Method for producing biologically active polysaccharide RON substance

• US 5,332,667 A
• Filed: 09/02/1993
• Issued: 07/26/1994
• Est. Priority Date: 02/08/1989
• Status: Expired due to Fees

• Current Assignee: Sapporo Breweries Limited (Sapporo Holdings Limited)
• Original Assignee: Sapporo Breweries Limited (Sapporo Holdings Limited)
• Inventors: Mitani, Yutaka, Watanabe, Nobuhiro, Kado, Hisao, Yoneta, Yasuo, Takeo, Suguru
• Primary Examiner(s): Lilling, Herbert J.
• Application Number:
  US08/116,210
• Time in Patent Office: 327 Days
• Field of Search: 435/101, 435/183, 435/252.1, 435/822, 536/1.1, 536/55.1
• US Class Current: 435/101
• CPC Class Codes: C08B 37/0009 alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
• CPC Class Codes: C12P 19/08 Dextran
• CPC Class Codes: Y10S 435/822 using bacteria or actinomycetales

Abstract

A biologically active polysaccharide RON substance which is excellent in such biological activities as antitumor activities, is produced by using a microorganism belonging to the genus Leuconostoc, which has an activity of producing the RON substance.

Claims

1. 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 α

-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;

(α

)_D²⁵ =+190°

˜

+220°

(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 α

-glucan;

(14) ¹³ C-NMR spectrum;

the substance shows characteristic chemical shift values of α

-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.

3. The process for producing a biologically active polysaccharide RON substance according to claim 1, wherein the microorganism having an ability of producing the physiologically active polysaccharide RON substance is Leuconostoc mesenteroides subsp. dextranicum of a strain selected from the group consisting of strain BL-75 (FERM BP-2242) and strain 46-1 (FERM BP-2670).

5. The process for producing a biologically active polysaccharide RON substance according to claim 1, wherein the culturing is conducted at a temperature of 15°

to 45°

C. and at a pH of 5 to 7.

7. The process for producing a biologically active polysaccharide RON substance according to claim 1, wherein the culturing is conducted at a temperature of 20°

to 30°

C., a pH of 5 to 7 and for a time period of 5 to 96 hours.

9. The process for producing a biologically active polysaccharide RON substance according to claim 1, wherein the tumor is selected from the group consisting of syngeneic Meth-A tumor, Lewis lung carcinoma, Melanoma B-16, Sarcoma-180 and Erlich ascites tumor.

11. The process for producing a biologically active polysaccharide RON substance according to claim 7, wherein the culturing is conducted in the presence of a nitrogen source selected from the group consisting of yeast extract, peptone, gluten meal, soybean powder, corn steep liquor, dry yeast, meat extract, ammonium sulfate and urea and an inorganic substance selected from the group consisting of phosphoric acid salts, magnesium salts, manganese salts, iron salts, cobalt salts and sodium salts.

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

(a) causing sucrose to act on a processed culture broth of a microorganism belonging to the genus Leuconostoc, 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 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 α

-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;

(α

)_D²⁵ =+190°

˜

+220°

(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 α

-glucan;

(14) ¹³ C-NMR spectrum;

the substance shows characteristic chemical shift values of α

-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.

4. The process for producing a biologically active polysaccharide RON substance according to claim 2, wherein the microorganism having an ability of producing the physiologically active polysaccharide RON substance is Leuconostoc mesenteroides subsp. dextranicum of a strain selected from the group consisting of strain BL-75 (FERM BP-2242) and strain 46-1 (FERM BP-2670).

6. The process for producing a biologically active polysaccharide RON substance according to claim 2, wherein the process is conducted at a temperature of 15°

to 45°

C. and at a pH of 5 to 7.

8. The process for producing a biologically active polysaccharide RON substance according to claim 2, wherein the process is conducted at a temperature of 20°

to 30°

C., a pH of 5 to 7 and for a time period of 5 to 96 hours.

10. The process for producing a biologically active polysaccharide RON substance according to claim 2, wherein the tumor is selected from the group consisting of syngeneic Meth-A tumor, Lewis lung carcinoma, Melanoma B-16, Sarcoma-180 and Erlich ascites tumor.

12. A biologically pure culture of microorganism, Leuconostoc mesenteroides subsp. dextranicum strain BL-75 (FERM BP-2242) or strain 46-1 (FERM BP-2670).

13. A biologically pure culture of claim 12, wherein said microorganism is Leuconostoc mesenteroides subsp. dextranicum strain BL-75 (FERM BP-2242).

14. A biologically pure culture of claim 12, wherein said microorganism is Leuconostoc mesenteroides subsp. dextranicum strain BL-46-1 (FERM BP-2670).