HETEROPOLYSACCHARIDES PRODUCED BY BACTERIA AND DERIVED PRODUCTS, THEIR PREPARATION, AND THE LYOPHILIZED CULTURE OF THE BACTERIA

Abstract: A novel polysaccharide S-60 is disclosed composed of principally carbohydrate, 10-15% protein, and 3-4.5% acetyl groups as the O-glycosidically linked ester. The carbohydrate portion contains ∼12% uronic acid (based on wt. gum) and the neutral sugars rhamnose and glucose, the latter having an approximate molar ratio of 1.5 to 1. This polysaccharide is produced by an unnamed Pseudomonas species, ATCC 31461, in a suitable fermentation medium. Deacetylated poiysaccharide S-60 is also disclosed. It has valuable properties in both the clarified and non-clarified form and is useful as an agar substitute or as a shapeable room deodorant.

Description: POLYSACCHARIDE S-60, BACTERIAL iENTATION PROCESS FOR ITS PREPARATION AND METHOD OF PREPARATING IT This invention relates to polysaccharides, their production and compositions containing them. It is known that heteropolysacLarides can be produced by certain microorganisms. Some of such heteropolys2ccllarides function as hydrophilic colloids and because of their viscosity and rheological properties have been used as thickening agents for aqueous systems. As with other fields of technology, research has continued with the objective of discovering new heteropolysaccharides having useful properties as thickening suspending andjor stabilizing agents. The novel heteropolysaccharide of the present invention is produced by the action of a specified bacterium on a selected carbon souce arid ferinentation medium ingredients under controlled conditions. The heteropo lysaccharide of this s invention is a high-molecular-weight polysaccharide containing primarily carbohydrate residues and a minor amount of protein. It is sometimes referred to as a "gum" but it is believed that the heteropolysaccharide terminology is more accurate and precise. In the following description of our invention, it will sometimes be referred to as Heteropolysaccharide 60, or S-60. In accordance with the invention, the novel compound is prepared by fermentation of a suitable nutrient medium with a hitherto undescribed organism, based on extensive taxonomic studies, which is an unnamed Pseudomonas species. An unrestricted permanent deposit of this organism was made with the American Type Culture Collection on November 21, 1978 under Accession No. ATCC 31461. Various classification keys for the genus Pseudomonas and the culture description of Pscudomonas species are found in the 7th Editicn of Berey's Manual (Breed et al., (1957) and the 8th Edition of Lergey's WJanual (Doudoroff et al., (1974), as well as by other schools in various publications; Faugh and Gilardi, 1974, Pseudomonas, manual of Clinical Microbiolorw, 2nd ed., Lennette et al., Eds., pp. 250-269. American Society for Microbiology, Washington, D.C.; Weaver et al., 1972, Identification of Unusual Pathogenic Gram-Negative Bacteria, E.O. King, Center for Disease Control, Atlanta; Iizuka et al., 1963, Attempt at Grouping the Genus Pseudomonas, J. Gen. Appl. Microbiology 9:73-82; and Hendric et al., 1966, Identification of Certain Pseudomonas Species, Identification Methods for Microbiologists, Part A, Gibbs et al., Eds., pp. 1-7, Academic press, New York. These keys and descriptions were searched for a Pseudomonas species having morphological and cultural characteristics similar to those of ATCC 31461. The following considerations make the assignment of a new Pseudomonas species justified and necessary. Description of the Strain 1. Characteristics of Cell Slorphology Single cells, straight or often curved, generally 0.6-0.8 by 2.0-3.0 Wm, often with tapered end. The older cultures become larger and longer (0.8-1.0 by 7 3Am), misshaped cells and pleomorphism appear, especially on media with limited amount of carbohydrates. On the contrary, cells keep rather consistent rod shapes when grown on media with carbohydrates, but again, most cells become large and pleomorphism develops during prolonged incubation. Gram-negative, non-capsulated, poly-B-hydroxybutyrate and polyphosphate granules are seen especially in cultures of nitrogen-deficient media. Blotile by polar multitrichous flagellation; one to four flagella are inserted at the polar end and occasionally subpolar insertion may be seen. 2. Characteristics of Colonial Morphology On nutrient agar plates, small (0.8-1.1 mm in diameter) and large (3.2-3.5 mm in diameter) colonies appear. They are yellow carl'oenoid pigmented, smooth, round, and convex to pulvinate. Large colonies often have a concentric wrinkle. The surface of these colonies has a hard but not viscid texture and entire colonies are removed if pushed by a loop. On YM agar plates, only one type of relatively large (H6-7 mm in diameter) yellow, round, smooth, slimy and convex colonies appear. Slimy elastic membranes form on the surface of these colonies and whole surface membranes (of colonies) can be removed. The secondary growth may occur around the edge of the original colonies. The color of these colonies is darker yellow towards the center than the edge and concentric color formation appeared. In addition to the intracellular yellow carotenoid pigment(s), diffusible brown pigment developed as a result of autooxidation after prolonged incubation. This phenomenon is more easily recognized on Nutrient agar. No fluorescent pigment was produced. 3. Physicological and Biochemical Characteristics The growth range of the strain S-60 is about 200C to 410C. No growth occurs at 40C. 3.0% NaCR is sufficient to inhibit the growth and the strain is capable of growth at pHs between 5 and 11. Acid, but no gas, is produced from almost all carbohydrates but not from polyalcohols. Urease may be produced. MR, VP, and indole tests were all negative. Argine dihydrolase, lysine and ornithine decarboxylase are not produced. Acid and reduction occurs in litmus milk. Lipolytic egg yolk reaction is negative. Gelatin is weakly hydrolyzed but not casein, starch, alginate, pectin, cellulose, chitin and DNA. 4. Susceptibility to Antibiotics The strain is very susceptible to Kanamycin, Neomycin, Chlortetracycline, Erythromycin, but not Streptomycin and Penicillin. 5. Nutritional Characteristics Organic growth factors are not required and ammonium salts serve as sole nitrogen source. At least 35 organic compounds were utilized, those being most carbohydrates other than D-ribose, starch, 2 ketoglucdnate, and mucate. In addition, acetate, caproate, caprylate, pelargonate, succinate, azelate, L-malate, DL-8-hydroxybutyrate, pyruvate, ethanol, n-proponal, p-hydroxybenzoate, phenylacetate, L-aalanine, L-threonine, L-leucine, DL-isoleucine, Laspartate, L-glutamate, and L-tyrosine were utilized. 6. The G+C Content of the DNA Evaluation of the D2JA resulted in the mole a to be ray63 (by Tm). Table 1. Biochemical and Other Miscellaneous Tests Employed for the Strain S-60 Oxidase: Kovac's + (weak) Hydrolysis of: Pathotech + (weak) Gelatin + (weak) Casein Catalase + Starch OF medium: Oxidative + Tween 80 + Fermentative - Pectin Gas from glucose - Al ginate H2S production: TSI - Cellulose from cystine + Chitin Ammonium from peptone + DNA ss-Galactosidase (OTPG) + (API) Esculin + Arginine dihydrolase Lysine decarboxylase - Growth on various media: Ornithine decarboxylase - efB agar Tryptophan deaminase - MacConkey agar Phenylalanine deaminase - SS agar Urease +/+ Mannitol salt agar Indole - TCJ3S agar MR test - Tinsdale tellurite VP test - blood agar + Nitrate reduction - Pseudosel agar Nitrite reduction Denitrification - Pigment production: N2-fixation: King A medium Growth in Burk's medium + King B medium Nitrogenase activity Malonate (Oxidation) - Dye reaction: Phosphatase + Congo red Haemolysis (sheep blood) - Nite blue Litmus milk: acid, reduction only reduction only TSI: Slant color no change 3-icetolactose production - Butt color no change Butt color no change Survival at 60 C for Gas 30 min. Egg Yolk Reaction FERl:ENTATICN CO;DITIOt;S: Heteropolysaccharide S-60 is produced during the aerobic fermentation of suitable aqueous nutrient media under controlled conditons via the inoculation with the organism of the unnamed Pseudomonas species. The media are usual media, containing source of carbon, nitrogen and inorganic salts. In general, carbohydrates (for example, glucose, fructose, maltose, sucrose, xylose, mannitol and the like) can be used either alone or in combination as sources of assimilable carbon in the nutrient medium. The exact quantity of the carbohydrate source or sources utilized in the medium depend in part upon the other ingredients of the medium but, in general, the amount of carbohydrate usually varies between about 28 and 4% by weight of the medium. These carbon sources can be used individually, or several such carbon sources may be combined in the medium. In general, many proteinaceous materials may be used as nitrogen sources in the fermentation process. Suitable nitrogen sources include, for example, yeast hydrolysates, primary yeast, soybean meal, cottonseed flour, hydrolysates of casein, corn steep liquor, distiller's solubles or tomato paste and the like. The sources of nitrogen, either alone or in combination, are used in amounts ranging from about 0.05% to 0.2E by weight of the aqueous medium. Among the nutrient inorganic salts which can be incorporated in the culture media are the customary salts capable of yielding sodium, potassium, ammonium, calcium, phosphate, sulfate, chloride, carbonate, and like ions. Also included are trace metals such as cobalt, manganese, iron and magnesium. It should be noted that the media described in the examples are merely illustrative of the wide variety of media which may be employed, and are not intended to be limitative. The fermentation is carried out at temperatures ranging from about 25eC to 350C; however, for optimum results it is preferable to conduct the fermentation at temperatures of from about 280C to 320 C. The pIi of the nutrient media for growing the Pseudomonas culture and producing the polysaccharide S-60 can vary from about 6 to 8. Although the novel pooysaccharide S-60 is produced by both surface and submerged culture, it is preferred to carry out the fermentation in the submerged state. A small scale fermentation is conveniently carried out by inoculating a suitable nutrient medium with the culture and, after transfer to a production medium, permitting the fermentation to proceed at a constant temperature of about 300C. on a shaker for several days. The fermentation is initiated in a sterilized flask of medium via one or more stages of seed development. The nutrient medium for the seed stage may be any suitable combination of carbon and nitrogen sources. The seed flask is shaken in a constant temperature chamber at about 300C for 1-2 days, or until growth is satisfactory, and some of the resulting growth is used to inoculate either a second stage seed or the production medium. Intermediate stage seed flasks, when used, are developed in essentially the same manner; that is, part of the contents of the flask from the last seed stage are used to inoculate the production medium. The inoculated flasks are shaken at a constant temperature for several days, and at the end of the incubation period the contents of the flasks are recovered by precipitation with a suitable alcohol such as isopropanol. For large scale work, it is preferable to conduct the fermentation in suitable tanks provided with an agitator and a means of aerating the fermentation medium. According to this method, the nutrient medium is made up in the tank and sterilized by heating at temperatures of up to about 1210C. Upon cooling, the sterilized medium is inoculated with a previously grown seed of the producing culture, and the fermentation is permitted to proceed for a period of time as, for example, from 2 to 4 days while agitating and/or aerating the nutrient medium and maintaining the temperature at about 300C. This method of producing the S-60 is particularly suited for the preparation of large quantities. The product is recovered from the fermentation medium by precipitation with a suitable alcohol, such as isopropanol. PHYSICAL AND CHEMICAL PROPERTIES OF THE POLYSACCHARIDE S-60 The heteropolysaccharide produced by an unnamed Pseudomonas species is composed of about principally carbohydrate, 3-4.5% acetyl groups as the O-glycosidically linked ester, and 10-15% protein. The carbohydrate portion of the S-60 polysaccharide contains uronic acid (rv12% b.o. wt. gum) and the neutral sugars glucose and rhamnose. The approximate molar ratio of rhamnose to glucose to 1.5 to 1. The acetyl content of 4.5% was treating a 0.2% aqueous solution of S-60 gum with an alkaline, hydroxylamine reagent followed by treatment with an acidic ferric chloride reagent [S. Hestrin (1949) J. Biol. Chem. 180 249-261]. The neutral sugars of polysaccharide S-60 were determined by dissolving ten mg. of the product in 2 ml 2N H2SO4, and the mixture is heated at 1000C for 4 hours. The resulting solution is cooled, neutralized with barium hydroxide and the pH is brought to 5-6 with solid carbon dioxide. The resulting precipitate of barium sulfate is removed by centrifugation and the supernatent is concentrated to a syrup under reduced pressure. The sugars in the hydrolysate are tentatively identifed by gasliquid chromatography of their aldononitril acetate derivatives on a Hewlett-Packard Model 5750 chromatograph using 3% by weight OV-225 on 80/100 mesh Gas Chrome Q at 2100C. The sugars are identified and quantitated by comparison with authentic standards [J.K. Baird, M.J. Holroyde, and D.C. Ellwood (1973) Carbohydr. Res. 27 464-467]. The various neutral sugars of the polysaccharides were also characterized by use of descending paper chromatography on Whatman No. 1 chromatography paper using as the solvent the upper layer of pyridine: ethyl acetate:water (2:5:5). Chromatograms were stained using silver nitrate dip and acid analine phthalate spray reagent. Component sugars were identified by co-chromatography with sugar standards and by the specific-color reaxtion with the anali phthalate reagent. The uronic acid content of the polysccharide was determined by two separate methods. In-one method the sample was decarboxylated with 19% hydrochloric acid and the liberated carbon dioxide was trapped in standard sodium hydroxide and determined by back titration [B.L. Browning (1967) Methods of Wood Chemistry II, 632-633] and by the carbazole colorimetric method [T. Bitter and H.M. Muir (1962) Anal. Biochem. 4 330-334]. Paper electrophoresis was used for the separation and tentative identification of the uronic acids present in the neutralized acid hydrolysate described above. Aliquots of this and known uronic acid standards were applied to Camag electrophoresis paper No. 68-011 and electrophoresis was carried out for 2.0 hours in a pH 2.7 buffer using a Camag Model HVE electrophoresis apparatus. Chromatograms were air dried and stained with silver nitrate dip reagent to locate the uronic acids being separated. Two major and one minor uronic acid spots were found. One of the major spots migrated with the same mobility as glucuronic acid (RGlcA = 1.0) while the other major spot (RGlcA = 0.85) and the minor spot (RglcA = 0.73) had lower mobility. Under these same conditions the relative mobility of known uronic acids are: Rm Glucuronic acid 1.0 Mannuronic acid 0.96 Galacturonic acid 0.65 Guluronic acid 0.63 An infrared spectrum of native S-60 was made on dried material in a KBr pellet. The heteropoly saccharide evidenced peaks at: 3400 cm-l, 2950 cm-l, 1740 cm-l, and 1620 cm-l indicating hydroxyl groups, methylene groups, carbonyl groups and carboxylic acid salts. It is incompatible with methylene chloride dye, and is substantially insoluble in N,N-dimethylformamide, soluble in DMSO or formamide. A sample of S-60 shows the following elemental analysis: t; - 2.00%, C - 42.62%, H - 5.80%. The polysaccharide S-60 imparts viscosity to an aqueous medium when dissolved in water in low concentrations. Because of this, its sensitivity to shear and overall rheology, it is useful as a thickening, suspending and stabilizing agent in aqueous systems; for example, as an additive to textile printing pastes or in formulating low drift aqueous herbicide compositions, salad dressings, thickened puddings, and adhesive compositions. After heating and cooling, it forms a weak very elastic gel. DEACETYLATED, NON-CLARIFIED S-60 When the dry polymer or the fermentation broth is heated at high pH (using e.g., sodium carbonate or sodium hydroxide to pH 10) and high temperatures 90-1000C for 10 minutes to 45 minutes deacetylation readily occurs. The resultant deacetylated polysaccharide S-60 forms firm non-elastic or brittle gels, useful in many industrial and food applications. The composition of the deacetylated S-60 is principally carbohydrate, ¯ /V178 protein, and 0% acetyl. The carbohydrate portion comprisesW1390 uronic acid and the neutral sugars rhamnose and glucose in the approximate molar ratio 1.5:1. One use for the deacetylated gum results from the fact that as a rigid, brittle gel, it can be molded, and used as a rigid structure, which after treatment with a suitable solution such as fragrance, finds applicability as a room deodorant or an air freshener, or the like. The deacetylated gum can also be used in gel electrophoresis, as a gelling agent for use in microtomes in electronmicroscopy. The native and deacetylated gums can also be used as suspending agents for barium in radiology, confectionery products, and as impression materials in tool making, dentistry and criminology. An infrared spectrum of deacetylated S-60 made on dried material in a KBr pellet evidenced peaks at: 3400 cm 1, 2950 cm 1, 1740 cm 1, 1650 cm and 1610 cm 1. A sample of deacetylated S-60 shows the following elemental analysis: N - 2.67%, C 41.9, H - 6.07%. DEACETYLATED, CLARIFIED 5-60 The composition of the clarified, deacetylated S-60 is as follows: 2% protein, 0% acetyl, and carbohydrate, the latter being ¯ 22% uronic acid and the neutral sugars rhamnose and glucose in the approximate molar ratio 1.5 to 1. An infrared spectrum of clarified, deacetylated S-60 made on dried material in a KBr pellet evidenced peaks at: 3400 cm 1, 2950 cm-1, and 1600 cm A sample of deacetylated, clarified S-60 shows the following elemental analysis: N -0.42%, C - 36.85%, H - 5.62%. The sample has a specific rotation: 25 589 45 The deacetylated clarified gum is especially useful as an agar substitute in microbiological culture media for various clinical and non-clinical microorganisms using a wide variety of culture media. The concentration of deacetylated clarified gum necessary to replace agar is dependent upon the medium used, but is within the range of about 0.5 to about 1.25% (weight volume). Growth characteristics of microorganisms are quite similar to those on standard agar-based media. The following detailed examples illustrate representative aspects of this invention. EXAMPLE 1 Fermentation Procedure for Producing Heteropolysaccharide S-60 A. Culture Maintenance The unnamed Pseudomonas organism, ATCC 31461, grows quite well on NA or YM agar, which are used routinely for culture maintenance. The incubation temperature is 30"C. The organism produces a yellow-orange carotinoid pigment and a brown diffusable pigment by 2-5 days' incubation. B. Seed Preparation Flask seeds are prepared in YM broth incubated at 300C. When inoculated with a fresh plate culture, the YM broth cultures give good growth and gum formation by 24 hours. The fermentation seed medium is the same as final fermentor medium, using one-gallon fermentors as seed vessels. C. Final Fermentor Medium The sodium- and potassium-salt form of the gum are prepared in a different media; both of which are described below. The organism has a definite K+ requirement which must be added to the sodium fermentation medium. (3% dextrose can also be used). Also, 0.2 corn steep liquor is added in both media. Sodium Salt Potassium Salt 3.0% Glucose 3.0% Glucose 0. 01% MgSO4.7H2O 0.01% MgSO4.7H2O 0.09% NII4NO3 0.09% NH4NO3 O.05E Promosoy 0.05% Promosoy (soy protein concen trate) 1 ml/L HoLe salts 1 ml/L HoLe salts 1 ppm Fe++ 1 ppm Fe++ 0.05% Ela2HnO4 0.05% K2HPO4 L Na2-HPO4 0.05% K2HPO4 10 ppm K+ pH control=DaOH pH control=KOH oLe salts are a trace element solution containing, tartrate, magnesium molybdate, CoCl3, ZnCl2, CuC12, boric acid, manganese chloride and ferrous sulfate. When a low calcium product is desired, either of the media above are used with deionized water. Fermentation is complete aq. 50 hours; beer viscosity usually is 5000-8000 cps. D. Recovery Because of the gelling nature of the product, good fiber formation usually does not occur with ambient precipitation. However, we have found that by pasteurization at 90-950C for 10-15 minutes (during which the thick beer heat-thins considerably) excellent fibers can be obtained by precipitation of the beer using two volumes of 99% isopropanol per volume of beer without cooling. Average yields of 1.5% gum are obtained with 3% glucose in the 20L and 70L fermentors. E. Drying The product is recovered and dried at 50-550C for up to one hour in a forced-air tray dryer. F. Product Quality One-percent viscosities of the K+ salt are usually in the range of 3000 cps and for the lowcalcium sodium salt, approximately 7000 cps. EXAMPLE 2 Deacetylation and Clarification of the Heteropolysaccharide S-60 Clarification of the gum, while not necessarily for all uses, is of value when the gum is used as an agar substitute. Clarification can be accomplished before deacetylation (in the native state) or after deacetylation. Since deacetylation uses hot caustic, and clarification is done hot, the two procedures are easily and conveniently combined. Both deacetylation and clarification can be done with the beer or the dry polymer. For deacetylation, if the beer is used, the pH is adjusted to 10 with KOH, the solution heated to 900C for 15 minutes, the pH adjusted to 7 with dilute H2S04, then Caul2 is added to a 0.2% concentration and cooled. A hard brittle gel results. The general procedure for both deacetylation and clarification follows: A. A 2% solution of beer or gum is heated to 900C B. The pH is adjusted to 10 with KOOK. C. The temperature of the beer or solution was maintained at 90-95cC for 15 minutes. D. The pH is adjusted to 6-8 with dilute HC1 or H2SO4. Ten Ten gns/liter of Super Aid were added to the material to be filtered. F. The material was filtered through a pressure filter unit (pre-heated) with approximately a 6 mm bed of Super Aid and approximately 20-30 psi, using a filter unit with an area of 136 cm2. G. The filtrate is precipitated with isopropanol immediately to prevent gelation and the fibers dried at 500C for one hour or less. When no deacetylation is necessary, the above procedure is followed, except that the pH is not raised; rather than holding at 900C, the solution is immediately filtered, and then recovered. Clarification is always done on the potassium form; KC1 can be added to a solution of previously made product as necessary. EXAMPLE 3 lleteropolvsaccharide S-60 Gelling Characteristics A compilation of data comparing the native gum and the deacetylated gum, both in the K+ form and in the Ca++ form, with carrageenan and agar follows Type Gel Nature Melts Sets Hysteresis Native S-60 Very Elastic 65-7o0C 65-700C None Deacetylated S-60 Gel Brittle 90 C 31-216 C 45-60 C Ca2+Gel Brittle 900C 45-50 C 45-5G C Kappa Carrageenan Brittle 40-95 C 25-750C 15-20 C Agar* Brittle 60-97 C 32-390C 60 C *Bacteriological grade specs: gelling temperature range 330-390C, melting temperature 700C minimum. (Whistler's "Industrial Gums") Minimum Gelling Concentration Kappa Carrageenan - 0.3% Agar Deacetylated S-60 - 0.05% (calcium gel) Note above that there is a wide range of temperatures given for setting and melting of all the various types of gels. For agar, the variations are primarily due to tT'e of seaweed while for kappa carrageenan the potassium ion concentration determines the gel characteristics. The gels of deacetylated S-60 are primarily characterized by the degree of deacetylation. With only slight deacetylation the gels set at higher temperatures and are more elastic; in fact, a wide range of gel types from very elastic to very brittle is possible, depending on the degree of deacetylation. The gels appear to be more similar to agar than to kappa carageenan, primarily because of the large hysteresis between setting and melting temperatures. It should be emphasized that they are difficult to melt and the gel-sol transition is difficult to observe. On the other hand, the gelling temperatures can be easily defined since the gels set sharply within a few degrees from incipient gelation to solid gel. EXAMPLE 4 DEACETYLATED, tJON-CLARIFIED S-60 S-60 fermentation liquor is heated to 900C and the pH adjusted to 10 by addition of 25% KOH. The temperature is maintained for 15 minutes, followed by neutralization with concentrated HC1. This liquor is then drained from the fermentor while hot and recovered with 2 volumes of 99% IPA. The fibers of deacetylated S-60 are collected, dried at 550C for one hour in a forced air tray drier, and then milled to a powder. EXAMPLE 5 CLARIFICATION OF DEACETYLATED S-60 Deacetylated heteropolysaccharide S-60 as prepared in Example 4 is reconstituted to a 1% concentration in deionized water using a Lightnin mixer for one hour followed by mixing on an Arti Barinko with heating to 500C. The solution is then centrifuged at 10,000 R.P.M. (GSA head) for 20 minutes in a Sorvall RC2-B refrigerated centrifuge keeping the temperature above 400C. The supernatant is decanted off and sequentially filtered through Gelman type AN Hydrophilic Acropor membranes (293 mm) of porosities of 5W, 3W, 1.2p and 0.8W. The filtrate is added to approximately 3-4 volumes of 99% isopropanol, the fibers collected and dried briefly in the forced air tray drier at 550C, followed by milling to a powder. The product is the deacetylated, clarified S-60 gum of this invention. EXAMPLE 6 Agar Replacement Using Deacetylated Clarified S-60 Several different media are prepared as follows: Nutrient Agar (A) 0.8% Nutrient Broth (Difco) 1. Agar (Difco) (B) 0. 8% Nutrient Broth (Difco) 0.2% KC1 0.9% S-60 Trypticose Soy Agar (A) 2.75% Trypticose Soy Broth (BBL) 1.5 % Agar (Difco) (B) 2.75% Trypticose Soy Broth (BBL) 0.2% KC1 0.9% S-60 Potato Dexrose Agar (A) 2.4% Potato Dextrose Broth (Difco) 1.5% Agar (B) 2.4% Potato Dextrose Broth (Difco) 0.2% KC1 0.9% S-60 YM Agar (A) 2.1t YM Broth (Difco) 1.5% Agar (Difco) (B) 2.1% Broth 0.2% KC1 0.9% S-60 Brain Heart Infusion Agar (A) 3.7% 3111 Broth (Difco) 1.5% Agar (B) 3.7% 3111 Broth ( Difco) 0.2% KC1 0.9 S-60 Burk's Agar EMI22.1 <tb> (A) <SEP> (B) <tb> 0.0584% <SEP> K2HP04 <tb> 0.0225% <SEP> KH2PO4 <SEP> <tb> 0D0174% <SEP> K2S04 <SEP> <tb> 0.01649 <SEP> MgCl2.6H9O <SEP> Same <SEP> as <SEP> (A) <tb> O. <SEP> 0064% <SEP> CaCl2. <SEP> 21H20 <SEP> <tb> 0.0005% <SEP> FeCl3. <SEP> 6520 <SEP> <tb> 0.00002% <SEP> Na2MoO4.2H2O <SEP> <tb> 0.0116% <SEP> NaCl <SEP> <tb> 1.0% <SEP> Glucose <tb> 1.5% Agar (Difco) 0.2% KC1 0.9% S-60 Deionized water was used for all media. The ingredients were combined (except for Burk's) and autoclaved for 15-20 minutes at 1210C and 15 psi, cooled to 550C, and poured into sterile petri dishes. The ingredients for Burk's were combined, except for the glucose, which was autoclaved separately, and added to the medium after autoclaving. After these nutrient plates had solidified and allowed to incubate at ambient temperature for 24 hours to check for sterility, they were streaked with the following fourteen cultures: : Agromyces ramosus ATCC 25173 Arthrobacter globiformis ATCC 8010 Aureobasidium pullulans NRRL YB-3861 Azotobacter indicus var myxogenes strain S-7 ATCC 21423 Azotobacter vinelandii ATCC 9047 Beijerinckia lacticogenes ATCC 19361 Erwinia cartovora ATCC 8061 Escherichia coli strain EG-47 Klebsiella pneumoniae strain S-53 Nocardia salmonicolor ATCC 21243 S-60 Streptococcus faecalis Trichoderma longbrachiatum ATCC 13631 Zoogloea ramigera ATCC 25935 The plates were incubated at 300C for 3-5 days and then examined for growth. Good growth was observed for all strains on media made with S-60 and little difference in colonial morphology was noted between media made with S-60 instead of agar. These results indicate that S-60 is an excellent replacement for agar in microbiological media. The gel point for all media containing S-60, except BHI medium and TSA, was 420C. The gel point for 3111 agar and TSA was 520C. Agar typically gels at 42-440C. EXAMPLE 7 Preparation of Molded Scented Gels Using Deacetylated S-60 (A) 1.50% Polysaccharide S-60 0.75% Sodium Carbonate 0.025% Methyl p-hydroxybenzoate 3.00% Rose fragrance 4.00% Isopropanol 2.00% Ethylene glycol 88.50% Water The native polysaccharide S-60 is blended with sodium carbonate and preservative and dissolved in water at 700C. The solution is then further heated to 900C and held at that temperature for ten minutes to deacetylate the polysaccharide. After cooling to 600C, the fragrance dispersed in the solvents is added and the mixture placed in the usual air freshener plastic molds. When the mixture cools to 380C, gelation occurred and a firm, self-supporting gel with good fragrance releasing properties results. (B) A dry deacetylated polysaccharide S-60 is prepared from the fermentate beer by adjusting the pH to 10.0 with dilute sodium hydroxide and heating to 900C for fifteen minutes. The solution is neutralized to pH 7 with dilute hydrochloric acid, precipitated in two volumes of isopropanol, dried, and milled. A solid air freshener gel is prepared from the deacetylated product in the following manner: 3.0 grams of the deacetylated polysaccharide S-60 are blended with 1.5 grams potassium chloride and 0.15 grams methyl p-hydroxybenzoate preservative and added to 177 ml water. The solution is heated to 900C to dissolve; cooled to 60 C, and a blend of 6.0 grams peppermint oil fragrance, 8.0 grams isopropanol, and 4.0 grams ethylene glycol is added. The solution is placed in plastic molds and cooled to ambient temperature. A strong, brittle gel with heavy mint fragrance forms. The gel can be unmolded easily and retains its shape without sagging.

Claims: IZHAT IS CLAItE D IS: 1. Heteropolysaccharide S-60, containing 10 1 protein, 3-4.5E acetyl and principally carbohydrate, the carbohydrate portion containing 12% uronic acid, and rhamnose and glucose in the approximate molar ratio of 1.5 to 1, said heteropolysaccharide being incompatible with methylene chloride dye, being substantially insoluble in N,N-dimethylformamide and soluble in DMSO or formamide, and having an infrared spectrum characterized by peaks at 3400 cm-l, 2950 cm-l, 1740 cm-l, 1620 cm-l. 2. A process for producing heteropolysaccharide S-60 of Claim 1 comprising incubating the unnamed Pseudomonas organism, ATCC 31461, in a fermentation medium containing a carbon source, a source of potassium ions, a source of nitrogen, trace inorganic elements, and water at a temperture of 2C-320C and pH of 6-8 for 40-60 hours, and recovering the gum by precipitation with a suitable lower alcohol. 3. The process of Claim 2 where the nitrogen source is corn steep liquor and the alcohol is isopropanol. 4. The lyophilized culture of an unnamed Pseudomonas species, ATCC 31461. 5. Deacetylated non-clarified heteropolysaccharide S-60, containing 17% protein, carbohydrate, and an acetyl content of 0%, the carbohydrate portion comprising 13% uronic acid and the neutral sugars rhamnose and glucose in the approximate molar ratio 1.5 to 1. 6. Deacetylated clarified heteropolysaccharide S-60, containing 2% protein, carbohydrate, and an acetyl content of 0%, the carbohydrate portion comprising 22% uronic acid and the neutral sugars rhamnose and glucose in the approximate molar ratio 1.5 to 1. 7. A process for preparing the compound of Claim 5 which comprises heating a 1-5% aqueous solution of the heteropolysaccharide S-60 at a pH of about 10, at a temperature of 90-1000C for from 10 minutes to 45 minutes, and recovering the product thereby produced. 8. A microbiological culture medium which comprises about 0.5-5 deacetylated heteropolysaccharide S-60 and a nutrient. 9. A medium of Claim 8 where the deacetylated S-60 is clarified. 10. A scented gel composition which comprises about 0.1-5 deacetylated heteropolysaccharide S-60, water and a lower alkanol.

Inventors: COLEGROVE, GEORGE T., KANG, KENNETH S., VEEDER, GEORGE T.
Applicants: MERCK & CO. INC.
Application year: 1979
Main classification: C12P21
IPC codes: A61L9, C08B37, C12P21, C12N1, A23G3