JPH0224281B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel polysaccharide, and more particularly to a novel polysaccharide produced from bacteria belonging to the genus Enterobacter.

As a result of repeated research on the production of polysaccharides by microorganisms, the present inventors found that bacteria belonging to the genus Enterobacter produce an interesting polysaccharide that is extremely water-soluble and does not lose viscosity even in the presence of salt. They discovered this and completed the present invention.

The polysaccharide of the present invention can be produced, for example, by the following method.

Bacteria belonging to the genus Enterobacter and having the ability to produce the polysaccharide can be cultured in a nutrient medium, and the resulting polysaccharide can be produced from the resulting solution by precipitation and purification according to conventional methods.

Microorganisms that can be used in the present invention include Enterobacter nichidenii (Feikoken Bacteria No. 5928).
(No.) is given as a typical example. The above-mentioned Microbiological Research Institute No. 5928 represents the accession number of the Institute of Microbial Technology, Agency of Industrial Science and Technology.

The mycological properties of this bacterium are as follows.

1 Morphological properties Morphology: Monobacillus Motility without forming spores: Yes Size: 1.0-1.5 x 1.5-2.0μ Gram staining: Negative Acid-fastness: Negative Flagella: Peritrichia 2 Growth status in each medium (1) Meat juice liquid culture Turns cloudy (2) Litmus milk Turns red (3) Meat juice agar plate culture Round, edges entirely smooth, slightly raised White (4) Meat juice agar slant culture Thready, smooth, white (5) Glucose meat juice agar culture Round, smooth, white (6) Flesh gelatin puncture culture No liquefaction (7) Gelatin puncture culture No liquefaction 3 Physiological properties (1) Optimal growth temperature 37℃ (2) Growth PH 5-10 Optimum growth PH 7-8 (3) Oxygen requirement Facultative anaerobic (4) Indole production negative (5) Hydrogen sulfide production negative (6) Sulfate reduction positive (7) Denitrification reaction positive (8) Methyl Red test negative (9) V-P Reaction Positive (10) Decomposition of starch Negative (11) Catalase Positive (12) Oxidase Negative (13) Utilization of citric acid Positive (14) Utilization of inorganic nitrogen source (NH ₄ ) ₂ SO ₄ , NaNO ₃
(15) Urease positive (16) O-F test Fermentation (17) Pigment production No pigment produced in King A medium No fluorescent pigment produced in King B medium (18) Utilization of carbon compounds Mannose (+ ), rhamnose (+), sorbitate (+), fructose (+), maltose (+), raffinose (+), glycerin (+),
Xylose (+), starch (+), galactose (+), arabinose (+), glucose (+), ribose (+), sucrose (+), lactose (+), mannite (-), malonic acid (+) ), Inosytsu (+), Trehalose (+),
(+: Grows, generates acid/gas,
-: No growth) (19) Phenylalanine deamination reaction negative (20) Growth in the presence of KCN negative (Based on the above mycological properties, Virgie's Manual of Determinative Bacteriology ( Bergey's Manual of
Determinative bacteriology) 8th edition. ) This strain belongs to the Enterobacteriaceae family because it is a Gram-negative bacillus, fermentative, positive for catalase, negative for oxidase, and positive for nitrate reduction.

In addition, methylred negative, V-P reaction positive, phenylalanine deamination reaction negative, nitrate reduction positive, urease positive, optimal growth temperature 37℃,
It has the characteristic of negative growth in the presence of KCN.
There is no family that satisfies all these characteristics, but KCN
Conforms to the tribe Klebsielleae, except for growth in the presence of When this bacterium is searched for as being included in the family, the genus that has properties similar to this bacterium is considered to be the genus Enterobacter due to its properties such as being motile, positive for sorbitol utilization, and negative for red pigment production.

As mentioned above, this bacterium differs from known species of the genus Enterobacter and from other known species in important bacteriological properties. Therefore, it is appropriate to recognize this bacterium as a new species of the genus Enterobacter, and give it the species epithet Nichidenii.

When culturing this bacterium, approximately 20 to 100 of commonly used substances (e.g. glucose, sucrose, starch decomposition products, etc.) are used as carbon sources in the nutrient medium.
g/, as a nitrogen source, yeast extract, peptone, Daysteller's Soluble, ammonium sulfate, sodium nitrate, etc. are used as a nitrogen source of about 1 to 10
g/, plus a phosphate source as inorganic salts of approx.
0.3-2g/, Magnesium source approx. 0.1-2g/
, potassium source approximately 0.3-2 g/, calcium source approximately 0.05-10 g/, sulfate source approximately 0.1-2 g/,
Prescribe. Examples of these inorganic salts are
Examples include KH ₂ PO ₄ , K ₂ HPO ₄ , MgSO ₄ and CaCO ₃ . The above-mentioned microorganisms are inoculated into a medium prepared by adding these components to tap water, and cultured with aerobic shaking using a conventional method.

After completion of the culture, the accumulated polysaccharides can be collected by various methods depending on the purpose of use. for example,
The culture solution is filtered or centrifuged to separate the bacterial cells, and the polysaccharides produced by adding alcohol such as isopropanol are precipitated from the solution.
After dissolving this in water, it is purified by a method such as reprecipitation with a quaternary ammonium salt or adsorption with an ion exchange resin to obtain the desired polysaccharide.

It is known that some bacteria belonging to the Enterobacteriaceae family produce polysaccharides, and Toshiyuki Watanabe, Masaharu Seguchi et al. Esherichia) has been reported to produce a polysaccharide consisting of glucose: mannose: rhamnose = 5:3:2 (molar ratio). In addition, Asahi Misaki et al. (Biochemistry, Vol. 39 (1967), p. 542) reported that Aerobacter contains galactose: glucuronic acid:
It has been reported that polysaccharides consisting of mannose = 2.5:0.8:1 (molar ratio) are produced.

The polysaccharide of the present invention differs from these known polysaccharides in terms of constituent sugars, chemical and physical properties, and has the following characteristics.

(1) The constituent sugars are glucose, mannose, and galactulonic acid, and their composition ratio (molar ratio) is glucose: mannose: galactulonic acid = 1:
0.5-1.0: 0.3-0.8.

(2) It is acetylated with a degree of acetylation of approximately 0 to 1.0, and when an ether extract of the hydrolyzate is subjected to gas chromatography and analyzed for organic acids, acetic acid is detected.

(3) Shows a molecular weight of 10 ³ to 10 ⁷ when measured by gel filtration method.

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

(5) Insoluble in organic solvents such as methanol, ethanol, acetone, and ether, but soluble in water (this aqueous solution is colorless and transparent).

(6) The infrared absorption spectrum has the following peaks.

790cm ^-1 , 860cm ^-1 , 1250cm ^-1 , 1400cm ^-1 ,
1600cm ^-1 , 1710cm ^-1 (7) The viscosity of a 5% w/v aqueous solution at 30℃ is BM
When measured with a type viscometer at 30r.pm, 10 ² ~
10 ⁵ cp.

(8) 10% w/v sodium chloride, or 10% w/v
In the presence of aqueous calcium chloride solution, the viscosity does not decrease. The viscometer and measurement conditions were the same as in (7).

The polysaccharides of the present invention can be widely used in various food and industrial fields as thickeners, excipients, gelling agents, emulsion stabilizers, printing pastes, sizing pastes, flocculants, etc. .

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

Example 1 50g of glucose, 5g of yeast extract per medium
g, K ₂ HPO ₄ 0.5 g, KH ₂ PO ₄ 0.5 g, MgSO ₄・
Liquid medium containing 7H ₂ O 0.3g and CaCO ₃ 10g (PH 7.0
One platinum loop of Enterobacter nichinidenii (Feikoken Bacteria No. 5928) was inoculated into a 500ml Sakaguchi flask containing 100ml (sterilized in an autoclave at 120°C for 20 minutes).
Shake at 200 r.pm on an orbital shaker and at 30°C.
Cultured for 96 hours. After completion of the culture, the culture solution was centrifuged to remove the bacterial cells, and isopropanol was added thereto to form a sediment. Take the precipitate and heat it at 105℃
After drying for 24 hours, 8 g of polysaccharide was obtained.

The above polysaccharide was hydrolyzed with 90% formic acid at 100℃ for 16 hours, and then with 2N trifluoroacetic acid at 100℃ for 5 hours.
When the sugar obtained by time hydrolysis was subjected to paper chromatography and gas chromatography, the Rf value and retention time showed that the constituent sugars were glucose: mannose: galactyuronic acid = 1:
It was 0.56:0.72. The degree of acetylation was 0, and the molecular weight determined by gel filtration was ¹⁰³ .

Example 2 Enterobacter nichidenii
5928) per medium, 50 g of glucose, 2 g of polypeptone, 0.5 g of K ₂ HPO ₄ , 0.5 g of KH ₂ PO ₄ ,
Liquid medium containing 0.3 g of MgSO ₄ 7H ₂ O and 10 g of CaCO ₃ (sterilized at 120°C for 20 minutes in a PH7.0 autoclave)
One platinum loop was inoculated into a 500 ml Sakaguchi flask containing 100 ml, and cultured in the same manner as in Example 1. During cultivation, the pH of the medium was maintained at 7.0 by addition of 1N NaOH.
After culturing, 20 g of polysaccharide was obtained in the same manner as in Example 1.

The sugar obtained by hydrolyzing the above polysaccharide in the same manner as in Example 1 was subjected to paper chromatography and gas chromatography, and the constituent sugars were glucose: mannose: galactyuronic acid = 1:0.92:0.34. It was hot. The degree of acetylation was 0.86 and the molecular weight was ¹⁰⁷ .

[Brief explanation of drawings]

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

Claims (1)

[Claims] 1. A polysaccharide having the following properties. (a) The constituent sugars are glucose, mannose, and galactyuronic acid, and the molar ratio is glucose:mannose:galactyuronic acid=1:0.5-1.0:
0.3 to 0.8, (b) degree of acetylation is 0 to 1.0, (c) exhibits a molecular weight of 10 ³ to 10 ⁷ when measured by gel filtration, (d) Moritsch reaction, phenol sulfuric acid reaction, Anthrone. Shows positive in each color reaction of sulfuric acid reaction. (e) Insoluble in organic solvents such as methanol, ethanol, acetone, and ether, but soluble in water. (f) The infrared absorption spectrum has the following peaks: 790cm ^-1 , 860cm ^-1 , 1250cm ^-1 , 1400cm ^-1 ,
1600cm ^-1 , 1710cm ^-1 (g) The viscosity of a 5% w/v aqueous solution at 30℃ is BM
When measured with a type viscometer at 30r.pm, 10 ² ~
10 ⁵ cp. (h) 10% w/v sodium chloride, or 10% w/v
v In the presence of an aqueous solution of calcium chloride, the viscosity does not decrease. The viscometer and measurement conditions were the same as in (g).