades, only a few have emerged as industrially important biopolymers with significant commercial value. The main constraints to full commercialization are the lack of structure-property relationship and polysaccharide production as well as their cost, mostly related to substrate cost and downstream processing.To expand the utilization aspects,many research interests focus on the isolation and identification of novel bacterial exopolysaccharides, which may have particular properties.Bacillus ,mucilaginosus is a silicate bacterium, which can utilize the limited nutrition to produce exopolysaccharide. Most of studies have focused on its degradation of soil mineral compounds (silicates, apatites and phosphorites) with the release of mobile potassium and water-soluble phosphorus, thereby improving the plant nutrition. In addition, B.mucilaginosus can be used as a bioflocculant and biosorbent in wastewater treatment to remove various contaminants. A highly acceptable hypothesis is that the exopolysaccharide secreted by B. mucilaginosus plays an important role during the process of weathering and flocculation. It has also been found that the exopolysaccharide possess the bioactivity that can promote the repair of gastric ulcer of mice and the upgrowth of immune organs in chickens.However, to the best of our knowledge, the chemical structure of the exopolysaccharide from B. mucilaginosus is still unexplored. Studying the chemical structure and physicochemical property of exopolysaccharide will be helpful to understanding the relationship of structure and property, thus expanding its potential application. In our study, the exopolysaccharide(BMPS) from B. mucilaginosus was isolated and purified from the culture broth by optimized method. Its primary structure and solution properties in aqueous solution were characterized and its function as the material in wound repair had been preliminary studied simultaneously.The main results of this study were summarized as follows:(1) Investigating the effect of dilution ratio, temperature, pH and the amount of NaCl on the viscosity of fermentation broth, the method of dilution after the addition of 3% NaCl could reduce the viscosity of broth. Under this condition, the elimilating of bacterial and protein by centrifugation and filtration were compared. Filtration had better effect on elimination for bacteria and protein than centrifugation. The anount of diatomite also had influence on the elimination of bacteria and protein as well as the revovery of polysaccharide.As a result, by addition of 3% (w/v) NaCl and then diluted for 3 times, and previously absorbed by 10 g/L diatomite, the solution was filtrated in vacuum with diatomite for three times to remove cells and proteins, the recovery of polysaccharide was 76.4%. The filtrate was then concentrated by Labscale TFF System. It was found that with the membrane of 200 kDa and operating pressure of 0.1 MPa, the total revoery of polysaccharide was up to 73.2%with the amount of endotoxin below 0.25 EU/mL. The crud polysaccharide was obtained by freeze-drying. An acidic hetero-exopolysaccharide BMPS was purified by ion-exchange and size-exclusion chromatography from crud polysaccharide. The molecular weight was measured to be 2.67×106 Da.(2) The contents of uronic acid and O-acetyl for BMPS were 14.3% and 4.9%,respectively and there was no N, P, S existed in BMPS. The monosaccharide of BMPS was composed of Glc, Man, G1cA and an unknown uronic acid with a molar ratio of 3.2: 2: 0.5:0.3. A neutral oligosaccharide BMPS-H with an average molecular weight of 1771.43 Da was obtained under mild hydrolysis from low molecular weight BMPS. Monosaccharide analysis indicated that BMPS-H contained glucose and mannose in a molar ratio of 1.5: 1. Based on methylation analysis and NMR spectroscopy, the following structure of BMPS-H was established:?-D-Glc (1-[4)-?-D-Man (1?4)-?-D-Glc (1?]m?3)-?-D-Glc (1-[4)-?-D-Man (1-4)-?-D-Glc (1?]n-?-D-Glc OAc OAc(3) BMPS displayed typical polyelectrolyte behavior in pure water and the intrinsic viscosity dramatically decreased with the addition of NaNO3. The relative stiffness parameter(B-value) was estimated to be 0.018, indicating the semi-flexibility of the BMPS backbone.The dependences among the [?] and <s2>z1/2 on Mw in the range of Mw from 267 x 104 to 37.3 x 104 Da, as well as the ratio of geometric to hydrodynamic radius revealed that BMPS exhibited an extended coil geometry in 0.1 mol/L NaNO3 aqueous solution. The molecular size and shape of BMPS was further characterized by using Yamakawa-Fujii-Yoshizaki theory and AFM. The value of ML (663.5 nm-1) and q (9.5 nm) indicated that BMPS existed as a semi-flexible chain again. The average chain diameter (0.6 nm) fitted by Yamakawa-Fujii-Yoshizaki theory was in accordance with the result (0.68 nm) of AFM.(4) BMPS solutions developed non-Newtonian shear-thinning (pseudoplastic) behavior even at concentrations as low as 0.01% which was concentration dependent. The flow behavior of BMPS was fitted with the Williamson model to obtain the zero-shear rate viscosities. The coil overlap concentration of BMPS in water was determined to be approximately 0.6% by the intersection of two exponential equation that the concentration dependence of zero shear-rate viscosity for BMPS. The viscoelastic behavior was observed with solutions within 1.4-2.7% consistent with the formation of entangled macromolecules in solution.(5) Investigating the influence of concentration of PVA and spinning voltage on the nanofiber, the optimized condition was: 8% for PVA and 14 kV of voltage. Different ratios of BMPS/PVA blends including 3/100, 5/100, 10/100 and 20/200 were electrospun into nanofibers by using 8% PVA aqueous solution under the voltage of 14 kV. It was found that both the viscosity and conductivity have a great influence on the spinning process and fiber morphology. With the increasing of the percentage of BMPS, the conductivity and viscosity of blends increased accordingly, thereby increasing the difficulty of electrospinning. The different ratios of BMPS/PVA nanofiber were crosslinked by glutaraldehyde for 10 min,which can change its water-soluble while keeping the morphology of fiber. Both the PVA nanofiber and BMPS/PVA nanofiber represented nontoxicity to NIH3T3 cells, and compared to PVA nanofiber, the BMPS/PVA nanofiber possessed a better biocompatibility.