Method for producing biologically active polysaccharide RON substance

A process for the preparation of a biologically active polysaccharide RON substance which comprises

• (a) culturing a microorganism belonging to the genus Leuconostoc, which has an ability of producing a physiologically active polysaccharide RON substance, said microorganism being at least one strain of Leuconostoc mesenteroides subsp. dextranicum, said strain selected from the group consisting of strain BL-75 (FERM BP-2242), strain NCFB 517 (FERM BP-2711), strain NCFB 531 (FERM BP-2712), strain NCFB 861 (FERM BP-2713), strain NCBF 864 (FERM BP-2714), strain 46-1 (FERM BP-2670), to form a polysaccharide RON substance having the following properties:
• (1) form:
• the substance is a white amorphous powder having no taste and no smell;
• (2) solubility:
• the substance is soluble in water, but the solution becomes milky white and viscous when the concentration is elevated;
• the substance is soluble in formamide and dimethylsulfoxide, but the substance is insoluble in alcohols, acetone, benzene, ethyl acetate, hexane, chloroform and carbon tetrachloride;
• (3) pH of aqueous solution:
• the aqueous solution is neutral to weakly acidic;
• (4) constituent saccharide:
• the substance comprises solely glucose;
• (5) elementary analysis values:
• the substance comprises 44.0 to 45.0% of C and 6.1 to 6.3% of H;
• (6) structure:
• the substance is an .alpha.-1,6-glucan and comprises a small amount of 3,6-branched side chains;
• (7) proteins:
• the substance comprises almost no proteins;
• (8) molecular weight:
• the substance does not permeate through a dialysis membrane and the molecular weight is presumed to be higher than 10,000 daltons;
• (9) specific rotation:
• (.alpha.).sub.D.sup.25 =+190.degree..about.+220.degree. (C=0.5, formamide);
• (10) color reaction:
• the substance is positive to an anthrone sulfuric acid reaction and a phenol sulfuric acid reaction, but negative to a biurette reaction, the Lowry-Folin reaction, the Elson-Morgan reaction and an iodine reaction;
• (11) melting point:
• the substance has no definite melting point;
• (12) ultraviolet absorption spectrum:
• the substance has no characteristic absorption;
• (13) infrared absorption spectrum:
• the substance shows a characteristic absorption of .alpha.-glucan;
• (14) .sup.13 C-NMR spectrum:
• the substance shows characteristic chemical shift values of .alpha.-1,6-glucan in the main signals;
• and
• (15) biological activity:
• the substance has an antitumor activity against mice having a transplantable tumor, and
• (b) collecting said substance from a culture broth.