JPH0445519B2 - - Google Patents

Description

[Detailed description of the invention]

It is known to be produced by complex polysaccharides or microorganisms. Some of the actions of this complex polysaccharide have been as a hydrophilic colloid and due to its viscosity and rheological properties, it has been used as a thickening agent in aqueous systems. Regarding other fields of science and technology, research continued with the aim of discovering new complex polysaccharides with properties useful as thickening, suspending and/or stabilizing agents. It is an object of the present invention to provide new complex polysaccharides having these desired properties. It is also an object to provide a method for the preparation of this new compound. It is also an object to provide formulations containing this novel complex polysaccharide as a thickening or suspending or stabilizing agent. Still further objects of the invention will become clear from the following description of the invention. The present invention relates to novel complex polysaccharides produced by bacterial action on selected carbon sources. The present invention also relates to a novel method for producing complex polysaccharides by culturing bacteria with selected carbon sources and culture medium components under controlled conditions. The complex polysaccharide of the present invention is a high molecular weight polysaccharide containing mainly carbohydrate residues and a small amount of protein. It is sometimes referred to as "gum," but the term complex polysaccharide is considered more accurate and precise. In the following description of the invention, the compound will sometimes be referred to as complex polysaccharide 60 or S-60. This novel compound is based on extensive taxonomic research and is based on a previously undescribed microorganism and unidentified member of the genus Pseudomonas.
species) can be produced by fermentation in a suitable nutrient medium. An unrestricted permanent deposit of this microorganism used in producing the complex polysaccharide was made with the American Type Culture Collection on November 21, 1978 under accession number ATCC 31461; January 1981
It was deposited on the 31st pursuant to the Budapest Treaty. Clues to the various classifications of the genus Pseudomonas and descriptions of the culture of the genus Pseudomonas are found in Pergey's
7th edition of Mannal (Breed et al. (1957)) and 8th edition of Bergey's Manual (Doudoroff et al. (1974)), as well as various publications by other schools; (Gilardi), 1974, Pseudomonas, Manul of Clinical Microbiology.
Clinical Microbiology, 2nd edition, edited by Lennete et al., pages 250-269, American Society for Microbiology, Washington, DC. C: Weaver et al., 1972, Identification of Unusual Gram-negative Bacteria.
Pathogenic Geam-Negatiue Bacteria, EOKing, Center for Disease Control, Atlanta; Iizuka et al., 1963, Attempt to classify the genus Pseudomonas.
Grouping the Genus Pseudomonas), J.Gem.
Appl.Microbiologg 9:7-82; Hendrie et al., 1966, Identification Methods for Microbiologists.
Microbiologists), Part A, edited by Gibbs et al., pages 1-7, Academic
Found in Academic Press, New York. From these clues and descriptions, we searched for Pseudomonas species with morphological and cultural characteristics similar to those of ATCC31461. The following considerations are necessary and justify the assignment of a new Psudomonas species. Description of the strain 1 Characteristics of cell morphology Unicellular, straight or often curved rod-shaped,
Typically 0.6−0.8 × 2.0−3.0 μm, often with tapered ends, larger and longer with longer culture periods (0.8−1.0 × >3 μm), with malformed cells and pleomorphism, especially carbohydrate restriction. Appears on the medium of amount. On the contrary, when grown on a carbohydrate-containing medium, the cells rather maintain a constant bacillary shape, but as the culture increases, most cells become larger again and become pleomorphic. Gram-negative, without capsule, and granules of poly-β-hydroxybutyrate and polyphosphate are seen especially when cultured on nitrogen-deficient medium. Motility due to a hyper-multicellular flagellated state, i.e. with 1 to 4 flagella attached to one end and sometimes close to the end (Subpolav)
It can be seen because it grows epiphytically. 2 Characteristics of Colony Morphology Small (0.8-1.1 mm in diameter) and large (3.2-3.5 mm in diameter) colonies appear on nutvient agar plates. Colonies have yellow carotenoid pigments and are smooth, round, convex to cushion-shaped. Large colonies often have concentric wrinkles. The surface of the colony is a hard, non-adhesive tissue, and the entire colony can be removed by pressing with a platinum loop. On YM agar plates, only one type of colony appears: relatively large (~6-7 mm in diameter), yellow, round, smooth, sticky, convex. A sticky, elastic membrane forms on the surface of the colony, and the entire membrane on the colony surface can be removed. Secondary growth forms around the periphery of the initial colony. The color of these colonies is darker yellow in the center than in the periphery, and concentric pigmentation is observed. In addition to intracellular yellow carotenoid pigments, diffusible brown pigments appear as a result of autoxidation during extended culture periods. This phenomenon is more easily observed on Nutrient agar. No fluorescent dye was produced. 3 Physiological and biochemical characteristics The growth range of the S-60 strain is approximately 20°C to 41°C. No growth occurs at 4°C. 3.0% NaCl
is sufficient to inhibit growth, and the strain has a pH of 5
It is possible to grow between and 11. Almost all carbohydrates produce acids and do not produce gas, but polyalcohols do not. Urease is produced. M.R., V.P.
and indole tests are all negative. Arginine, dihydrolase, lysine and ornithine decarboxylase are not produced. Acid production and reduction occur in litmus milk. lipolytic egg yolk reaction
reaction) is negative. Weakly hydrolyzes gelatin, but does not hydrolyze casein, starch, alginic acid, pectin, cellulose, chitin, or DNA. 4. Susceptibility to antibiotics The strain is highly susceptible to kanamycin, neomycin, chlortetracycline, and erythromycin, but not to streptomycin and penicillin. 5. Nutritional characteristics No organic growth factors are required and amium salts meet the requirements as the sole nitrogen source.
At least 35 organic compounds were utilized, namely loribose, starch, 2-ketogluconate, most carbohydrates other than mucate. Furthermore, acetate, caproate, caprylate, pelargonate, succinate, azelate, L-maleate, DL-β-hydroxybutyrate, pyruwate, ethanol,
n-propanol, p-hydroxybenzoate, phenyl acetate, L-α-alanine,
L-threonine, L-leucine, DL-isoleucine, L-aspartate, L-glutamate, and L-tyrosine were utilized. 6 GC content of DNA Evaluation of DNA resulted in a mol% of ~68 (from Tm).

【table】

【table】

[Table] Table 2 shows comparative data between the bacterial species and related species in the present invention.

Culture conditions: The complex polysaccharide S-60 is produced in aerobic culture of a suitable aqueous nutrient medium under controlled conditions by inoculation of an unidentified microorganism of Pseudomonas species. The medium is a conventional medium containing sources of carbon, nitrogen, and inorganic salts. Generally carbohydrates (e.g. glucose,
Fructose, maltose, sugar, xylose, mannitol, etc.) can be used alone or in combination as assimilable carbon sources in nutrient media. The exact amount of carbon source or carbon sources used in the medium will depend in part on the other components of the medium, but generally the amount of carbohydrate will typically vary between about 2% and 4% by weight of the medium. These carbon sources can be used individually or in combinations of several carbon sources in the culture medium. In general, many proteinaceous substances can be used as nitrogen sources during the cultivation process. Suitable nitrogen sources include, for example, yeast hydrolyzate, live yeast, soybean flour, cottonseed flour, casein hydrolyzate, corn steep liquor, soluble flour of distilled spirit residue, tomato paste, and the like. Nitrogen sources, alone or in combination, range from approximately 0.05% to 0.2% by weight of the aqueous medium.
It is used in amounts ranging from Nutrient inorganic salts that can be added to the culture medium are the usual salts that can be ions such as sodium, potassium, ammonium, calcium, phosphoric acid, sulfuric acid, chlorine, carbonic acid, etc. Trace metals such as cobalt, manganese, iron and magnesium are also included. It should be noted that the media described in the Examples are only examples of a wide range of media that can be used, and are not limiting. Cultivation is performed at a temperature range between approximately 25°C and 35°C, but for optimal results, temperatures between approximately 28°C and 32°C are recommended.
It is preferred to carry out the cultivation at temperatures up to .degree.
The pH of the nutrient medium for the growth of Pseudomonas and the production of polysaccharide S-60 can vary from about 6 to 8. The new polysaccharide S-60 can be produced by surface and submerged culture, but submerged culture is more suitable. Small-scale cultivation is conveniently carried out by inoculating the fungus into a suitable nutrient medium, transferring it to a production medium, and culturing it for several days on a sheaker at a constant temperature of about 30°C. Cultivation begins with the growth of an inoculum in a sterile flask containing a medium and goes through one or more stages. The nutrient medium for inoculum growth is a suitable combination of carbon and nitrogen sources. The seed flask is shaken at a constant temperature of about 30°C for 1 to 2 days or until satisfactory growth is obtained, and a portion of the resulting growth is used to inoculate the second stage seeds or production medium. used. If desired, intermediate seed flasks are propagated in essentially the same manner. That is, a portion of the contents of the seed flask from the previous step is used to inoculate the production medium. The inoculated flask is shaken at constant temperature for several days, and at the end of the incubation period the contents of the flask are recovered by precipitation with a suitable alcohol such as isopropyral alcohol. On a large scale, it is preferred to carry out the cultivation in suitable tanks equipped with a stirrer and means for aerating the culture medium. According to this method, the nutrient medium is prepared in a tank and sterilized by heating to a temperature of about 121°C. After cooling, the sterile medium is inoculated with the seeds previously grown in the production medium and the culture is incubated for e.g. 2 to 4 days while stirring and/or aerating the nutrient medium and keeping it at a temperature of about 30°C. Do it in a period. This method of producing S-60 is particularly suitable for mass production. The product is recovered from the culture medium by precipitation with a suitable alcohol such as isopropanol. Physical and chemical properties of polysaccharide S-60 A complex polysaccharide produced by an unidentified Pseudomonas bacterium is composed mainly of carbohydrates, 3-4.5% acetyl groups of O-glycosidic esters, and 10% protein. -15%. The carbohydrate portion of polysaccharide S-60 is uronic acid (~12%
(based on gum weight) and the neutral sugars glucose and camnose. The approximate molar ratio of rhamnose to glucose is 1.5:1. An acetyl content of 4.5% was determined by treating a 0.2% aqueous solution of S-60 resin with alkaline raw hydroxylamine reagent followed by acidic ferric chloride reagent (S. Hestrin).
(1949) J.Biol.chem.180, 249-261]. The neutral sugar of polysaccharide S-60 was determined as follows.
Dissolve 10 mg of product in ₁ ml of _2Nd SO and mix
Heat at 100℃ for 4 hours. The resulting solution was cooled, neutralized with barium hydroxide, and then adjusted to pH 5-5 with solid carbon dioxide.
Set it to 6. The resulting barium sulfate precipitate is removed by centrifugation, and the supernatant is concentrated under reduced pressure until it becomes syrupy. The sugar content of the hydrolyzate is 3% by weight OV-
Gas chrome Q80/100 mesh holding 225
Hewlett-Packard Model 5750 chromatograph used at 210°C.
These aldononitrile acetate derivatives are experimentally immobilized by gas chromatography. Sugars are identified and quantified by comparison with true standards [JKBaird, MJ Holroyde, DCEllwood (1973) Carbohydr.Res.27 ,
464-467]. The various neutral sugars of polysaccharides can still be analyzed using descending paper chromatography using Whatman No. 1 chromatography paper using the upper layer of pyridine:ethyl acetate:water (2:5:5:) as the solvent. It was characterized. The chromatograph was stained with silver nitrate immersion and phthalic acid-aniline spray reagent. Constituent sugars were determined by simultaneous chromatography with sugar standards and phthalic acid.
It was fixed by a specific color reaction with aniline reagent. The uronic acid content of polysaccharides was determined by two different methods. One method involves decarburizing a sample with 19% hydrochloric acid, capturing the liberated carbon dioxide with standard sodium hydroxide, and performing back titration [BL Browning (1967) Methods of Wood
Chemistry, 632-633] and by carbazole colorimetry [T. Bitter, HM Muir (1962) Mnal.
Biochem.4, 330-334] was determined. Paper electrophoresis was used for the separation and experimental identification of uronic acids in the above-mentioned neutralized acid hydrolyzate. An aliquot of this decomposition product and a known uronic acid standard were placed on Camag electrophoresis paper No. 60-011 and electrophoresed in a PH 2.7 buffer using a Camag model HVE electrophoresis apparatus. I spent time. The chromatogram was air-dried and stained with silver nitrate immersion reagent to determine the position of the separated uronic acid. Two large and one small spot were seen. One of the big spots is glucuronic acid (R _GlcA = 1.0)
The other large spot (R _GlcA = 0.85) and small spot (R _GlcA = 0.73) move with the same mobility as
had a smaller mobility. The relative mobilities of known uronic acids under these same conditions are as follows: Rm Glucuronic acid 1.0 Mannyuronic acid 0.96 Galacturonic acid 0.65 Guluronic acid 0.63 I followed him. Complex polysaccharides have ^hydroxyl groups, methylene groups, carbonyl ^groups , and carboxylic acid ^{groups .}
showed a peak. It does not accept methylene chloride dyes, is virtually insoluble in N,N-dimethylformamide, and soluble in DMSO or formamide. The S-60 sample shows the following elemental analysis values: N-
2.00%, C-42.62%, H-5.80%. Polysaccharide S-60 provides viscosity to aqueous solutions when dissolved in water at low concentrations. Because of this, its sensitivity to shear and its overall rheological properties, it can be used as a thickening, suspending and stabilizing agent in aqueous systems, e.g. in printing pastes in the textile industry, or in low flow aqueous herbicidal compositions, salads, etc. It is useful as an additive in the formulation of dressings, thick puddings, and adhesive compositions. After heating and cooling, it forms a weakly elastic gel. Deacetylated, unclarified S-60 dry polymer or culture solution to a high pH (e.g., PH10 using sodium carbonate or sodium hydroxide)
Deacetylation occurs easily when heated to a high temperature of 90-100°C for 10 to 45 minutes. The resulting deacetylated polysaccharide S-60 forms a hard, inelastic or brittle gel and is useful in many industrial and food related applications. The composition of deacetylated S-60 is mainly carbohydrates, ~17% protein, ~0 acetyl
%. The carbohydrate portion consists of ~13% uronic acids and the neutral sugars rhamnose and glucose in a molar ratio of 1.5:1. One use of deacetylated resin is that it is a hard and brittle gel that can be used to make molds and hard structures, so it can be applied to indoor deodorizers, air fresheners, etc. after being treated with a suitable solution such as perfume. Find out. Deacetylated resins are still used in gel electrophoresis and as gelling agents for microtomes when using electron microscopes. Natural and deacetylated resins are also used as barium in radiology, as suspending agents in confectionery, and as molding substances in tool making, dentistry, and criminology. The infrared absorption spectrum of deacetylated S-60 measured on dry matter using the KBr tablet method is 3400 cm ^-1 , 2950
It showed peaks at cm ^-1 , 1740 cm ^-1 , 1650 cm ^-1 and 1610 cm ^-1 . A sample of deacetylated S-60 shows the following elemental analysis: N-2.67%, C-4189%, H-6.07%. Deacetylated, Clarified S-60 The composition of cleared, deacetylated S-60 is as follows: ~2% protein, 0% acetyl, and carbohydrate, the latter ~22% uronic acid with an approximate molar ratio of 1.5 : Consists of 1 neutral sugar rhamnose and glycose. The infrared absorption spectrum of cleared, deacetylated S-60 measured on dry matter using the KBr tablet method was 3400 cm ^-1 ,
A peak of 1600 cm ^-1 was observed at 2950 cm ^-1 . A sample of deacetylated, cleared S-60 shows the following elemental analysis: N - 0.42%, C - 36.85%, H - 5.62%. The sample exhibits the following specific rotation: [α] ²⁵ ₅₈₉ = -45° Deacetylated clear resins are particularly useful as agar substitutes in microbiological culture media for a variety of clinical and non-clinical microorganisms using a wide variety of culture media. The concentration of deaceterized clear resin required to transform into agar depends on the medium used, but is approximately
It is in the range of 0.5 to about 1.25% (weight per volume ratio). The growth characteristics of the microorganisms are quite similar to those of standard agar-based media. The following detailed examples describe representative bacteria of the invention. Example 1 Cultivation process A for production of complex polysaccharide S-60 Subculture Unidentified Pseudomonas
The fungus ATCC3146 grows very well on NA or YM agar, so these are routinely used for subculturing. The culture temperature is 30°C. The microorganisms produce yellow-orange carotenoid pigments and brown soluble pigments over 2-5 days of culture. B. Production of inoculum The inoculum for the flask is made from YM medium cultured at 30°C. When inoculating a new plate culture,
Cultivation in YM medium gives good growth and resin formation by 24 hours. The seed medium for fermentation using a one gallon fermenter as the seed culture vessel is the same as the final fermenter medium. C. Final Fermenter Media The sodium- and potassium-salt forms of the resin are made in different media; they are both discussed below. Microorganisms require a certain amount of K ^+, which must be added to the sodium-type culture medium. (3% dextrose can also be used. Sodium salt Potassium salt 3.0% glucose 3.0% glucose 0.01% MgSO ₄ 7H ₂ O 0.01% MgSO ₄ , 7H ₂ O 0.09% NH ₄ NO ₃ 0.09% NH ₄ NO ₃ 0.05% Promosoy
0.05% Promosy (soy protein concentrate) 1ml/lHoLe salts 1ml/lHoLe salts 1ppmFe ⁺⁺ 1ppmF ⁺⁺ 0.05%Na ₂ HPO ₄ 0.05%K ₂ HPO ₄ 10ppmK ⁺ PH control = NaOH PH control = KOH HoLe salts is a solution of trace elements including tartaric acid, magnesium molybdate, CoCl ₃ , ZnCl ₂ , CuCl ₂ , boric acid, manganese chloride, and ferrous sulfate. When aiming for a low calcium product, any of the above media may also be used with deionized water. Cultivation is completed in 50 hours; the viscosity of the culture medium is usually 5000-
It is 8000cps. D Recovery Due to the gelling nature of the product, good fiber formation does not normally occur upon natural precipitation. However, pasteurization at 90-95°C for 10-15 minutes (during which time the concentrated culture is heated - becomes noticeably diluted).
It was found that excellent fibers were obtained by precipitation from the culture medium without cooling using 99% isopropanol in an amount twice the volume of the culture medium. 20 and 70 fermenters with an average yield of 1.5% resin are obtained with 3% glucose. E. Drying Collect the product and dry in a forced air tray dryer at 50-55°C for up to 1 hour. F Product Quality The 1% viscosity of K ⁺ salts is typically in the range of 3000 cps, and for low calcium sodium salts approximately
It is 7000cps. Example 2 Deacetylation and Clarification of Complex Polysaccharide S-60 Although not necessary in all uses, clarification of the resin is valuable when the resin is used as an agar substitute. Clarification can be performed before (native) or after deacetylation.
Since deacetylation is carried out with a hot alkali and clarification is carried out in hot conditions, the two methods are easily and conveniently combined. Both deacetylation and clarification can be performed in either culture broth or dry polymer.
For deacetylation, if a culture medium is used, the PH
Adjust the solution to 10 with KOH and heat the solution at 90℃ for 15 minutes.
Adjust the pH to 7 with dilute H ₂ SO ₄ , add CaCl ₂ to a concentration of 0.2%, and cool. A hard and brittle gel is obtained. The general procedure for both deacetylation and clarification methods is as follows: A. Heat a 2% solution of broth or resin to 90°C. B Set the PH to 10 with KOH. C. Maintain the temperature of the culture medium or solution at 90-95°C for 15 minutes. DPH is brought to _6-8 with dilute HCl or _H2SO4 . E Add 10 g of Super Aid to the material to be filtered. F. Apply this material to approximately 6 mm of Super Aid using a filter section with an area of 136 ^cm3 .
Pass through a pressure filter section (preheated) at a pressure of about 20-30 psi. Solution G is immediately precipitated with isopropanol to prevent gelation and the fibers are dried at 50°C for 1 hour or less. When deacetylation is not required, the method described above is followed as is except for raising the pH: keep at 90°C and immediately strain and collect the solution. Clarification is always carried out in the potassium salt form: KCl
can be added to the previously made product solution if necessary. Example 3 Gel characteristics of complex polysaccharide S-60 Below is a compilation of data comparing both K ⁺ and Ca ⁺⁺ forms of the pristine resin and deacetylated resin with carrageenan and agar. show:

TABLE Note as above that there is a wide range of temperatures for solidification and melting of all the various types of gels. For agar, the variation is mainly due to the type of seaweed, and for Katsupa carrageenan, the potassium ion concentration determines the gel characteristics. Deacetylated S-60 gels are primarily characterized by the degree of deacetylation. With a small amount of deacetylation, the gel hardens at higher temperatures and is more elastic: in fact, a wide range of gels from elastic to stiff are possible depending on the degree of deacetylation. Kel is more similar to agar than to Katsupa carrageenan, mainly due to the large hysteresis between solidification and melting points. They are difficult to dissolve,
It should be emphasized that it is difficult to observe gel-sol changes. On the other hand, the gel point can be easily defined since the gel sharply solidifies into a hard gel within a few degrees from the beginning of gelation. Example 4 Deacetylated unclarified S-60 S-60 culture broth is heated to 90°C and the pH is brought to 10 by addition of 25% KOH. Maintain temperature for 1 minute, then continue to thicken.
Neutralize with HCl. The culture is drained from the fermenter and harvested while hot with double volume of 99% IPA. The deacetylated S-60 was collected, dried in a forced air tray dryer at 55°C for 1 hour, and ground into powder. Example 5 Clarification of deacetylated S-60 Deacetylated complex polysaccharide S-60 produced in Example 4
Arti-
Mix in Barinko. The solution was incubated in a Sorvall RC2-B cryocentrifuge for 20 minutes while keeping the temperature above 40°C.
Centrifuge at 10,000 R.PM (GSA head).
Decant the supernatant and remove the wells of 5μ, 3μ, 1.2μ,
0.8μ Gelman type
Pass through ANHhydrophilioAcopor membrane (293 mm). Add about 3-4 times the amount of the liquid to 99% isopropanol, collect the fibers, briefly dry at 55℃ with a forced air tray dryer, and grind into powder. The product is the deacetylated clarified S-60 resin of this invention. Example 6 Substitution of Agar Using Deacetylated Clarified S-60 Several different media are made as shown below:

【table】

Table: Deionization was used for all media. Ingredients combined (excluding Burk's) at 121°C, 15psi
The mixture was autoclaved for 15-20 minutes, cooled to 55°C, and poured into a sterilized Petri dish. Purk's ingredients are combined except for glucose, autoclaved separately, and added to the post-autoclave medium. Once these culture plates had solidified and were incubated at room temperature for 24 hours to test for sterilization, they were streaked and inoculated with the following 14 bacterial strains: Agromyces ramosus
ramosus) ATCC25173 Arthrobaterglobifomis
ATCC8010 Aureobasidium pullulans NRRL YB−3861 Azotobacter indicus
indicus) var myxogenes S-7
Strain ATCC21423 Azotobacter Vinelandii ATCC9047 Beijerinckia lacticogenes ATCC19361 Eiwinia
cartovora) ATCC8061 Escherichia coli EG-47 strain Klebsiella pneumoniae
pueumoniea) S-53 strain Nocordia salmonicolor (Nocordia
salmonicolor) ATCC21243 S-60 Streptococcus faecalis Trichoderma longhrachiatum ATCC13631 Zoogloea ramigera
ATCC25935 plates were cultured at 30°C for 3-5 days and growth was examined. Good growth was obtained for all strains on the medium made with S-60, and there was almost no difference in colony morphology between the medium made with S-60 instead of agar and the one on agar. These results indicate that S-60 is an excellent substitute for agar in microbiological media. The gel point of all media containing S-60, except BHI media and TSA, was 42°C.
The gel point of BHI agar and TSA was 52°C. Agar typically gels at 42-44°C. Example 7 Production of molded fragrance gel using deacetylated S-60 (A) 1.50% Polysaccharide S-60 0.75% Sodium carbonate 0.025% Methyl p-hydroxybenzoate 3.00% Rose scent 4.00% Isopropanol 2.00% Ethylene glycol 88.50 % Water The native polysaccharide S-60 is mixed with sodium carbonate and preservatives and dissolved in water at 70°C. The solution is further
Heat to 90°C and hold at that temperature for 10 minutes to deacetylate the polysaccharide. After cooling to 6°C, the fragrance dispersed in the solvent is added and the mixture is placed in a conventional air freshener plastic mold. Once the mixture has cooled to 38°C, gelation occurs and a hard, free-standing gel with aromatic properties is obtained. (B) Dry deacetylated polysaccharide S-60 is prepared from a culture solution by adjusting the pH to 10.0 with dilute sodium hydroxide and heating at 90°C for 15 minutes. The solution was neutralized to pH 7.0 with dilute hydrochloric acid and precipitated with twice the amount of isopropanol.
Dry and grind. A solid air freshener gel is made from the deacetylated product in the following manner: 3.0 g of deacetylated polysaccharide S-60 with 1.5 g of potassium chloride and 0.15 g of methyl p-hydroxybenzoate preservative.
Mix with 177ml of water. Heat the solution to 90℃ to dissolve it, cool it to 60℃, and add 6.0% oil fragrance.
g, isopropanol, 8.0 g, and ethylene glycol, 4.0 g. Pour the solution into a plastic mold and cool to room temperature. A strong hard gel is formed with a strong and fleeting odor. The gel can be easily deformed and retains its shape without sagging. The aspects of the present invention can be summarized as follows. 1 Contains 10-15% protein, 3-4.5% acetyl and mainly carbohydrates, ~12% carbohydrate portion
S-60 is a complex polysaccharide consisting of uronic acid, rum sauce and glycose in a molar ratio of approximately 1.5:1.
and the polysaccharide is incompatible with methylene chloride dye, substantially insoluble in N,N-dimethylformamide and soluble in DMSO or formamide, 3400 cm ^-1 , 2950 cm ^-1 , 1740 cm ^-1 ,
The complex polysaccharide S-60 is characterized by having an infrared absorption spectrum characterized by a peak at 1620 cm ^-1 . 2 Unidentified Pseudomonas
A microorganism of the genus ATCC 31461 was cultured in a fermentation medium containing a carbon source, a potassium ion source, a nitrogen source, trace inorganic elements, and water at 283°C and pH 6-8 for 40-60 hours, and the gum was prepared by precipitation with an appropriate lower alcohol. 2. The method for producing complex polysaccharide S-60 according to item 1, which comprises recovering the complex polysaccharide S-60. 3. The production method according to item 2, wherein the nitrogen source is corn stave liquor and the alcohol is isopropanol. 4 Unidentified Pseudomonas
Freeze-dried culture of the fungus, ATCC 31461. 5 17% protein, 0% acetyl content, carbohydrate moiety is 13% uronic acid, and its molar ratio is
Deacetylated unclarified complex polysaccharide S-60 containing carbohydrates consisting of the neutral sugars rhamnose and glucose in a ratio of 1.5:1. 6 2% protein, acetyl content 0%, carbohydrate portion 22% eronic acid, approximate molar ratio
Deacetylated clarified complex polysaccharide S-60 containing carbohydrates consisting of the neutral sugars rhamnose and glucose in a ratio of 1.5:1. 7 Add a 1-5% aqueous solution of complex polysaccharide S-60 to a pH of approx.
10.Heat at a temperature of 90-100℃ for 10 minutes to 45 minutes,
6. A method for producing a compound according to claim 5, which comprises recovering the product thereby produced. 8 Approximately 0.5-5% deacetylated complex polysaccharide S-60
microbiological culture medium consisting of and nutrients. 9. The culture medium according to item 8, wherein the deacetylated S-60 is clarified. 10 about 0.1-5% deacetylated complex polysaccharide S-60,
A fragrance gel composition consisting of water and a lower alkanol.

Claims (1)

[Claims] 1 2% protein, acetyl content 0%, carbohydrate portion 22% uronic acid, approximate molar ratio
It contains carbohydrates consisting of the neutral sugars rhamnose and glucose in a ratio of 1.5:1 and is characterized by peaks at 3400 cm ^-1 , 2950 cm ^-1 and 1600 cm ^-1 (KBr) and [α] ²⁵ ₅₈₉ = Deacetylated clear complex polysaccharide S-60 characterized by having a specific optical rotation of -45°. 2 2% protein, 0% acetyl content, carbohydrate portion 22% uronic acid, approximate molar ratio
It contains a carbohydrate consisting of the neutral sugars rhamnose and glucose in a ratio of 1.5:1 and is characterized by peaks at 3400 cm ^-1 , 2950 cm ^-1 and 1600 cm ^-1 (KBr) and [α] ²⁵ ₅₈₉ = Deacetylated clear complex polysaccharide S-60 with specific optical rotation of -45°
A method for culturing microorganisms, characterized in that about 0.5-5% and nutrients are used.