Preliminary Studies On An Extracellular Polysaccharide From A Strain Of Bacteria

Recently, in the field of research and application of microbial extra polysaccharides, great attentions have been increasingly paid to polysaccharides of biological activity which are natural products. Polysaccharides of biological activity have many functions, for example, enhancing the organism of immunity, activity of radioresistance, antineoplastic activity, antisenscence, and so on. As a result, it is a profound project to research polysaccharides of biological activity.In the researching, we found that a strain(Rhizobium sp. N613) could produce abundant extra polysaccharide which is called REPS. And now inland and abroad literature do not have any report about REPS. This paper focused on the pharmacology of REPS, studying the cultivation substrate and cultivation condition of the strain, studying the batch fermentation and fed-batch fermentation in 10L fermentation tank, fabricating kinetic models of batch fermentation, and primary researching on downstream processing. The detailed contents were as followed:1. Three dosage of REPS (40, 20, 10 mg /(Kg·d)) were given by intraperitoneal (ip) administration for ten days. Indexes of thymus and spleen in normal mice, velocity and index of carbolic particle clearance, the antibody titer of serum against SRBC, and the stimulating index of lymphocyte transformation were determined after ten days. Compared with the control group, the group of high dose was the most significant and the results were 0.32, 0.65, 3.41×10^-2, 5.10, 2.23 and 1.36 respectively, which were 1.52, 1.35, 1.54, 1.11, 1.18 and 1.36 times of the control group respectively. The results indicated that REPS could enhance non-specific immunity, humoral immunity and cellular immunity in the normal mice. In addition, mice with S180 tumor were also given REPS in the same dose and method with what have mentioned above. Compared with the control group, the highest rate of tumor inhibition could reach 45%. And the results of the routine blood examination indicated that the mechanism of inhibiting S180tumor might relate to the immune function of mice.2. The optimal cultivation medium and cultivation conditions of the strain (Rhizobium sp. N613) with a high yield of REPS were studied by means of the orthogonal design. From the shake-flask experiments, the optimal cultivation medium consisted of the following composition(g/L): glucose 20, (NH4)2SO4 1.0, juice of bean sprout 200, phosphate 0.4, MgSO4 0.4. The optimal cultivation conditions were: the primary pH 7.0, 30°C, lOOmL in 250mL total volume.3. The batch fermentation was conducted in 10L fermentation tank and the fermentation kinetics of REPS was designed. The lag phase was 0 h ⁶ h, exponential phase 6 h -20 h, stationary phase 20 h³0 h, and decline phase was after 30 h. The maximal yield of REPS reached 8.2g/L. The kinetic models for cell growth, REPS production and substrate consumption were obtained based on Logistic equation and Luedeking-piret equation. The kinetic models were as follows:X(/) = 0.377exp(0.3130/ { 1 - (0.377/14.4)[ 1 -exp(0.3130] >P(t) = 0.273[Z(0 - 0.377] + 0.021 x(14.4/0.313)ln { 1 - (0.377/14.4) [1 - exp(0.313?)] >Sit) = 30.3 - 1.38[Jf(0 - 0.377] - 0.04x 14.404/0.3131n { 1 - (0.377/14.4)[l -exp(0.3130] >4. The fed-batch fermentation was conducted in 10L fermentation tank. The maximal yield of REPS reached 12.6g/L, which was 1.5 times of the batch fermentation.5. The primary technology of downstream processing was established. The recovery rate of REPS was above 95 per cent and the purity reached 99.2 per cent.