KR20040071964A - Alteromonas sp. 00SS11568 and method for preparation of extracellular polysaccharide therefrom - Google Patents

Abstract

The present invention relates to a strain of Alteromonas sp. 00SS11568 that secretes extracellular polysaccharides and a method for producing extracellular polysaccharides (exopolysaccharides) therefrom.

The extracellular polysaccharide produced by the present invention is a heteropolysaccharide having a glucose and galactose ratio of 3: 1, and has a molecular weight of 4.4 × 10 ⁵ Da and excellent emulsification stability and can be used in various industrial fields.

Description

Strains of Oss 11568 of the genus Alteromonas and a method for producing extracellular polysaccharides therefrom [Alteromonas sp. 00SS11568 and method for preparation of extracellular polysaccharide therefrom}

The present invention relates to a strain of genus 00SS11568 that secretes extracellular polysaccharides and a method for producing extracellular polysaccharides (exopolysaccharides) therefrom.

In this specification, several publications are cited by reference, which references only the author and year in the specification, the details of which are listed immediately before the claims.

Polysaccharides secreted extracellularly by microorganisms protect microorganisms from other organisms, act as a defense against antibodies, neutralize toxic substances in the surrounding environment, or form complexes with metal ions and prevent evaporation of intracellular moisture in dry environments. It is a substance that performs many functions related to the survival of microorganisms (Sutherland, 1983).

However, extracellular polysaccharides secreted by microorganisms have a wide range of functions such as gel formation ability, emulsification stability, surface tension control ability, water absorption ability, adhesion ability, lubrication ability, and film formation ability, depending on conditions. (Fu and Tseng, 1990; Irene et al. , 1990: Low et al., 1998; Marra 1990). In addition, extracellular polysaccharides with heavy metal adsorption capacity (Norberg and Persson, 1984) and anti-tumor activity (Oda et al. , 1983) have been reported. In particular, microbial extracellular polysaccharides have a high potential for industrial use. It can improve productivity by improving, it is possible to mass production by continuous culture using fermentation tank in a short time, and it is easy to separate and recover the extracellular polysaccharides produced.

Therefore, studies on such microorganisms and microalgae extracellular polysaccharides are being actively conducted, and in particular, the microorganisms known as microbial extracellular polysaccharides are the genus of zooglan and Pseudomonas produced by the genus Zoogloea sp. Psedomonas sp.), V. Fisher Lee (Vibrio fisheri), cyano test in (Cyanothece sp.) and the Alte Monastir town and the extracellular polysaccharide produced from such nucleoside di (Altermonas macleodii), production of extracellular polysaccharides from Korea coast It is known that the marine microorganisms are not as many as the genus of Zoogloea sp . KCCM 10036 ( Jang et al ., 1998).

Accordingly, the present inventors have made a thorough study for the discovery of microorganisms capable of producing new extracellular polysaccharides different from the conventional microbial extracellular polysaccharides. With the discovery of a new microorganism that secretes extracellular polysaccharides based on biofilms formed in variously distributed facilities, the present invention has been completed.

That is, an object of the present invention is to provide a strain of genus 00SS11568 marine microorganisms.

Another object of the present invention is to provide a method for producing extracellular polysaccharides from the above strains.

Still another object of the present invention is to provide an extracellular polysaccharide produced from the above strain.

Other objects and functions of the present invention will become apparent to those skilled in the art from the configurations and functions of the following invention.

1 is a molecular biological phylogenetic diagram of the genus 00SS11568 strain.

Figure 2 is a scanning micrograph of the genus 00SS11568 strain.

Figure 3 is a graph showing the growth of cells and the production of extracellular polysaccharides by the temperature of the genus 00SS11568 strain genus Alteromonas.

Figure 4 is a graph showing the growth of cells and the production of extracellular polysaccharides by the pH of the genus 00SS11568 strain of Alteromonas.

5 is a graph showing the growth of cells and the production of extracellular polysaccharides by the culture medium of the genus 00SS11568 strain.

6 is a graph showing the growth of cells and the production of extracellular polysaccharides by the inoculation amount of the genus 00SS11568 strain.

7 is a graph showing cell growth and production of extracellular polysaccharides by the inorganic nitrate source of genus 00SS11568 strain.

8 is a graph showing the growth of cells and the production of extracellular polysaccharides by the organic nitrate source of the genus 00SS11568 strain.

9 is a graph showing the growth of cells and the production of extracellular polysaccharides by the yeast extract of the genus 00SS11568 strain.

10 is a graph showing cell growth and production of extracellular polysaccharides by KH ₂ PO ₄ of the genus 00SS11568 strain.

11 is a graph showing cell growth and production of extracellular polysaccharides by MgSO ₄ of the genus 00SS11568 strain.

12 is a graph showing cell growth and production of extracellular polysaccharides by CaCl ₂ of the genus 00SS11568 strain.

FIG. 13 is a graph showing cell growth and production of extracellular polysaccharides by trace elements of the genus 00SS11568 strain.

14A is a graph showing the carbon source of the genus 00SS11568 strain, and FIG. 14B is a graph showing cell growth and production of extracellular polysaccharide by glucose.

Figure 15 is a graph showing the viscosity, pH, cell growth and production of extracellular polysaccharides of the genus 00SS11568 strain with M-11568 medium using M-11568 medium in a 5L incubator.

16A-16C are scanning micrographs of p-11568 (FIG. 16A: 1% p-11568, FIG. 16B: 0.1% p-11568, FIG. 16C: 0.05% p-11568).

Figure 17 shows monosaccharide composition by thin layer chromatography of p-11568 (1: xylose, 2: glucose, 3: p-11568, 4: galactose, 5: glucosamine).

18 is a graph showing the molecular weight of p-11568.

Figure 19a is an analysis graph by the calorimeter of p-11568, Figure 19b is an analysis graph by the differential scanning calorimeter.

20 is a graph showing the change of shear stress with respect to the shear rate of p-11568 1% aqueous solution.

21 is a graph showing the change in shear stress with respect to the shear rate according to the concentration of p-11568.

22 is a graph showing the change in shear stress with respect to the shear rate with the temperature of p-11568 1% aqueous solution.

FIG. 23 is a graph showing the change of shear stress with respect to the shear rate according to the pH of p-11568 1% aqueous solution.

24 is a graph showing the change in shear stress with respect to the shear rate according to the NaCl concentration of p-11568 1% aqueous solution.

25 is a graph showing the change in shear stress with respect to the shear rate according to the CaCl ₂ concentration of 1% aqueous solution p-11568.

FIG. 26 is a graph showing the change of shear stress with respect to the shear rate according to the heat treatment of p-11568 1% aqueous solution.

FIG. 27 is a solidification photograph after 121 ° C. treatment of 1% aqueous solution of p-11568. FIG.

FIG. 28 is a graph showing shear stress versus shear rate with xanthan gum and gellan gum in a 1% aqueous solution of p-11568.

FIG. 29A is a graph showing intrinsic viscosity by Huggins equation using Cannon-Fenske capillary viscometer of p-11568, and FIG. 29B is a graph showing intrinsic viscosity by Karemer equation.

The present invention relates to a strain of genus Alteromonas 00SS11568 (KCTC10324BP) that secretes extracellular polysaccharides and a method for producing extracellular polysaccharides therefrom.

The extracellular polysaccharide produced by the present invention is a heteropolysaccharide having a glucose and galactose ratio of 3: 1, and has a molecular weight of 4.4 × 10 ⁵ Da, is dissolved in an aqueous solution, and has excellent emulsion stability and can be used in various industrial fields. .

In the present invention, a sample was taken after visually observing a biofilm formed in variously distributed facilities in the coastal area centering on the coastal inhabited area around the four major rivers where the inflow of living sewage and other pollutants in the south coastal coastal area is high.

After extracting 00SS11568 genus Alteromonas, a strain that secretes extracellular polysaccharides from the collected samples, extracellular polysaccharides were produced therefrom.

The strain was deposited with the Gene Bank in Korea Research Institute of Biotechnology on August 7, 2002, and was given accession number KCTC10324BP.

Looking at the method for producing extracellular polysaccharide according to the present invention.

First, an optimal medium for producing extracellular polysaccharides is prepared.

The composition of this optimum medium consists of inorganic nitrogen, organic nitrogen, inorganic phosphoric acid, MgSO ₄ , CaCl ₂ , trace elements and carbon sources.

As inorganic nitrogen, KNO ₃ , NH ₄ H ₂ PO ₄ , (NH ₄ ) ₂ SO ₄ , NH ₄ Cl, NH ₄ NO _3, etc. may be used, and among them, (NH ₄ ) ₂ SO ₄ is most preferably used. Do.

As organic nitrogen, yeast extract, yeast extract, pepton, tryptone, malt extract, malt extract, beef extract, urea, etc. can be used in 0.5% by weight. Preference is given to using extracts and yeast extracts.

As inorganic phosphoric acid, KH ₂ PO ₄ or K ₂ HPO ₄ may be used in an amount of 0.1 wt% based on the total weight of the medium composition.

In addition, it is preferable to use MgSO ₄ as 0.4% by weight and CaCl ₂ as 0.07% by weight based on the total weight of the medium composition, and the trace elements are FeSO ₄ , CuSO ₄ , MnSO ₄ , ZnSO ₄ , CoCl ₂ · 6H ₂ O And ZnSO ₄ are most preferred.

As the carbon source, glucose is used at 7% by weight based on the total weight of the medium composition.

Prepare the medium consisting of the above composition at 25 ℃ initial pH 9, the medium amount is 25ml, the inoculation amount of the strain is incubated at 2.5% by weight of the total weight incubation.

The extracellular polysaccharides secreted from the genus 00SS11568 strain according to the present invention incubated as described above was named "p-11568".

The present invention will be described in detail with reference to the following Examples. However, these examples are only for illustrating the present invention in more detail, the present invention is not limited to these examples.

Example 1 Pure Separation of Strains

Samples were taken after visual observation of the biofilms formed in the facilities distributed in the coastal areas, centering on the coastal inhabited areas around the Four Rivers where the inflow of sewage and other pollutants in the coastal areas of the south coast is high. The sample collected was suspended in 1 ml of ZoBellb (Table 1) liquid medium and plated again on a ZoBell solid medium, incubated at 25 ° C. for 7 days, and the strain having viscous was first purified. Thereafter, the isolated strains were inoculated again with 1 platinum each in SZoBell medium, and cultured at 25 ° C. and 120 rpm for 7 days, and the supernatant was separated by centrifugation of the culture solution. Then, extracellular polysaccharides were precipitated by adding twice the amount of ethanol to the supernatant, recovered, dried, and weighed to finally select 00SS11568, a strain having a high recovery rate of extracellular polysaccharides. .

Ingredient ZoBell badge SZoBell Badge Glucose - 20g / ℓ peptone 5 g / ℓ 5 g / ℓ Yeast extract 1g / ℓ 1g / ℓ FeCl ₄ 0.01 g / l 0.01 g / l Salt water 750 ml 750 ml Distilled water 250 ml 250 ml pH 7.2 7.2

Test Example 1 Identification of Production Strain

① Morphological characteristics

Morphological characteristics of the genus 00SS11568 strain are shown in Figure 1 and Table 2. 00SS11568 strains are Gram-positive, bacilli, with a size of 0.5-1㎛ and lacking motility. The color of colonies changed from light yellow to light brown depending on the number of days of culture. The shape changed to distributed. In particular, the viscosity was very high on agar medium (Table 2).

Chacteristics 00SS11568 Relation to oxygen Aerobic Morphology Rods Growth at 45 ℃ - Oxidase + Gram stain + Size 0.5-1.0㎛ Oxidase + Catalase +

② Substrate utilization

The carbohydrate availability of the 00SS11568 strain was examined for 95 substrates using the BIOLOG GN Microplate method, and the results are shown in Table 3. The selected strain was incubated at 25 ° C. for 2 days using TSA (Trypticase soy agar, Difco) solid medium containing seawater, and the grown cells were sterilized in suspension (MgCl ₂ ㆍ 6H ₂ O 5g / ℓ, CaCl ₂ 2H ₂ O 1g / L, NaCl 25g / L), the turbidity of the suspension solution was adjusted to 0.2 by OD ₆₆₀ using a BIOLOG spectrophotometer, the wells of BIOLOG GN and GP microplates ) Were inoculated with 150 μl bacterial suspension. Inoculated microplates were incubated at 30 ° C. for 24-48 hours and measured at 690 nm using a BIOLOG microstation plate reader. In the case of saccharides, in the case of sugars, dextrin, adonitol, cellobios, fructose, galactose, maltose, glucose, mannitol and mannose (mannose) and sucrose were used as substrates, of which the substrate use specificity for maltose was high.

In addition, arabinose, glycerol, inositol, lactose, melezitose, psicose and turanose were not used as substrates.

Organic acids include methyl pyruvate, L-lactic acid, succinic acid, L-aspartic acid, and glycyl-L-glutamic acid. acid) and alanineamide, L-alanine, L-alanylglycine, and L-asparagine as amino acids. The availability of the nucleic acid precursors, inosine and uridine, was observed.

Character 00SS11568 Cellobios + D-galactose + D-glucose + myo -inositol - maltose ++ Mannitol + Sucrose + dextrin + Adonitol + (-) Arabinose - Fructose + Glycerol - Lactose - Meletchiose - Mannose + Psychoos - Turanose -

③ 16S rDNA analysis

16S rDNA gene of the 00SS011568 strain was amplified to determine the base sequence, and the results of molecular analysis were shown in SEQ ID NO: 1 and FIG. Strains incubated in ZoBell solid medium were inoculated in 2 ml ZoBell liquid medium, incubated at 25 ° C for 24 hours, and then centrifuged (12,000 xg, 15 min. 4 ° C) to recover the cells. Wizard Genomic DNA was recovered from the recovered cells. Genomic DNA was extracted using Purification kit (Promega). 16S rDNA was amplified by PCR using Genomic DNA as a template, and primer pairs used were 27F and 1522R (Table 4). PCR conditions were 0.2 μM primer, 1.5 mM MgCl ₂ , 0.2 mM dNTPs, 0.1% Boehringer Mannheim (BSA), 10 ng DNA template, and 2.5 U Taq DNA polymerase (Promega) were added. The volume was 50 μl dose. Reaction conditions were treated for 5 minutes at 94 ° C, denatureation for 1 minute at 94 ° C, annealing for 1 minute at 55 ° C, and extension for 2 minutes at 72 ° C for 35 minutes. After the final expansion was carried out for 7 minutes. PCR products were agarose gel (agarose gel) electrophoresis confirmed the expected size (1.5 kbp) and purified using the Wizard PCR Preps DNA Purification kit (Promega). 16S rDNA sequences were determined using ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystem) and automated sequencing device (Model 377, Applied Biosystem, USA) directly from PCR products. Primers for sequence determination were used 27F, 518R, 357F, 802F, 1241F, 1522R (Table 4). The determined 16S rDNA sequence is about 1,500 bp in length and contains a portion of the 16S rDNA gene. 16S rDNA sequencing was estimated using the BLAST search of the GeneBank database to estimate the approximate taxonomic position of each strain and to obtain the respective base sequence data to be compared. Alignment was performed using the SeqAid program against the prearranged sequences of the systematically closest group of strains isolated from RDP. Alignment was performed for systematic analysis, and PHYLIP (version 4.57c) was used for systematic analysis. As a result of phylogeny analysis, 00SS11568 was relatively new to other strains belonging to the genus Alteromonas.

primer Specificity Sequence (5'-3 ') part Reference 27F BACTERIA 5'-AGT GTT TGA TCM TGG CTC AG-3 ' 8-27 Giovanoi, 1991 357 F BACTERIA 5'-GCA TCC TAC GGG AGG CWG CAG-3 ' 337-357 Amann et al., 1990 803F BACTERIA 5'-GGA TTA GAT ACC CTG GTA-3 ' 785-802 Lee et al., 1993 1241F BACTERIA 5'-ACA CAC GTG MTA CAA TGG-3 ' 785-802 KAto et al., 1997 518R UNIVERSAL 5'-GTA TTA CCG CGG CTG CTG-3 ' 534-518 KAto et al., 1997 1522R BACTERIA 5'-AAG GAG GTG ATC ACN CCR CA-3 ' 1541-1522 Giovanni, 1991

F: Forword, R: Reverse, site is as E. coli numbering,

M = C: A, W = A: T, R = A: G, N = A: C: G: T

④ Microstructure observation by electron microscope

After culturing 00SS11568 in ZoBell liquid medium for 12 hours, washed three times in 0.05M sodium cacodylate buffer (pH 7.2) and centrifuged (5000 xg, 2 min) to recover the cells and the primary fixation of modified Kamovsky After fixing for 2-4 hours at 4 ° C. in Modified Kamovsky's fixative solution, washing with 0.05 M sodium cacodylate buffer three times at 4 ° C. for 10 minutes, and the second fixation was 1% tetracycline. Osmium (Osminum tetroxide) was fixed at 4 ℃ for 2 hours. After the second fixation, the sample was washed three times again with distilled water, dehydrated with ethanol series (30, 50, 70, 80, 90, 100, 100, 100%) for 10 minutes, and then twice with 15 minutes with HMTD. Treatment was followed by lyophilization. Thereafter, gold coating was performed using an ion sputter and the microstructure was observed at 7,500 magnification using a scanning electron microscope. As a result, the morphological characteristics of 00SS11568 was found to be 0.5-1 μm in size bacilli (FIG. 2).

As a result of identifying 00SS11568 strain having excellent production of extracellular polysaccharide, the strain was found to be a genus of marine organisms, Alteromonas sp . Accordingly, the present inventors named this strain 00SS11568 in the genus Alteromonas, and deposited the gene bank in Korea Research Institute of Biotechnology on August 7, 2002, and received accession number KCTC10324BP.

Example 2 Production of Extracellular Polysaccharides

① Composition of optimal medium for producing extracellular polysaccharides

The composition of the optimal production medium for producing extracellular polysaccharides using the genus Alteromonas 00SS11568 was investigated by the following method.

Ⓐ Pre-culture

Plated in ZoBell solid medium and incubated for 3 days at 25 ℃. The grown colonies were inoculated in a 30 ml ZoBell liquid medium using a loop and incubated at 25 ° C. and 150 rpm for 48 hours.

Ⓑ bacterial growth measurement

Absorbance (A ₆₆₀ ) was measured using a spectrophotometer (UV-VIS 2401PC, Japan).

Ⓒ extracellular polysaccharide measurement

The culture solution was centrifuged (8,000x g , 15 minutes) to remove the cells, and two times of ethanol was added to the culture medium from which the cells were removed to precipitate polysaccharides, followed by centrifugation (3,000 x g , 15 minutes). Polysaccharides were recovered. The recovered polysaccharides were dried under reduced pressure, and then dried to measure extracellular polysaccharides.

Ⓓ investigation of physical factors

In order to analyze the physical factors required for the production of extracellular polysaccharides, the results of the investigation on temperature, initial pH, medium volume and inoculation amount are shown in Table 5.

Physical factors Condition Extracellular Polysaccharides (mg / l) Temperature 25 ℃ 3.14 g / ℓ Initial pH pH 9 3.61 g / ℓ Discharge 25 ml 5.3 g / l Inoculation amount 2.5% 4.57g / ℓ

㉮ optimum temperature

30 ml of SZoBell medium was added to a 250 ml Erlenmeyer flask, inoculated with 1 wt% of the 00SS11568 strain isolated in Example 1, followed by shaking culture at 120 rpm in an incubator controlled at 10, 25, 30, and 35 ° C., respectively. 3 shows the results of cell growth and the production of extracellular polysaccharides. The production of p-11568 by temperature was 3.14 g / L at 25 ° C, and the production of p-11568 was the best, and the cell growth was 0.189 (A ₆₆₀ ) at 30 ° C. Therefore, 25 ℃ was the culture temperature for the production of p-11568. This is in general agreement with the characteristics of mesophilic strains. As a result, the optimum temperature of microbial polysaccharide-producing microorganisms currently commercially produced is 28-30 ° C. (Mitsuda et al. , 1981; Iwamuro et. al. , 1981). In addition, the optimal culture temperature for the production of xanthan gum is the most important factor in the production of polysaccharides. The growth of the production strain was good at 25 ℃, but the rate of biosynthesis of xanthan gum was reported to be high at 30 ℃. (Shu et al. , 1990).

발 Initial pH

Into a 250 ml Erlenmeyer flask, 30 ml of SZoBell liquid medium was inoculated, inoculated with 1 wt% of the 00SS11568 strain isolated in Example 1, and then the pH was adjusted to 6, 7, 8, 9, 10 with 1N HCl, 1N NaOH, 4 shows the results of cell growth and the production of extracellular polysaccharides after shaking culture at 120 rpm and 25 ° C. The production of p-11568 according to the initial pH was 4.53g / l at pH 9 and the production of p-11568 was the best, and the cell growth was also 0.214 (A ₆₆₀ ) at pH 9, the highest growth of cells was observed in all treatments. It became. Therefore, pH 9 was investigated as the initial pH for p-11568 production. For most polysaccharides, the optimal pH varies with the microorganism, but is generally reported as a neutral pH (Anderson et al. 1972; Williams et al. , 1978), but for 00SS11568 the growth of microorganisms at pH 9 and p The production of -11568 is good, suggesting that polysaccharide production by pH is possible.

㉰ optimal volume

25, 50, 75, 100, and 150 ml of SZoBell liquid medium were put into a 250 ml Erlenmeyer flask, respectively, and inoculated with 1 wt% of the 00SS11568 strain isolated in Example 1, followed by shaking culture at 120 rpm and 25 ° C. 5 shows the results of the cell growth and the production of extracellular polysaccharides. The production of p-11568 was the best in the production of p-11568 from 25ml to 5.3g / l, and the cell growth was highest from 0.1ml (A ₆₆₀ ) to 100ml. . Therefore, 25 ml was determined as the optimal medium for p-11568 production.

㉱ Optimum dose

30 ml of SZoBell liquid medium was added to a 250 ml Erlenmeyer flask, and the 00SS11568 strain isolated in Example 1 was inoculated at 1.0, 2.5, 5.0, 7.5, and 10.0 wt%, respectively, and then shake-cultured at 120 rpm and 25 ° C. Figure 6 shows the results of the cell growth and the production of extracellular polysaccharides. The production of p-11568 according to the initial inoculation amount was 4.57 g / L at 1.0%, and the production of p-11568 was the best at the 1% initial inoculation amount. In the case of 1.0% inoculation, the cell growth was 0.1 (A ₆₆₀ ), and cell growth was lower than that of 5.0% inoculation, but the cell growth rate was high at 1.0% inoculation. However, in the case of 2.5% inoculation, the production and the growth of the p-11568 were 4.32g / L for the production of p-11568 and 1.81 (A ₆₆₀ ) for the growth of the cells. Therefore, 2.5% inoculum was selected as the initial inoculum for p-11568 production, as it was assumed that it would be easy to induce changes in production factors under the conditions of polysaccharide production and cell growth. . In the production of metabolites by microorganisms, the initial dose is an important factor in the productivity of the target product (Sarkar et al. , 1985).

Ⓔ inorganic nitrogen

To the 250 ml Erlenmeyer flask was added 0.5 g / l of inorganic nitrogen KNO ₃ , NH ₄ H ₂ PO ₄ , (NH ₄ ) ₂ SO ₄ , NH ₄ Cl, and NH ₄ NO ₃ , respectively, based on 25 ml of ZoBell medium. 00SS11568 cells isolated in Example 1 were inoculated to be 2.5% by weight, and cultured in a shaker (120 rpm, 30 ° C.), followed by cell growth and the production of extracellular polysaccharides. The production of p-11568 by the treatment of inorganic nitrogen source (NH ₄ ) ₂ SO ₄ was 6.59g / ℓ, which was better than the control 1.67g / ℓ, and the cell growth was 0.064 (A ₆₆₀ ) for (NH ₄ ) ₂ SO ₄ . It was lower than the control 0.078 (A ₆₆₀ ) and low cell growth was observed in the pretreatment. The pH of the pretreatment was 8.3, higher than the initial pH.

Ⓕ Organic Nitrogen

In a 250 ml Erlenmeyer flask, organic nitrogen, yeast extract, pepton, tryptone, malt extract, beef extract, and urea based on 25 ml of ZoBell medium Were added each 0.5% by weight, inoculated so that the 00SS11568 cells isolated in Example 1 to 2.5% by weight, shaking cultured at 120rpm, 25 ℃ condition and the results of the investigation of cell growth and the production of extracellular polysaccharides. 8 is shown. The production of p-11568 by organic nitrogen treatment was 3.31g / ℓ in yeast extract, which was the best than control 1.67g / ℓ, and the cell growth was 0.15 (A ₆₆₀ ) for 1.0% yeast extract than control 0.078 (A ₆₆₀ ). In particular, the highest cell growth rate was found to be 0.179 (A ₆₆₀ ) in the malt extract, and higher cell growth than the control group 0.078 (A ₆₆₀ ) was observed. Thus, 1% (NH ₄ ) ₂ SO ₄ increased the production of p-11568 at Y _{p / x} = 1.98 compared to the control. The pH of yeast extract was higher than the initial pH of 8.8 in all treatments. Therefore, the yeast extract was selected as an organic carbon source. Also, in order to determine the concentration of the yeast extract selected as the organic nitrogen source, the concentration of the yeast extract was adjusted to 0.1, 03, 0.5, 0.7, and 1.0%, and the inoculation was carried out so that the cells were 1.0%. After culturing in a shaker, cell growth, viscosity, and the production of extracellular polysaccharides are shown in FIG. 9. The production of p-11568 by organic nitrogen source treatment was 7.93 g / l at 0.5 wt%, better than that of control 1.87 g / l, and cell growth was 0.152 (A ₆₆₀ ) for 0.5% yeast extract than control 0.078 (A ₆₆₀ ). It was high and showed the highest cell growth rate among all treatments. Thus, 0.5% yeast extract was selected at the organic carbon source concentration. Iwamuro et al. (1983) reported that polysaccharide production was increased by the addition of organic nitrogen sources in each of the strains of Porodisculus pendulus , but polysaccharide production was no longer improved when yeast extracts of 4.0 g / l or more were added.

Ⓖ Inorganic Phosphate

Into a 250 ml Erlenmeyer flask, inorganic phosphoric acid KH ₂ PO ₄ and K ₂ HPO ₄ were added at a concentration of 0.05, 0.1, and 0.5 g / L, respectively, based on 30 ml of ZoBell medium, and 2.5 weights of 00SS11568 cells isolated in Example 1 were added. After inoculating to%, shaking cultured at 120 rpm and 25 ° C., the results of the cell growth and the production of the extracellular polysaccharide were shown in FIG. 10. Production of inorganic phosphate treatment distinct p-11568 is a KH ₂ PO ₄ was better than for the control 3.17g / ℓ at 0.1g / ℓ to 5.47g / ℓ, the cell growth was 0.1g / ℓ KH ₂ 0.152 (A in PO _{4 660} ), which was higher than the control group (0.07 (A ₆₆₀ )), showing the highest cell growth rate among all treatments. Therefore, 0.5% KH ₂ PO ₄ increased the production of p-11568 at Y _{x / p} = 1.73 compared to the control, pH was 5.0. Therefore, 0.1% KH ₂ PO ₄ was used as the inorganic phosphoric acid source concentration. Phosphoric acid plays an important role in RNA synthesis and cell wall formation during microbial growth. In the case of Azotobacter vinelandii , alginate production is affected by the initial concentration of inorganic phosphate (Horan et al. , 1981), and Xanthomonas sp. In this case, it is reported that by limiting the inorganic phosphate promotes the production of polysaccharides (Kennedy et al ., 1984).

Ⓗ MgSO ₄

MgSO ₄ was added at a concentration of 1, 2, 4, and 6 g / L, respectively, based on 25 ml of ZoBell medium in a 250 ml Erlenmeyer flask, and the 00SS11568 cells isolated in Example 1 were inoculated to be 2.5 wt%, 120 rpm, 25 11 shows the results of the cell growth and the production of the extracellular polysaccharide after shaking culture under the condition of ℃. The production of p-11568 by MgSO ₄ treatment was the best at 0.435 to 4.35 g / l, and the highest cell growth was observed at 0.33 (A ₆₆₀ ). Thus, 0.4% MgSO ₄ was selected as the Mg ²⁺ source. Mg ²⁺ regulates the enzymatic activity during the synthesis of extracellular polysaccharides and sulfates, increases the permeability of enzymes, reduces the sulfur-containing essential components of cells, and cofactors of various enzymes ( Cofactors have been reported (Kang et al. , 1979).

CaCl ₂

CaCl ₂ was added at a concentration of 0.1, 0.3, 0.5, 0.7, 1.0 and 2.0 g / L, respectively, based on 25 ml of ZoBell medium in a 250 ml Erlenmeyer flask, and the inoculated so as to obtain 1.0 wt% of the 00SS11568 cells isolated in Example 1 The results of the cell growth and the production of the extracellular polysaccharide after shaking culture at 120 rpm and 25 ° C. are shown in FIG. 12. The production of p-11568 by CaCl ₂ treatment was the best at 0.07% 3.9g / l, and the cell growth was 0.112 (A ₆₆₀ ). Therefore, 0.07% CaCl ₂ was selected as the Ca ²⁺ source.

Ⓙ Trace element

To the 250 ml Erlenmeyer flask, FeSO ₄ , CuSO ₄ , MnSO ₄ , ZnSO ₄ , and CoCl ₂ · 6H ₂ O were added at a concentration of 0.001 g / L, respectively, based on 25 ml of ZoBell medium, and the 00SS11568 cells isolated in Example 1 were added. Inoculated to 1.0% by weight, after shaking culture under the conditions of 120rpm, 25 ℃ the cell growth and the production of extracellular polysaccharides are shown in Figure 13 the results. P-11568 for each trace element was 1.6g / l FeSO ₄ , 2.17g / l CuSO ₄ , 1.82g / l MnSO ₄ , 3.12g / l ZnSO ₄ , 1.72g / l CoCl ₂ · 6H ₂ O ZnSO ₄ treatment was better than 1.67g / ℓ in ZnSO ₄ treatment, ZnSO ₄ showed the highest value of 0.439 (A ₆₆₀ ). However, ZnSO ₄ increased to Y _{p / x} = 1.85 in p-11568 production. The addition of FeSO ₄ showed better cell growth than the control, but the production of polysaccharides was similar to that of the control, and the addition of KCl and MnSO ₄ were similar to the control, growth and polysaccharide production. On the other hand, when ZnSO ₄ was added, polysaccharide production and cell growth were the best.

Ⓚ carbon source

To a 250 ml Erlenmeyer flask, xylose, fructose, glucose, galactose, mannose, sucrose and starch, each of carbon sources, based on 25 ml of ZoBell medium, were added at a concentration of 2.5% by weight (w / v), respectively. Inoculated so that the 00SS11568 cells isolated in Example 1 to 1.0% by weight, after shaking culture under conditions of 120rpm, 25 ℃ the cell growth and the production of extracellular polysaccharides are shown in Figure 14 the results. The production of p-11568 by carbon source treatment was 1.28 g / l xylose, 2.28 g / l fructose, 3.36 g / l glucose, 0.07 g / l galactose, 1.03 g / l mannose, 2.99 g / l starch, starch 4.91 g / l was better than the control 1.67 g / l and the cell growth was shown as glucose 0.63 (A ₆₆₀ ). Thus, glucose was selected as the carbon source. For starch, p-11568 production and cell proliferation were the highest, but this was excluded because it was due to out-of-medium elution of starch, not cell proliferation or polysaccharide production. The growth of cells was good in fructose, glucose, sucrose and starch, and the production of polysaccharides is thought to be produced in proportion to the cell weight. .

In order to investigate the concentration of glucose suitable for the production of extracellular polysaccharides, fructose was added at a concentration of 1, 3, 5, 7, 10% by weight (w / v), respectively, and 2.5 weights of 00SS11568 cells isolated in Example 1 were obtained. Inoculation was carried out to%, shake cultured at 120rpm, 25 ℃ condition and the results of the cell growth and the production of extracellular polysaccharides are shown in Figure 14a and 14b. The production of p-11568 by fructose treatment was the best at 5.33 g / l at 7%. There are early studies using lactose in the production of xanthan gum and galactoglucan, the most commonly used polysaccharides of glucose and sucrose as carbon sources for the production of polysaccharides, and others (Lee et al. , 1991). It is also possible to find an example in which soluble starch is used as a carbon source. Mitsuda et al. (1981) reported that glucose is the most effective carbon source for Bacillus strains. Souw et al. (1979) reported that the yield of xanthan gum is high when the concentration of sucrose and glucose is 40 g / l.In general, the optimal concentration of carbon source for the production of microbial polysaccharides is reported to be about 10-80 g / l depending on the strain used. (Williams et al. , 1978).

Ⓛ p-11568 Production Optimum

Optimum conditions for the production of p-11568 were 5% by weight glucose, 0.5% by weight yeast extract, 0.5 g / L (NH ₄ ) ₂ SO ₄ , 0.1 g / L KH ₂ PO ₄ , ₄ g / L MgSO ₄ , CaCl ₂ 0.7 g / l, FeSO ₄ 0.001g / l, CuSO ₄ 0.001g / l, MnSO ₄ 0.001g / l, ZnSO ₄ 0.001g / l, CoCl ₂ .6H ₂ O 0.001g / l and seawater The medium composition prepared so that the inoculation amount of OOSS11568 strain of genus Alteromonas at a pH of 9 and a temperature of 25 ° C. was 2.5% by weight was named “M-11568” in the present invention, and M-11568 was designated as p- It was the optimal medium composition to produce 11568 (Table 6).

Furtherance density Glucose 5% Yeast extract 0.5% (NH4) ₂ SO ₄ 5 g KH ₂ PO ₄ 0.1 g MgSO ₄ 4 g CaCl ₂ 0.7 g ZnSO ₄ 0.001 g Salt water 1,000 ml pH 9.0

Hourly polysaccharide production under optimal conditions of ⓜ p-11568

In order to investigate the productivity of M-11568 for the production of p-11568, 30 ml of M-11568 was placed in a 250 ml Erlenmeyer flask, and inoculated to 2.5 wt% of 00SS11568 cells of the genus Alteromonas isolated in Example 1. After incubation in a shaker (120 rpm, 25 ℃), cell growth, viscosity and the production of extracellular polysaccharides were investigated. Cell proliferation peaked at 1.89 (A ₆₆₀ ) at 168 hours after incubation and then decreased, and production of p-11568 was investigated at 4.65 g / l after 48 hours incubation.

② Production of polysaccharides according to incubation time in 5 ℓ batch incubator

Strains were incubated for 24 hours in M-11568 medium at 1% in 3 L working volume using a 5 L batch incubator to incubate the strains. At this time, the culture temperature was 25 ℃, the aeration amount was set to 1.5vvm at 200rpm. The result of p-11568 production over time is shown in FIG. 15. Polysaccharide production increased with incubation time, and peaked after 72 hours of culture. Under optimal conditions, the maximum polysaccharide yield of approximately 19.2 g / l was obtained, and cell growth increased up to 72 hours, but cell growth decreased after 72 hours. This phenomenon is considered to be because oxygen is not smoothly transferred by maintaining the high viscosity of the culture medium itself due to the increased viscosity of the polysaccharide secreted into the culture medium.

Test Example 2 Characteristics of Extracellular Polysaccharides

In order to investigate the characteristics of the extracellular polysaccharides produced by the 00SS11568 strain isolated in Example 1, after separating and purifying the extracellular polysaccharides, p-11568 produced from the culture medium, molecular weight, monosaccharide composition, microstructure, protein content, elements Analysis, thermal resolution, solubility, emulsion stability and physical properties were investigated.

① Separation and Purification

Separation of p-11568 was performed by diluting 00SS11568 culture medium with distilled water twice, adding NaOH to a final concentration of 0.1 N, stirring for 40 minutes with Rotary shacker (S-20, HANA, Korea), and then centrifugal separator (Sorvall, USA). ) Centrifuged at 12,000 × g for 30 minutes to remove the cells first, and then the supernatant was 100% cold, which is twice as much as the culture medium from which the cells were removed at 12,000 × g using a continuous centrifuge (Sorvall, USA). Ethanol (cold ethanol) was added and cold-washed at 4 ° C for 24 hours to precipitate extracellular polysaccharides. Precipitated polysaccharides were washed twice with 70% cold ethanol, dissolved in distilled water and neutralized with 0.1 N HCl, and 3 times distilled water of 3 times the standard amount was continuously added at 4 ° C. to give VIVA Flower (VF20P4, Satorius, USA), and then 2 times 100% cold ethanol was added thereto, followed by cooling at 4 ° C. for 24 hours to precipitate extracellular polysaccharides to recover polysaccharides. After completion of the first dialysis, extracellular polysaccharides were lyophilized and separated into crude polysaccharides. The extracellular polysaccharides of the selected strains were used as the extracellular polysaccharides. In addition, the purified sample for the component and structural analysis of the extracellular polysaccharide for the analysis of p-11568 purified the crude polysaccharide previously isolated. 1 g of copolysaccharide was dissolved in distilled water, and 0.3% of CPC (cetyl pyridinium chloride) was added thereto, followed by slow mixing for 30 minutes with a shaker, followed by precipitation. Thereafter, the precipitated mixture was again centrifuged at 9,000 x g for 20 minutes using a centrifuge (Sorvall, USA) to remove the supernatant and the precipitate was dissolved in 10% NaCl again. This was further added to 2 times 100% cold ethanol, cooled at 4 ° C. for 24 hours, precipitated polysaccharides to remove ethanol, centrifuged to recover extracellular polysaccharides, and then dissolved in distilled water and dissolved in dialysis membrane (D0655, Sigma, USA). Dialysis was carried out in running water for 2 days, and then dialyzed with 3 distilled water at 4 ° C. for 1 day and lyophilized. Thereafter, the purified polysaccharide was purified through gel permeation chromatography. Sepadex G-200 was charged to a column (1.5 × 75 cm) and the sample was dissolved in 0.4M NaCl buffer and injected into the column. The same buffer was used to elute at a flow rate of 0.5 ml / min. Fraction collectors (Erla, Japan) were fractionated 2mL each and used for analysis. Table 7 shows the results for the content of p-11568 by separation and purification of p-1568. The yield of final p-11568 by gel filtration was 55.08%.

Purification steps Extracellular Polysaccharides (g / ℓ) yield(%) Crude extracellular polysaccharide 3 100 CPC sedimentation 2.4356 81.19 Ethanol precipitation 1.8368 61.23 Gel filtration 1.6526 55.08

② microstructure of p-11568

Scanning electron microscopy (JSM-5410LV, JEOL, Tokyo, Japan) was used to observe the morphological characteristics of p-11568. After dissolving p-11568 in distilled water to 0.1%, lyophilized, and gold-coated using an ion deposition machine, the microstructure was observed at 15,000 magnification using a scanning electron microscope, and the results are shown in FIGS. 16A and 16B. . The microstructure of p-11568 was examined to be fibrous.

③ Monosaccharide composition

As a result of acid hydrolysis of 50 mg of purified polysaccharide to investigate p-11568 constituent sugar, TLC showed that the main components of reducing sugar constituting p-11568 were glucose and galactose, as shown in FIG. As a result of analysis by high performance liquid chromatography, the composition ratio was about 3: 1, and glucose was relatively high.

④ Molecular weight measurement of p-11568

Elution of textane (molecular weight 2 mDa, 500 kDa, 70 kDa: Sigma, USA) and p-KG03 as standard materials by gel filtration column chromatography using Sephadex G-200 The relationship between the time ratio and the log-scale molecular weight is shown in FIG. 18 when the molecular weight is determined by a standard curve. The molecular weight of p-11568 was found to be 4.4 × 10 ⁵ Da.

⑤ Protein quantification of p-11568

The protein content of p-11568 was quantified by measuring the absorbance at 750 nm using a protein assay kit (Sigma. USA) according to the Lowry method (1951), and BSA (Bovine Serum Albumin) was used as a standard. Used as. The protein content in 1% aqueous solution was found to be 2.27%.

⑥ Elemental Analysis of p-11568

Elemental analysis was carried out using Perkin-Elmer (240C) to investigate the elemental components contained in p-11568. Also, the acidic hydrolysis of solid ash with 1.0N HCl was carried out using atomic absorption spectrometer (Varian, Spectra AA- 30, USA) to investigate the metal ions present in the polysaccharides, the results are shown in Table 8. As shown in Table 8 below, p-11568 according to the present invention was composed of 32.8% C, 4.84% H, and 0.72% N, and was found to contain 1.2% S.

Component Volume(%) carbon 32.82 Hydrogen 4.84 nitrogen 0.72 mass 1.872

⑦ Thermal resolution of p-11568

When a substance changes from crystal to other form, it causes a physical change, which is expressed as a change in heat. To investigate the pyrolysis characteristics of p-11568 In order to investigate the pyrolysis characteristics of the purified polysaccharide (Thermal Analyzer, TGA 1000, Reomateric Scientific. UK), 2 mg of p-11568 was added and the temperature was increased to 20 ° C per minute. The result of measuring the micromass change of the polysaccharide by heating it up to 900 degreeC is shown in FIG. 19, The pyrolysis start temperature was 253 degreeC, and the pyrolysis temperature was 733 degreeC. In the case of differential scanning calorimeter, 25 mg of p-11568 was added after adding 5.0 mg of p-11568, and the sample was not added and the sample was added with DSC-4 (Perkin-Elmer, USA). The melting point of the polysaccharide and the endothermic heat amount of the polysaccharide were examined in Figs. 19A and 19B. The thermal decomposition initiation temperature of p-11568 was 273 ° C.

⑧ Solubility of p-11568

p-11568 is a creamy fibrous substance, and the results of the solubility in the solvent are shown in Table 9. Formaldehyde, 10N NaOH, D.W. The solubility is excellent in the back, but precipitates are formed insoluble in organic solvents such as benzene, acetone, DMSO, chloroform, ether, ethanol and methanol.

solvent characteristic ++ + - Formaldehyde ● Formic acid ● benzene ● Acetone ● DMSO ● 10N NaOH ● 5N HCl ● chloroform ● ether ● ethanol ● Methanol ● D.W. ●

++: very soluble, +: soluble,-: insoluble

유화 p-11568's emulsification stability

The results of examining the emulsion stability of p-11568 are shown in Table 10. 2 ml of corn oil was added to 3 ml of a 1% aqueous solution, followed by mixing to prepare an oil-in-water (O / W) emulsion. Xanthan gum is stable until 144 hours when compared to xanthan gum, gellan gum, sodium alginate, arabic gum and polysaccharide p-11568, which are commonly used as emulsifiers. P-11568 also remained in suspension for up to 96 hours, after which the liquid layer was separated. Gellan gum was suspended for up to 12 hours, and then the liquid layer was separated. Sodium alginate and gum arabic did not occur. Therefore, it is thought that polysaccharide p-11568 can be used as an emulsion stabilizer.

Polysaccharides Oil painting stability 12 72 96 144 168 240 p-11568 ++ ++ + - - - Xanthan Gum ++ ++ + + - - Gellan gum + - - - - - Sodium alginate - - - - - - Arabian sword - - - - - -

++: completely emulsified, +: Partially emulsified,

-: separated.

Test Example 3 Physical Properties of Extracellular Polysaccharides (p-11568)

① Property coefficient

p-11568 is a non-Newtonian fluid and has been investigated as having a pseudoplastic by the power-low model. p-11568 has a consistency index (K) of 4% and a flow index of 1-11%. behavior index (η) was 0.42 (FIG. 20).

② Property change by concentration

In the polysaccharide solution, the shape of the curve showing the change in viscosity with respect to the change in shear rate shows a logarithmic decrease, but the shape of the curve showing shear stress increases with increasing shear rate. Increasing curves show different shapes. P-11568 is a solution of 0.1, 0.25, 0.5, 0.75 and 1.0% (w / v) concentration to give a change in concentration at 25 ℃ to measure the shear stress according to the shear rate is shown in Figure 21. As the shear rate increases, the shear stress increases, and as the polysaccharide concentration increases, the shear stress increases proportionally. These results show that the high viscosity requires a lot of force to cut the polysaccharide itself. This feature is typically seen when the hyperbolic polysaccharide solution is a pseudoplastic plastic non-Newtonian solution. It is assumed that p-11568 is a water soluble gum having the pseudoplastic plastic non-Newtonian solution.

③ Property change by temperature

The physical properties of the polysaccharide were observed by gradually increasing the temperature to 10, 25, 40, and 60 ° C. using a 1% polysaccharide aqueous solution of p-11568, and then cooled to 25 ° C. to examine the physical properties thereof. . As the temperature of p-11568 increases, the shear stress with respect to the shear rate decreases, which is thought to be the result of decreasing the viscosity of polysaccharides with increasing temperature. However, when the temperature was lowered to 25 ° C., the shear stress against the shear rate increased again. From these properties, the polysaccharide of p-11568 seems to have the characteristics of a reversible fluid.

④ Property change by pH

As a result of investigating the change in the physical properties of the polysaccharide by the control of each pH of p-11568 1.0% aqueous solution from pH 2 to pH 12 as shown in FIG. The shear stress was slightly decreased near pH 2, but the shear stress at pH 12, a strong alkali, was higher than the shear stress at pH 7. It seems to have relatively stable physical properties over the entire range. In general, most polysaccharide solutions produced by microorganisms have been reported to change their physical properties depending on pH (Heyn et al. , 1974; Tako, et al. , 1977). Their viscosity also changed as the pH changed based on the pH region showing the highest viscosity. The polysaccharides of p-11568 can also be expected to develop new applications because they are used in food and other industrial applications because of the properties of polysaccharides that maintain stable properties over a wide range of pH.

⑥ Changes in physical properties due to salts (NaCl, CaCl ₂ )

The results of comparing the change of shear stress by treating each salt in a concentration of 0.25, 0.5, 1.0, and 2.0% in a 1% solution of p-11568 polysaccharide are shown in FIGS. 24 and 25. In the case of NaCl, the shear stress was increased by increasing the concentration from 0.25% to 1.0%, and the shear stress was decreased by increasing the NaCl concentration in the NaCl added from 2.0% to 5.0%. In the case of CaCl ₂ , shear response increased with respect to the pretreatment concentration. In addition, the shear stress of CaCl ₂ treatment group was higher than that of NaCl, which is a monovalent cation. Therefore, Tako showed a synergistic effect due to the higher shear stress upon the addition of monovalent cations. (1989).

⑦ Effect of heat treatment

In the physical properties of p-11568, the shear stress decreased with increasing temperature, but after cooling, the characteristics of reversible fluid increased. In addition, p-11568 was treated at 60 ° C., 80 ° C., 100 ° C., and 121 ° C., respectively, and then cooled to 25 ° C. to examine the properties of the changed properties after high temperature treatment. . After the high temperature of 80 ℃ or more and cooled to 25 ℃ showed almost the same value as the shear stress of 25 ℃ showing the characteristics of excellent reversible fluid (reversible fluid). p-11568 could not present the data due to the gelation phenomenon in 121 ℃ treatment, which is a very unique physical properties, it is determined that it will be an important feature in the application development and application in various industries (Fig. 27).

⑧ Change of physical properties by mixing with other polysaccharides

The properties of p-11568 were mixed with 0.5% gellan gum, xanthan gum and the same amount of p-11568. Comparing the intrinsic properties of 1% p-11568 with 1% xanthan gum and 1% gellan gum aqueous solution is shown in FIG. 28. As the shear rate of all three polysaccharides increased, the shear stress increased, and all of the three polysaccharides were water-soluble gums that were characterized by pseudoplastic non-Newtonian solutions. In particular, p-11568 had higher shear stress according to shear rate than gellan gum. As a result of comparing the three polysaccharide viscosities according to the shear rate, the viscosity of p-11568 was higher than that of gellan gum according to the shear rate. It seems to be.

⑨ Intrinsic viscosity of p-11568

In order to observe the properties of the conformation of the biopolymer chain, the intrinsic viscosity of the dilute solution [η] was measured using the Cannon-Fenske capillary viscometer using the Huggins equation and the Karemer equation. Shown in 29b. The intrinsic viscosity by the Huggins equation of p-11568 was 16.86 dℓ / g and the intrinsic viscosity by the Karemer equation was 16.89 dℓ / g.

Concentration (g / dℓ) η (sec) η _sp (-) η _red (dℓ / g) η _r (-) η _inh (dℓ / g) 0.2 45.77 0.372 18.588 1.372 15.805 0.3 53.85 0.314 20.468 1.614 15.958 0.4 62.25 0.866 21.648 1.866 15.594 0.5 77.54 1.324 16.48 2.324 16.866 One 126.08 1.779 27.789 3.779 13.294 2 321.37 8.641 43.207 9.641 11.33

As described above, 00SS11568 (KCTC10324BP) of the genus Alteromonas of the present invention is a marine microorganism that secretes viscous polysaccharides and can use p-11568, an extracellular polysaccharide secreted by them, as a novel functional biopolymer, and has emulsion stability. It was excellent in that it could be used as a biological material for industrial material production.

<Reference>

Jang Jae-hyuk, Bae Seung-kwon, Kim Bong-jo, Ha Soon-deok, Gong Jae-yeol. 1998. Effect of Fermentation Conditions on the Production of Useful Polysaccharides from Marine Bacterium Zoogoloea sp. Korean Journal of Biotechnology and Bioengineering, 13: 303-307.

2. Anderson, N., and FW Doane. 1972. Agar diffusion method for negative staining of microbial suspensions in salt solutions. Appl. Microbiol . 24: 495-496

Fu, JF, and YH Teeng. 1990, Construction of the Jactose utilizing Xanthomonas campestris and production of xanthan gum from whey. Appl. Environ. 56: 919-923.

Heyn, ANJ 1974.The infrared absorption spectrum of dextran and its bound water., Biopolymer, 13: 475-506.

5. Irene, BM, PE Jansson, and B. Lindberg. Structural studied of the capsular polysaccharides from Streptococus pneumoniae type 7A. Carbohydrate Reserch, 198: 67-77.

6.Iwamuro, Y., M. Aoki, K. Mashiko and Y. Mikami. 1983. Molecular weight viscosity relationships of pendulan from Porodisculus pendulus . J. Ferment. Technol., 61: 505-510.

Kang, KS, and IW Cottrell. 1979. In : "Microbial Technology", Bull, A. and DC Ellwood (eds.), Academic press. Vol. I, p 417-481.

8. Kennedy, JF, and IJ Bradshew. 1984. In: "Progress in industrial microbiology", Bushell, ME (ed.), Elsevier . 19: 319-365

9.Low, D., JA Ahlgren, D. Horne, DJ McMahon, CJ Oberg, and JR Boradbert. 1998. Role of Streptococcus thermophilus MR-1C capsular exopolysaccharide in cheese moisture retention. Appl. Environ. 64: 2147-2151

10. Margaritis, A., and JE Zajic. 1978. Mixing, mass transfer, and scale-up of polysaccharide fermentation. Biotechnol. Bioeng. 20: 939-1001.

Marra, M. 1990. Structure characterization of the exocelluar polysaccharides from Cyanospira capsular . Carbohydrate Reserch. 197: 338-344.

12. Oda, M., H. Hasegawa, S., Komatsu, M., Kambe, and F., Tsuchiya. 1983. Anti-tumer polysaccharide from Lactobacillus sp., Agro. Biol. Chem., 47: 1623-1625.

13. Noberg, AG, and H. Persson. 1984. Accumulation of heavy-metal ions by Zoogloea ramigera . Biotech. Biochem. 115: 239-246.

14. Shu, CH, and ST Yang. 1990. Effects of temperature on cell growth and xanthan production in batch cultures of Xanthomonas campestris . Biotechnol. Bioeng. 35: 454-468.

15. Sarkar, JM, GL Hennebert and J. Mayaudon. 1985. Optimization and chracterization of an extracellular polysaccharide produced by Glomerella cingulata . Biotechnol. Lett., 7: 631-636.

16. Souw, P., and AL Demain. 1979. Nutritional studies on xanthan production by Xanthomonas campestris NRRL B1459. Appl. Environ. Microbiol., 37: 1186-1192.

17. Tako, M., T. Nagahama and D. Nomura. 1977.Some rheological properties of the viscous polysaccharide produces by coryneform bacteria strain C-8. Nippon Nogeikagaku kaishi, 51: 389-395.

18. Tako, M., A. Sakae and S. Nakamura 1989. Rheological properties of gellan gum in aqueous media. Agri. Biol. Chem., 53: 771-776.

19. Sutherland, IW 1983. Extracellular polysaccharides. In : Biotechnolohgy. Vol. 3. Verlag Chemie. Weinheim .. p 533-574.

20. Williams AG, and JWT Wimpenny . Exopolysaccharide production by Pseudomonas NCIB 11264 grown in continuous culture. J. Gen. Microbol. 104: 47-57.

<110> Korea Ocean Research and Development Institute <120> Alteromonas sp. OOSS11568 and method for preparation of          extracellular polysaccharide therefrom <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1537 <212> DNA <213> Alteromonas sp. <400> 1 agagtttgat ngagattcat tttggcacat attgaccggc gtcggcgggc ctaacacatg 60 caagtcgatc ggtaacattt atagcttgct aagagatgac cagtggtgga cgggtgagta 120 atgcttggga acttgccttt gcgaggggga taacagttgg aaacgactgc taatgccgca 180 taatgtcttg ggaccaaacg gggcttaggc tccggcgcaa agagaggccc aagtgagatt 240 agctagttgg taaggtaacg gcttaccaag gcgacgatct ctagctgttc tgagaggaag 300 atcagccaca ctgggactga gacacggccc agactcctac gggaggcagc agtggggaat 360 attgcacaat gggggaaacc ctgatgcagc catgccgcgt gtgtgaagaa ggccttcggg 420 ttgtaaagca ctttcagttg tgaggaaaag ttagtagtta atacctgcta tccgtgacgt 480 taacaacaga agaagcaccg gctaactccg tgccagcagc cgcggtaata cggagggtgc 540 gagcgttaat cggaattact gggcgtaaag cgcacgcagg cggtttgtta agctagatgt 600 gaaagccccg ggctcaacct gggatggtca tttanaactg gcagactaga gtcttggaga 660 ggggagtgga attccaggtg tagcggtgaa atgcgtagat atctggagga acatcagtgg 720 cgaaggcgac tccctggcca aagactgacg ctcatgtgcg aaagtgtggg tagcgaacag 780 gattagatac cctggtagtc cacaccgtaa acgctgtcta ctagctgtgt gtgcctttaa 840 ggcttgccta acgnaaactt accccattag ttaaccggct ggggaggttc cggcgccagg 900 gttaaactta aatgaattgg ccggggcccg cacaagccgg ggaacatgtg gtttaattcg 960 atgcaacgcg aagaacctta cctacacttg acatgctgag aagttactag agatagtttc 1020 gtgccttcgg gaactcagac acaggtgctg catggctgtc gtcagctcgt gtcgtgagat 1080 gttgggttaa gtcccgcaac gagcgcaacc cttgtcctta gttgccagcc ttaagttggg 1140 cactctaagg agactgccgg tgacaaaccg gaggaaggtg gggacgacgt caagtcatca 1200 tggcccttac gtgtagggct acacacgtgc tacaatggca tttacagagg gaagcgagac 1260 agtgatgtgg agcggacccc ttaaagaatg tcgtagtccg gattggagtc tgcaactcga 1320 ctccatgaag tcggaatcgc tagtaatcgc aggtcagaat actgcggtga atacgttccc 1380 gggccttgta cacaccgccc gtcacaccat gggagtggga tgcaaaagaa gtagttagtc 1440 taaccttcgg gaggacgatt accactttgt gtttcatgac tggggtgaag tcgtaacaag 1500 gtaaccctag gggaacctgc ggctggatca cctcctt 1537

Claims (6)

A strain of genus Alteromonas 00SS11568 (KCTC10324BP) that secretes extracellular polysaccharides (exopolysaccharides). The extracellular polysaccharide obtained by culturing the genus 00SS11568 (KCTC10324BP) strain in the culture medium containing inorganic nitrogen, organic nitrogen, inorganic phosphoric acid, MgSO ₄ , CaCl ₂ and glucose. The extracellular polysaccharide according to claim 2, wherein the extracellular polysaccharide is p-11568. 4. The extracellular polysaccharide according to claim 3, wherein p-11568 is a heteropolysaccharide having a glucose to galactose ratio of 3: 1 and a molecular weight of 4.4 x 10 ⁵ Da. Method of producing extracellular polysaccharides from S. a. Genus 00SS11568 (KCTC10324BP) strain inoculated in M-11568 medium, and cultured at 25 ℃, pH 8 conditions. The method of claim 5, wherein the M-11568 medium comprises inorganic nitrogen, organic nitrogen, inorganic phosphoric acid, MgSO ₄ , CaCl _2, and glucose.