JPS5821402A - Novel polysaccharide - Google Patents

Abstract

PURPOSE:A novel polysaccharide that is composed of glucose, mannose, glucuronic acid and mannuronic acid with a specific acetylation degree and molecular weight, thus being suitably used as a thickener or diluent, because of its keeping the viscosity constant even in the presence of a salt. CONSTITUTION:A new strain in Enterobacter, Enterobacter nichidenis (FERM- 5928), is inoculated to a medium and cultured aerobically by the shaking method to produce the objective new polysaccharide that is composed of glucose, mannose, glucuronic acid and mannuronic acid at a molar ratio of 1/(0.7-1.0)/(0.5- 0.7)/(0.1-0.3), with an acetylation degree of 0-1.0, a molecular weight of 10<3>- 10<7>, according to the gel filtration method, and being positive to the Molish reaction, phenol-sulfuric acid reaction and anthrone-sulfuric reaction, insoluble in solvents such as methanol, acetone or ether and soluble in water.

Description

[Detailed description of the invention]

- The present invention relates to novel polysaccharides, and more particularly to novel poly-Sts produced from bacteria belonging to the genus Enterobacter. The present inventors conducted research on the production of polysaccharides by microorganisms and found that bacteria belonging to the genus Enterobacter are extremely water-soluble and do not lose their viscosity even in the presence of salt. The present invention has been completed based on the discovery that polysaccharides of interest can be produced. The polysaccharide of the present invention can be produced, for example, by the following method. Bacteria belonging to the genus Nidterobacter and capable of producing the polysaccharide can be cultured in a nutrient medium, and the resulting polysaccharide can be produced from the filtrate by precipitation and purification according to conventional methods. Among the microorganisms that can be used in the present invention, Enterobacter
tar n1 ohidanis) (Feikoken Bacteria Accession No. 5928) is cited as a typical example. The Microbiological Research Institute's reception number in the above column represents the reception number of the Institute of Microbial Technology, Agency of Industrial Science and Technology. Ho's mycological properties are as follows. f Morphological properties ′ Morphology: Single rod koji, motile without forming spores or spores; Size: 1.θ~1.5 x 1.5~2.OP Durham staining: Negative acid-fast ~: # Sexual flagella: Pericyria 2 Growth status in each medium (1) Meat juice liquid culture becomes cloudy

[2] Lidomus milk turns red (3) Meat juice agar medium
Smooth, round, white (4) Glucose broth agar medium Smooth, round, white (5) Gelatin puncture culture No liquefaction 3 Physiological properties (1) Optimal growth temperature 37″C (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) Nitrate reduction Positive (7) Denitrification reaction Positive (8) Methyl red test Negative (9 ) V-P reaction Positive (10) Decomposition of starch Negative (ll) Catalase
Positive (12) Oxidase Negative (
13) Utilization of citric acid Positive (14) Utilization of inorganic nitrogen source (Using N ~ 5o41.NaN0.. (15) Release Positive (16) O-F
Test Fermentative (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 (+), sorbitol (+), fructose ( +),
Maltose (T), raffinose (+), glycerin ('+), xylose (+), starch (+), galacto, -su (+), ara and north (+), glucose (+)! , ribose (+), sucrose (g), lactose (
+), mannitol ←), malotsic acid ←) (+j grows. Acid/gas generated, -: No growth) (19) Phenylalanine deamination reaction Negative (20
) K Growth in the presence of CN Negative This strain has 0 fermentative bacteria, Katara, Zeta positive, Oxida,
-se negative, nitrate reduction positive enterobacteria, belonging to the family Interobacteriaeeae. Furthermore, it has the following characteristics: methyl red negative, v-p reaction positive, phenylalanine deamination reaction negative, nitrate reduction positive, urease positive, optimal growth temperature 37''c, and growth negative in the presence of KCN.All of these characteristics. There is no satisfying family, but except for growth in the presence of KCN, it matches the ha Klebsislleae.If you search for Arashi as being included in group ■, the genus with the properties closest to Arashi is: It is thought to belong to the genus Enterobacter based on its properties such as being motile, positive for sorbitol use, and negative for red pigment production.As mentioned above, visiting the zoo is important for bacteriological research, as well as for the known rights of the genus Enterobacter and other known rights. Therefore, it is appropriate to recognize the plant as a new strain of Enterobacter maple.
is). When culturing Oriental orchards, approximately 20 to 100 g of commonly used substances (e.g., glucose, lactose, starch decomposition products, etc.) are used as carbon sources in the nutrient medium; yeast extract, peptone are used as nitrogen sources. , Desti Cheese Soluble. Approximately 1-10g/! using ammonium sulfate, 11um sodium nitrate, etc. as a nitrogen source! , furthermore, as inorganic salts, about 0.3 to 2 g of a phosphate source to about 0.1 to 2 of a magnesium source.
g/he potassium source approx. 0.3 to 2 g/4 calcium source approx. 0
．． 05-10g//, sulfate source approx. 0.1-2g/l
Prescribe. Examples of these inorganic salts are KH, PO
4I, -HPO4r, Mg5Qp, CaCO3
Examples include. Inoculate the above microorganisms into a medium prepared by adding 1 cup of tap water to these ingredients, and use the standard method aerobically! l
Cultivate the smell. After completion of the culture, the accumulated polysaccharides can be collected by any appropriate method depending on the intended use. For example, bacterial cells are separated by filtration or centrifugation of the culture solution, and polysaccharides produced from the solution are precipitated by adding alcohol such as inpropanol, which is dissolved in water and dissolved. Purification is performed by methods such as reprecipitation with a quaternary ammonium salt and adsorption with a certain ion exchange resin to obtain the desired multi-It compound. It has been known for some time that a number of bacteria belonging to the Enterobacteriaceae family produce polysaccharides, and Toshiyuki Watanabe, Masaharu Seguchi, et al.
0) p. 281) is by Escherichia (Ptshsric
hia) is glu:l-7,:mannose:rhamnose=
It has been reported that a polysaccharide having a molar ratio of 5:3:2 is produced. In addition, Asahi Misaki et al. (Biochemistry. Vol. 39 (1967), p. 542)
erobacter) is galactose: glucurontm
:Mannose = 2.5:0.8:1 (molar ratio) is reported to be produced. The polysaccharide of the present invention has constituent sugars, chemical and physical properties,
It has different characteristics from these known polysaccharides. (1) m acid sugar it'I sylcose. The composition ratio of mannose, glucuronic acid and mannuronic acid (0 molar ratio) is glucose: mannose: glucuronic acid: mannuronic acid = 1: 0.7-1.0: 0.5-0.7:
It is 0.1-0.3. (2) It is acetylated with a degree of acetylation of 0-, 1.0, and when the ether extract of the hydrolyzate is subjected to gas chromatography and the parent acid is analyzed, acetic acid is detected. . (3) The gel shows the molecular weight of l〆~to7 by measuring the molecular weight by a proper method. (4) Shows positivity in each of the color reactions of Maurylesch reaction, phenol sulfuric acid reaction, and Anthrone sulfuric acid reaction. (5) It is insoluble in organic solvents such as methanol, ethanol, acetone, and ether, and has Loe j, tg in water (this aqueous solution is colorless and transparent). (6) When measuring the infrared absorption spectrum using the film method,
The infrared absorption spectrum shown in the attached Figure @1 is shown. (7) The viscosity of a 54s w/v aqueous solution at 30 lζ is 10" to 1ap when measured with a BM type viscometer * 30 r-p, m. (8) 10% w/v Sodium chloride, or 104
In the presence of w/v calcium tetrachloride aqueous 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 used as thickeners, excipients, gelling agents, emulsion stabilizers, and in various fields of food and industrial applications.
Paste for textile printing, paste for sizing. It can be widely used as a flocculant. Next, the present invention will be explained in further detail with reference to Examples. Example 1 50 g of Citx sugar and 5 g of #mother extract per 11 medium. K, HPOy 0.5 g, KH, PQ, p O,
5g, Mg5Q, -7H,QO, 3g. 100 m of a liquid medium containing 1 g of CaC (in a pH 7.0 autoclave, incubated at 120°C for 20 minutes)
One platinum loopful of Enterobata chinensis (Feikoken Bacteria Receipt No. 5928) was inoculated into a 500 ml Sakaro flask containing 200 r, p, on an orbital shaker.
m, de l! ! % and cultured at 37°C for 96 hours. After the cultivation was completed, the culture solution was centrifuged to remove the bacterial cells, and Impropanol was added to the suspension to form a sediment. The precipitate was collected in an oven and dried at 105°C for 24 hours to obtain 8 g of polysaccharide. The above polylj was hydrolyzed with 90% formic acid at 100°C for 16 hours, and then with 2N) refluoroacetic acid at 100°C.
The sugar obtained by hydrolyzing for 5 hours is processed into paper clover.
When subjected to gas chromatography, the Rf value and retention time revealed that the constituent sugars were gelose: mannose: glucuronic acid: mannuronic acid = 1
: 0.73 : 0.65 : 0. ．． It was 13. Furthermore, the number of acetylated thighs was 0, and the number of molecular wrinkles determined by the Gelza method was 1♂. Example 2 Medium 11 ladle a [50 g, polypeptone 2S. K-HPOge 0.5 g, KH2F0,0
．． 5 g, Mg5O,・7Hyo o oQ
g. A liquid medium containing 31 g of CaCo (pH 7,0 in an autoclave, 120″C, sterilized for 20 minutes) was placed in a 3/5
Enterobacter chinensis (FEI accession number 5928) was aseptically sown in a 1-volume JAF Amenku, and the temperature was 37'C, the number of revolutions was 600r, p, 'm, and aeration was carried out.
Aerobic culture was carried out for 96 hours under VVM aeration and stirring conditions. During the 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. When the sugar obtained by hydrolyzing the above polysaccharide in the same manner as in Example 1 was subjected to paper chromatography-i and Geiss chromatography, the constituent sugars were glucose: mannose: glucuronic acid: mannuron@
= 1. : 0.94 : 0.53 : 0.2
7, the degree of acetylation was o, s6, and the molecular track was 10. Patent Applicant Nittan Kahan Co., Ltd. December 28, 1980 1 Relationship between the case and the indicated case Patent Applicant 4 Attachment 8 of the amendment order (1) Page 2 of the specification, lines 6-7 “Entero Bnterobaater n1chids
Enterobacter nichidenii (Enterobacter nichidenii)
) amended to J (the song title has been changed as shown in the attached strain name notification). (2) Same book, page 5, lines 15-16 “Nichidenis (N1c
hidenis) J and certain [Nichidenii (N1c)
Hidenii) Correct as J. (3) 4I, page 9, line 18, and @page 10, line 20 to page 11, line 1, ``2↑Dennis'' is corrected to ``Nichideni''. 7. Inventory of attached materials (1) Notification of change of bacterial strain name (1 copy) Showa t7/Month/day 1 Indication of incident. Relationship to the case 7 Patent Jt1m Person 4 Next to page 11, line 13 of the specification dated January 5, 1982, attached to 7 Patent Jt1m Person 4, an item ``4. Brief explanation of drawings'' is added, and ``No. 1 The figure is an infrared absorption spectrum of the polysaccharide of the present invention.''

Claims (1)

[Claims] It is composed of glucose, mannose, glucuronic acid and mannuronic acid, with a molar ratio of 1 and an i of 0.7 to 1.
0: A polysaccharide which is acetylated with an acetylation degree of 0 to 1.0 with an acetylation degree of 0.5 to 0.7''=0.1 to 0.3, and a molecular weight of 10!I to 107 according to the Gehrch method.