| ?-glucans are a kind of polysaccharides that are not synthesized by human and animals.?-(1,3)-D-glucans have shown unique and various biological functional properties which made people pay more attention to them. However, most?-(1, 3)-D-glucans are hard to extract along with low yield and poor water solubility, which would inhibit their biological activities. Extracellular polysaccharides produced by bacterial fermentation are relatively easy to extract, while there have been very a few reports about water soluble?-glucans extracted from bacteria. Therefore, it would be very important of making studies on the strain producing water soluble extracellular?-(1,3)-D-glucan as well as the extraction processes, structure and properties of the?-(1, 3)-D-glucan, to take more advantages of the biological functions of?-(1,3)-D-glucan.A salt-tolerance new strain ZX09 was isolated from a soil sample from ocean coast of Shandong in our laboratory. The most notable features of this strain are that it could grow in an inorganic medium with 7% NaCl and produce a lot of water-soluble extracellular polysaccharide. According to morphologic, biochemical and physiological characteristic, the vegetative cells are cylindrical rods. The available carbon sources of ZX09 are sucrose, lactose, glucose, mannose, fructose and xylose, and unavailable ones are starch, meltose and cellulose. It can also grow in PDA medium and LB medium. 16S rDNA sequence analysis indicated that the strain was related to the genus Agrobacteria(Agrobacterium sp. ZX09).The culture broth was added to two volumes of 95% ethanol and crude productivity of extracellular polysaccharide, which was named Salecan, was extracted and then dried. The best fermentation conditions to produce more extracellular polysaccharide were studied, and the suitable Htm medium was:NaH2PO4 1g, KNO3 3g, MgCl2 0.2g, CaCl2 0.07g, FeCl2 0.0125g, MnSO4 0.003g, ZnCl2 0.0075g, Sucrose 30g, Water 1000 ml, pH 7.0-7.2. The viscosity of the fermentation broth could get the best at 48 h when it could produce more than 20 g Salecan from 1000 ml fermentation broth. When added the 48h fermentation broth in the new culture medium which was fermented for 48h again and continued this fermentation for 20 times, no contamination or decline of Salecan production was observed. The extracellular polysaccharide produced by Agrobacterium sp. ZX09 is stable in fermentation together with simple extraction processe and high yield.The chemical compositions of Salecan are sugar 77.13%, protein 6.20%, ash 10.28% and moisture 5.20%. Salecan was isolated and purified by deproteinization and DEAE-cellulose, and it was found that there was only one polysaccharide in Salecan. Molecular weight of this polysaccharide is about 2×106 Da. Thin-layer chromatography (TLC) analysis revealed that the purified polysaccharide was composed of D-glucose only, which was conformed by gas chromatographic analysis. No functional group was found in the polysaccharide molecular such as sulfated group and uronic acid. By examining Fourier Transform Infrared Spectroscopy, periodate oxidation,1H NMR and 13C NMR spectra, it was proven that the polysaccharide in Salecan was consisted of the following repeating unit:?3)-beta-D-Glcp-(1?3)-[beta-D-Glcp-(1?3)-beta-D-Glcp-(1?3)]3-alpha-D-Glcp-(1?3)-alpha-D-Glcp-(1?. It is a kind of (1?3)-?-D-glucan with noval structure.Salecan is water soluble and its solution has high viscosity. The rheological research provided an investigation of the rheological properties of Salecan solution over a wide range of shear rate (0.001 -1000 s-1), concentrations (0.3%,0.5%,1.0% and 1.5%), temperature (5 to 95?), heating and freeze-thaw, pH (1.0 -13.0), frequency (0.1 -100 rad/s) and salts. Salecan solutions showed a non-Newtonian pseudoplasticitic behavior at all concentrations. In the examination of flow properties as a function of shear rate, Salecan solutions exhibited a Newtonian plantau at the shear rate below 0.1 s-1 and then a region of shear-thinning. The viscosity became higher with the increase of the concentration, and the power law model well described the rheological behavior of the solutions. There would be a phase transition when the temperature was increased from 5 to 95?. Heating and Frozen didn't affect the solution viscosity at certain concentration, and over a wide pH range from 6.0 to 12.0, the viscosity almost kept invariant. With increasing frequency the storage and loss moduli G'and G" of Salecan solution increased continuously, and there was always G'>G" which showed an elastic behaviour. The presence of salts could promote viscosity and stability of Salecan solution. It can be seen that Salecan, with high viscosity and stable rheological properties of its solution, may be utilized in foods to give viscosity, body, and mouth feel as a new source of thickening agent.The toxic study in ICR mice was to investigate the acute and subchronic toxicity of Salecan. There were no deaths or any unusual behaviour in Salecan-treated mice. The oral LD50 of Salecan in ICR mice was greater than 3000 mg/kg body weight in acute toxicity test. In the 90-day feeding study, ICR mice were fed diets containing 0%,1.0%, 2.5% and 5.0% of Salecan. Based on the results from subchronic study, the overall health, body weight gain, food consumption and clinical pathology parameters (hematology, blood chemistry and organ histology) were comparable between the groups feed Salecan and the control group. No dose-related effects were observed in the treated animals. The only exception was the observation that blood glucose in female mice fed Salecan was lower than in the control group. In addition, the fecal matter from Salecan-fed mice exhibited increase water content versus the control animals. The results demonstrate that Salecan is non-toxic at the levels tested (at or below 5%) does not pose a health risk to animal use.In vitro the Salecan was indigestible by a-amylase, and strongly inhibited pancreatic a-amylase activity. Fasting mice fed on Salecan failed to increase blood glucose levels. Experimental diets containing the Salecan suppressed steep increase in blood glucose concentration. This biological property of Salecan shows that it would be use in functional food or medicine to decrease absorption of dietary carbohydrate and postprandial blood glucose level for diabetics.As a novel water soluble?-glucan, Salecan is simple to be extracted from fermentation broth with high yield, and it also has excellence rheological properties and biological activities, as well as edible safety. All the results from this study indicate that Salecan could be used in industries such as food and medicine. |
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