JPS5839441B2 - New polysaccharide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel polysaccharides, more specifically Azotobacter polysaccharides.
The present invention relates to a novel polysaccharide produced from bacteria belonging to the genus (Azotobacter).

Several bacteria belonging to the genus Acidobacter are known to produce polysaccharides;
, Gorin and J.F.T., 5pence rl.
Canadian Journal of Chem
istry vol. 144 (1966), pp. 993-998] is Acidobacter vinellandii (A.
ndii) produces a polysaccharide consisting of partially acetylated mannuronic acid and guluronic acid.

Also, J.D. Blake and F. Joffrey, CJ, D., Blake and N.

GeoffreylAustralian Journ
al of Chemistry Volume 123 (1970)
, p. 194] reported that Azotobacter vinelangii produces a polysaccharide consisting of galacturonic acid nigercose:rhamnose=43:2:1 (molar ratio).

The polysaccharide of the present invention has constituent sugars, chemical and physical properties,
Unlike these known polysaccharides, it has the following characteristics.

(1) The constituent sugars are glucose, mannose, and mannuronic acid, and their composition ratio (molar ratio): haclucose: mannose: mannuronic acid = 1:0.4-0.7:4-17
It is.

(2) It is acetylated with a degree of acetylation of approximately 0.1 to 1.0, and acetic acid is detected when an ether extract of the hydrolyzate is subjected to gas chromatography to analyze organic acids.

(3) Shows a molecular weight of 103 to 105 when measured by the 3.5-dinitrosalicylic acid method.

(4) Shows positivity in each color reaction of Molisch reaction, phenol sulfuric acid reaction, and Anthrone sulfuric acid reaction.

(5) When the infrared absorption spectrum is measured by the film method, the infrared absorption spectrum shown in the attached FIG. 1 is obtained.

(6) Insoluble in organic solvents such as methanol, ethanol, acetone, and ether; insoluble in water under acidic conditions; soluble in neutral or basic conditions (this aqueous solution is colorless and transparent)
It is.

(7) The viscosity of the 5% w/v aqueous solution at 30°C is 102 to 105 cp when measured with a BM type viscometer, 30 r, p, m.

Thus, the polysaccharide of the present invention can be obtained by culturing the polysaccharide-producing bacterium belonging to the genus Azotobacter, for example, Acidobacter vinelangii-IFO12018 (obtained from the Fermentation Research Institute (Yodoyo Ward, Osaka City)) under aerobic conditions in an aqueous medium. while bubbling nitrogen and/or nitrogen-containing gas for about 25 to 30 minutes.
C. for 72 to 120 hours, and the resulting polysaccharide can be produced from the p solution by precipitation and purification according to conventional methods.

The aqueous medium contains as an essential component about 30 to 180 mmol/l of at least one monosaccharide or trisaccharide substance, such as glucose, sucrose, starch hydrolyzate, etc., as a carbon source, and further contains as a trace component Approximately 0.1-2.5
mmol/l phosphate source, approximately 0.02-0.06 mmol
J mol/l molybdenum source, about 0.005-0.02 mmol/l iron source, about 0.5-2. CBIJ mol/l magnesium source, about 0.1-2.5 mmol/l potassium source, about 2.0-5.0 mmol/l sodium source, about 0.05-0.20 mmol/l Contains a calcium source and about 0.005-0.02 milJ mol/l sulfate source.

Examples of such trace components include KH2PO4゜K2HP.
O42Mg SO4s Na C1, Cac12,
Na2MoO4.

Examples include F e 804.

In particular, from the viewpoint of improving the yield of the target polysaccharide, the phosphate ion concentration in the medium should be adjusted to 0.1 to 2.5 mmol/1.
1 Preferably 0.2-1.0 mmol/11 medium pH
It is desirable that the value is 6.0 to 8.2, preferably 7.0 to 7.7.

The pH of the medium can be adjusted in a conventional manner by means that do not adversely affect the culture of bacteria.

For example, it can be adjusted by adding an alkaline solution, such as a sodium hydroxide solution, to the medium intermittently.

It has also been found that the presence of a substance that serves as an uncoupler in the medium significantly promotes the production of the target polysaccharide.

Examples of such substances include 2,4-dinitrophenol (DNP), 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF6847), 5-chloro-1-butyl-2'-chloro-4 '-nitrosalicylanilide (S-13), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP),
Carbonyl cyanide-m-chlorophenylhydrazone (
cccp), 4.5.6.7-tetrachloro-2-trifluoromethylbenzimitazole (TTFB), pentachlorophenol (PCP), hexachlorophene,
flufenamic acid, mefenamic acid, xanthomethine, 2-
Azido-4-nitrophenol (NPA) and formic acid, acetic acid, propionic acid, benzoic acid, succinic acid, tartaric acid,
Examples include various organic acids and their salts, such as malic acid, citric acid, isocitric acid, glutamic acid, ascorbic acid, and salts thereof.

These concentrations in the medium are appropriately selected depending on the substance actually used, but usually for organic acids or their salts it is about 2 to 2.
5 g/11 and others ranging from about 5 to 100 μ/l.

It is assumed that the uncoupler inhibits ATP production, eliminates the inhibition of hexokinase, and also promotes the oxidation of GDP mannose.

In addition, adjusting the phosphate ion concentration as described above reduces the amount of inorganic phosphate and ADP, inhibits glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, etc., and thereby inhibits glycolysis. It is presumed that this promotes the production of the target polysaccharide.

The polysaccharide of the present invention can be widely used in various fields as a thickener, excipient, gelling agent, emulsion stabilizer, printing paste, sizing adhesive, and flocculant.

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 In this example, the relationship between the phosphate ion concentration in the medium and the yield of the target polysaccharide was investigated.

Next, each component shown in Table 1 was mixed in a shaking flask and made up to 11 with water to prepare various aqueous media with various phosphate ion concentrations ranging from 0.005 to 5.0 mmol/l. did.

Azotobacter cultured on an agar slant at 30°C for 48 hours
・Inoculate 1 platinum loop of Vinerrangei-IFO12018 cells into each flask containing the medium, and place on an orbital shaker for 20 minutes.
While shaking at 0 r, p, m.

Cultured at 30°C for 96 hours.

During the culture, the medium pH was maintained at 75 by addition of 1N NaOH.

After the culture was completed, the culture solution in each flask was drained, and the polysaccharide produced with ingropanol was precipitated, dried, and weighed to determine the yield of polysaccharide in each flask.

The results are shown in Table 2. As is clear from the results in Table 2, the phosphate ion concentration in the medium is 0.1 to 2.5 mmol/
11 preferably shows a good yield in the range of 0.2 to 1.0 mmol/l.

The polysaccharide obtained at a phosphate ion concentration of 0.5 mmol/l was hydrolyzed with 90% formic acid at 100'C for 116 hours,
Furthermore, when the sugar obtained by hydrolysis with 2N trifluoroacetic acid at 100°C for 5 hours was subjected to paper chromatography and gas chromatography, the constituent sugars were glucose, mannose, and mannuronic acid. It was confirmed that the molar ratio is glucose: mannose: mannuronic acid = 1:0.4.
It was Nia.

The degree of acetylation was 0.9. The molecular weight was 104 by the 3.5-monodinitrosalicylic acid method.

Similarly, the polysaccharide obtained at a phosphate ion concentration of 0.2 mmol/l is glucose:mannose:mannuronic acid-
The molar ratio was 1:0.7:16.5, the degree of acetylation was 0.2, and the molecular weight was 9.5X10'.

In addition, the polysaccharide obtained at a phosphate ion concentration of 2.5 mmol/l is glucose: mannose: mannuronic acid = 1
20, shows a molar ratio of 6:4, and the degree of acetylation is 0
．． 5. The molecular weight was 2×103.

Example 2 In this example, the relationship between the pH of the medium and the yield of the desired polysaccharide was investigated.

In the same manner as in Example 1, various aqueous media were prepared by mixing the components shown in Table 3 below in a shaking flask, making up to 11 with water, and adjusting the pH to a predetermined value with IN NaOH.

In the same manner as in Example 1, Azotobacter vinelangii
IFO12018 was cultured for 96 hours.

During the cultivation, the pH of the medium was kept at a predetermined value by the addition of IN NaOH.

After culturing, the yield of polysaccharide in each flask was measured in the same manner.

The results are shown in Table 4. As is clear from this result, a good yield is shown when the medium pH is 6.0 to 8.2, preferably 7.0 to 7.7.

Example 3 In this example, the relationship between the addition of various organic acid salts and the yield of the desired polysaccharide was investigated.

In the same manner as in Example 1, the fractions shown in Table 5 below were mixed in a shake flask and made up to 1 liter with water to prepare an aqueous medium containing various organic acid salts.

As a control, a medium to which no organic acid salt was added was also prepared.

In the same manner as in Example 1, Azotobacter vinelangii
IFO12018 was cultured for 96 hours.

During the culture, the pH of the medium was adjusted to 7.5 by adding 1N NaOH.
was held at

After culturing, the yield of polysaccharide in each flask was measured in the same manner.

The results are shown in Table 6. As is clear from this result, the addition of the organic acid salt significantly promotes the production of the polysaccharide.

Example 4 In this example, the relationship between the concentration of organic acid salts and the yield of polysaccharides was investigated.

As in Example 3, the yield of polysaccharide was measured using sodium acetate as the organic acid salt and varying its concentration in the medium.

The results are shown in Table 7. Other organic acid salts showed similar trends, and the results show that good yields can be obtained at organic acid salt concentrations of about 2 to 259/l.

Example 5 In this example, 2,4-dinitrophenol (DNP)
The relationship between the addition of and the yield of the desired polysaccharide was investigated.

The yield of polysaccharide was measured in the same manner as in Example 4, except that DNP was used instead of st-IJium acetate and its concentration in the medium was varied.

The results are shown in Table 8.

As is clear from this result, D of about 5 to 100 ■/l
It can be seen that good yields can be obtained at different NP concentrations.

[Brief explanation of drawings]

FIG. 1 is an infrared absorption spectrum of the polysaccharide of the present invention.

Claims (1)

[Claims] 1 Consists of glucose, mannose and mannuronic acid, the molar ratio of which is glucone:mannose:mannuronic acid = 1:0.4-0.7:4-17, and the degree of acetylation is approximately 0.1-1.0. polysaccharide having a molecular weight of 103 to 105 by the 3,5-dinitrosalicylic acid method.