KR100695778B1 - Microorganism producing fucoidan and method for producing fucoidan using the same - Google Patents

Abstract

The present invention relates to the genus Caulobacter , which produces fucoidan. sp .) The present invention relates to a microorganism and a method for producing fucoidan using the same, and specifically to the genus Caulobacter producing fucoidan from seaweeds. sp .) The present invention relates to a method for producing fucoidan by treating microorganisms, cultures of the microorganisms, and fractions separated from the cultures with algae. Since the microorganism of the present invention, the culture or fraction of the microorganism is active to produce fucoidan from seaweed, there is an effect that can efficiently produce fucoidan with various physiological activities.

Fucoidan, Algae, Caulobacter, Enzymes

Description

Microorganisms producing fucoidan and method for producing fucoidan using same

1 is a result of measuring the sulfate content generated by the four isolated strains of seaweeds.

Figure 2 is the result of measuring the content of fucose produced by the four isolated strains disintegrate seaweed.

Figure 3 is genus Caulobacter sp .) The enzyme fraction obtained from the B2203 strain is a result of measuring the amount of sulfuric acid produced by decomposition of seaweed.

Figure 4 is genus Caulobacter sp .) The enzyme fraction obtained from the B2203 strain is a result of measuring the content of fucose produced by digesting seaweed.

Figure 5 is a genus of Microbacterium sp . This is the result of digesting seaweed (Kashima) with algae polysaccharide degrading agent isolated from A2203 strain.

The present invention relates to the genus Caulobacter , which produces fucoidan. sp .) The present invention relates to a microorganism and a method for producing fucoidan using the same, and specifically to the genus Caulobacter producing fucoidan from seaweeds. sp .) The present invention relates to a method for producing fucoidan by treating microorganisms, cultures of the microorganisms, and fractions separated from the cultures with algae.

Fucoidan is a generic term for sulfated fucose-containing polysaccharides having an average molecular weight of 15 to 500,000, comprising 1) fucose and sulfate groups, and 2) glucuronic acid, mannose, fucose, and sulfate groups. Sulfated fucosegluromannan containing, for example, sulfated fucose-containing polysaccharide-U described in WO 97/26896 publication (constitution sugars and molar ratios thereof are fucose: mannose: galactose: uronic acid: sulfuric acid group = about 10: 7 4: 5: 20), 3) Fucogalactan sulfate consisting of fucose and galactose, such as fucogalactan sulfate (constitution sugar and molar ratio thereof) described in International Patent Application No. PCT / JP00 / 00965 : Galactose = 1: 1 to 6), and 4) fucose and mannose (sulfate ester-fucose-mannose composition), and the like (Larsen, B., Acta . Chem . Scand . , 12) . : 1395, 1970). The fucoidan is particularly contained in large amounts of red algae or brown algae such as kelp and seaweed. Natural fucoidan exists as one of the components of macromolecules composed of alginic acid, protein and fucoidan in algae.

Fucoidan is known to have a variety of excellent activities such as anticancer action, antitumor action, anticoagulant action, immune enhancing effect, hepatocyte growth promoting action and antiallergic action. For example, Korean Patent Laid-Open Publication No. 2002-31411 discloses the use of fucoidan for treating cytokine production, diseases requiring nitrogen monoxide production, and allergic diseases, and Korean Patent Publication No. 1995-07868 discloses the use of Uses for the treatment of ulcers and inhibition of the fixation of pylori are disclosed, and Republic of Korea Patent Publication No. 2004-32992 discloses the use of fucoidan adhesion, arthritis and psoriasis treatment.

Attempts to separate and purify fucoidan from seaweed have been made in Japan since the 1980s, as the activities of various excellent fucoidans are known. In general, methods used to produce fucoidan from seaweed include hot water extraction, acid hydrolysis extraction and enzyme extraction.

Among the fucoidan extraction methods, acid hydrolysis extraction is most widely used. However, acid hydrolysis requires a neutralization step, and there is a fear that strong acid remains in the final product if neutralization is not adequate.

In addition, conventional methods typically undergo a pretreatment process to finely powder seaweeds to facilitate extraction of water soluble components, including fucoidan. However, when the algae is finely powdered, it has a problem that excessive solvent is required during the extraction process because of excessive adsorption of water due to the expansion of its surface area, and the yield of the solids is very low compared to the production cost due to the extremely low content of solids. There is a problem of low economy.

The present inventors have been researching to develop an effective method for extracting fucoidan from seaweed, and isolated and identified a novel microorganism having excellent ability to produce the enzyme that produces fucoidan from seaweed, and using the microorganism from the seaweed The present invention has been completed by confirming that fucoidan can be produced in high yield.

It is an object of the present invention to provide a Caulobacter sp . Microorganism which produces fucoidan from seaweed.

Another object of the present invention is the genus Caulobacter sp .) to provide a method for culturing microorganisms.

Another object of the present invention is the genus Caulobacter sp .) to provide a method for producing fucoidan from algae using microorganisms.

In order to achieve the object of the present invention, the present invention is a genus Caulobacter producing fucoidan from seaweed sp .) It provides a microorganism and a method for culturing the microorganism.

In addition, to achieve another object of the present invention Caulobacter genus to produce fucoidan from algae sp .) to provide a culture of the microorganism and a fraction separated from the culture or lysate of the microorganism.

Furthermore, in order to achieve another object of the present invention, there is provided a method for producing fucoidan from seaweed using the microorganism.

Hereinafter, the present invention will be described in detail.

In the present invention, to find a novel microorganism that produces fucoidan from algae. To this end, the present inventors collected samples from about 200 places in nature and separated the microorganisms from them, and measured the ability to produce fucoidan of each microorganism. The measurement was carried out by the microorganisms combined with alginic acid and lamyranine in algae in the polymer form of sulfated fucose-type polysaccharides, including sulfated fucan, sulfated fucose-galactose (sulfateester-fucose-galactose), and sulfated fucose mannan ( to determine whether it can be decomposed with fucoidans such as sulfate ester-fucose-mannose composition) and sulfated fucoseglumananane composition. For this purpose, the production of sulfate groups and fucose, which can be detected only by decomposition into fucoidan type, was measured.

As a result, four kinds of microorganisms isolated from about 200 samples had high sulfuric acid group content and fucose content, and these were named as A2104 strain, B3321 strain, B2203 strain and B2206 strain, respectively. Among the four strains, the B2203 and B2206 strains had relatively higher sulfate group content and fucose content in constituent sugars (see FIGS. 1 and 2).

The B2203 strain identified as having high fucoidan production ability was identified using a classification based on homology of the nucleotide sequence of 16S ribosomal DNA (rDNA; DNA encoding ribosomal RNA), and as a result, the B2203 strain was found in the genus Caulobacter. sp.) was identified as a new microorganism.

The new strain Caulobacter sp.) In order to confirm whether the fucoidan-producing ability of B2203 is a property of general Kaulobacterium microorganisms, the new strain Caulobacter from KCCM. sp.) B2203 and the homology of 95% or more bakteo cowl segment varnish (Caulobacter segnis (Accession Number: AB023427.1)), Caulobacter vibrioides (Accession No .: ATCC11764)), Caulobacter cresecntus (Accession No .: AJ227757.1)) were obtained in the same manner as above, and tested the production of fucoidan through the production of sulfate and fucose as described above Caulobacter distributed by KCCM sp.) strains did not produce fucoidan at all. Accordingly, the present inventors first identified novel microorganisms that produce fucoidan from seaweeds among Caulobacter sp. Microorganisms.

The new strain Caulobacter sp.) The bacteriological properties of B2203 are summarized as follows.

1) Action: Fucanated sulfate, fucogalactan sulfate (consisting of sulfate ester-fucose-galactose), sulfated fucosemanganate (consisting of sulfate ester-fucose-mannose) and sulfated fucose glutamate from algae produce fucoidan, such as -gluronic acid-mannose / xylose.

2) Morphological characteristics: Colorless rod-shaped, most of the stalk (stalk) is located in the center.

3) Physicochemical Properties

 Gram stain  negative NaCl   ≥ 0.05% required for growth  +   4% tolerated  - Sugar Utilization   Glucose  +   Galactose  +   Mannose  +   Fructose  -   Sucrose  + Starch hydrolysis  + Amino acid utilization  Alanine  +  Aspartate  +  Glutamate  +  Proline  - Utilization of primary alcohols  Methanol  -  Ethanol  -  Propanol  -  Butaol  - Orgnic growth factors needed  Vitamin b2  -  Biotin  -

Biochemical and culture characteristics were analyzed according to Burgy's Manual of Systematic Bacteriology.

The Kaulobacter genus according to the invention sp.) B2203 strain was deposited with the Korea Agricultural and Microbiological Conservation Center (KACC) on March 24, 2004, and deposited as accession number KACC 91095.

Kaulobacter genus of the present invention (Caulobacter sp.) The substrate capable of producing fucoidan by using microorganisms is not particularly limited, but seaweeds are preferred. The seaweed is kelp (Laminaria japonica), Seaweed (Undaria pinnatifida Sporophyll), Arbore Sense (Padina arborescens), Fucus Evanesense (Fucus evanescens), Himmansria (Himanthulia), Bifucaria (Bifurcaria) And Fukus Bezikulosus (Fucus vesiculosusBrown algae and ephipaea cordariales nematicus, includingPhaeophyceae Chordariales nemacystusRed algae, etc. are preferable, brown algae are more preferable, and kelp or seaweed ear is the most preferable.

Therefore, the invention in bakteo a cowl which produces the fucoidan from seaweed (Caulobacter sp.) In bakteo a microorganism, in particular the cowl (Caulobacter sp .) B2203 (KACC 91095) microorganisms are provided.

The microorganism of the present invention can be cultured in a large amount by a conventional method of culturing the microorganism of the genus Cowlerbacter.

As the culture medium, a medium containing a carbon source and a nitrogen source may be used. Preferably, a medium to which seaweed is added may be used. For example, it can be cultured in a medium containing peptone, yeast extract, magnesium sulfate, agar and seaweed powder. Cultivation of the microorganism is possible under the conditions of cultivating microorganisms of the genus Kaulobacter, for example, may be incubated for 24 hours to 80 hours at 20 ℃ to 40 ℃. More preferably, it is preferable to incubate at 30 ° C. for about 72 hours. In order to separate the culture of the microorganism into the culture medium and the lysate, it may be subjected to centrifugation or filtration, and this step may be performed by those skilled in the art as needed. The concentrated cells can be frozen or lyophilized according to a conventional method so as not to lose their activity.

The culture or lysate of the microorganism of the present invention may be fractionated by molecular weight by a method conventional in the art to obtain a fraction having fucoidan production activity. Preferably, fractions can be obtained for each molecular weight by gel-column chromatography.

The genus Caulobacter sp .) The fraction having the fucoidan production activity produced from the microorganism is not particularly limited to the kind as long as it can produce fucoidan. In one embodiment of the present invention, the genus Caulobacter sp .) To identify the fractions with fucoidan production activity produced from microorganisms, the enzymes were isolated intracellularly and extracellularly of the strain, followed by gel-column chromatography using five extracellular and intracellular according to molecular weight. Three total eight enzyme fractions were obtained. For the eight enzyme fractions obtained above, the fucoidan-forming ability according to each temperature (30 ° C and 50 ° C) and each pH (pH 4, pH 5, pH 6, pH 7, pH 8 and pH 9) should be liberated in the fucoidan type. The content of detectable sulfuric acid groups and fucose was measured according to known methods. The seaweed polysaccharide was prepared as a seaweed polysaccharide by seaweed polysaccharide, and then 1 vol% of each fraction was added to the seaweed polysaccharide to react for 24 hours. As a result, the sulfuric acid content of CFN-2 and CFX-2 was high in the total 8 enzyme fractions (see FIG. 3 and Table 4), and the fucose content of CFN-2 and CFN-3 was high (FIG. 4 and See Table 4).

Thus, the Caulobacter genus sp .) The fraction with fucoidan production activity produced from the microorganism may be, for example, the enzyme of the CFN-2 fraction, the enzyme of the CFX-2 fraction and the enzyme of the CFN-3 fraction.

Caulobacter sp .) The fraction with the fucoidan production activity produced from the microorganism is also present in the extracellular and intracellular fractions of the microorganism, so it is also present in the culture of the microorganism or in the lysate of the microorganism. In this case, the culture medium means the rest except for the microorganism when the microorganism is incubated by the invention or a known method according to the present invention, the crushed means that the microorganism is crushed by a known method such as ultrasonic waves.

The method for producing fucoidan from the algae according to the present invention is more specifically, (a) degrading algae; (b) removing alginic acid and salt from the seaweed degradation product; (c) treating the alginic acid and salt-free algae digestion with Kaulobacter sp . microorganisms or fractions having fucoidan production activity therefrom to obtain crude fucoidan solution; And (d) purifying the crude fucoidan solution to obtain fucoidan.

The above steps are described in more detail as follows.

(a) step: disintegrating algae

This step is to decompose the algae to contain water-soluble components. Seaweeds used in the method is preferably brown algae or red algae as described above, seaweed and kelp are most preferred. The algae can be decomposed by a known method, it is preferable to decompose by adding a seaweed polysaccharide degrading agent.

The algae polysaccharide decomposer refers to an enzyme or a composition containing the enzyme, which liquefyes a water-soluble polysaccharide component from seaweed, and decomposes a viscous polysaccharide from a polysaccharide complex composed of fucoidan, alginic acid, lamiranin, and the like in seaweed. For example, the algae polysaccharide may be isolated from Microbacterium sp. A2203 (KACC 91096), but is not limited thereto.

Seaweed polysaccharides isolated from the Microbacterium sp. A2203 (KACC 91096) can be obtained by the following process. Microbacterium sp. A2203 (KACC 91096) was incubated at 25 ° C. for 30 hours on a medium containing kelp or wakame powder (pH 7.0) and then centrifuged at 10000 rpm to precipitate the cells and recover the supernatant. do. This supernatant may be used as an algae polysaccharide, but may be used after additional separation as follows. That is, the recovered supernatant was removed from the primary cells using a 0.45 μm microfilter, and then a crude extract having a molecular weight of 100,000 to 1,000,000 was isolated to pH 4.8 buffer solution with biomax 10 (Phamacia co. Ltd, USA). By using the buffer exchange, the extract can be used as an algae polysaccharide.

Seaweeds such as brown algae have high salinity and high viscosity, so it is difficult to increase the extraction of liquids during the extraction of tablets at the production site. Therefore, by using the algae polysaccharide degrader of this step, it is possible to rapidly decompose the algae algae, liquefy the water-soluble polysaccharide component efficiently, and decompose viscous polysaccharides such as alginic acid in the extracted polysaccharide complex. Since the viscosity can be reduced, the extraction yield of fucoidan can be improved. The result of reacting the seaweed polysaccharide degrading agent of the present step with seaweed is shown in FIG. 5.

Prior to this step, 1 part by weight of seaweed is washed with 10 to 20 parts by weight of water to desalinate, and then washed with hot water of 90 to 100 ° C. Seaweeds, such as brown algae, have a high salt concentration, which leads to a high cost of removing salts after the concentration process. Also, when processed through the treatment of a seaweed polysaccharide degrading agent as described in the present invention, the effects of external salts such as sodium and chlorine ion This is because it needs to be minimized. In addition, it is preferable to perform the present desalting process in order to diversify the processing suitability, such as the difficulty of the powdering process due to deliquescent properties. As mentioned above, by washing with 90-100 degreeC hot water, the time which seaweeds take in water can be reduced.

(b) step: Alginic acid and salt removed Seaweed Degradate  Manufacturing steps

This step is a step of removing and desalting alginic acid from the seaweed degradation product prepared in step (a).

Removal of alginic acid can be carried out using known methods, such as by isoelectric precipitation of alginic acid or by adding salts which form precipitates with alginic acid, for example by adding ethyl alcohol and calcium chloride to precipitate the alginic acid and filtering the alginic acid. Can be removed.

The filtrate is recovered and ultrafiltered several times to demineralize and concentrate. At this time, the solids in the algae digested product from which alginic acid and salts were removed by adding purified water to smoothly decompose by the enzyme in step (c) was about 5 Brix (viscosity of about 60 cP) to 10 Brix ( Viscosity of about 80 cP).

(c) step: Jofukoidan  Getting steps

In this step, the alginic acid and salt-free seaweed digests are treated by treating Caulobacter sp . Microorganisms, cultures of the microorganisms, crushed microorganisms or fractions with fucoidan production activity generated from the microorganisms. It is a step of obtaining a fucoidan liquid. At this time, the reaction of the microorganism, the culture medium of the microorganism, the crushed product of the microorganism or the fraction having the fucoidan production activity generated from the microorganism is added to the alginic acid and salt-free seaweed digested products at 0.7% by volume to 1.0% by volume. desirable.

Fusulfated sulfated fucogalactan is a sulfated fucose-type polysaccharide that is present as a polysaccharide in combination with lamyranine by the action of the microorganism, the culture medium of the microorganism, the crushed product of the microorganism or the fucoidan production activity generated from the microorganism, It is liberated with fucoidans such as sulfated fucose mannan and sulfated fucose glumannan to obtain crude fucoidan liquid. The produced fucoidan may vary depending on the degradation time and the amount of enzyme, but is usually fucoidan with an average molecular weight of about 500,000.

Additionally, the crude fucoidan solution may be extracted under high temperature and high pressure to obtain low molecular fucoidan. The high temperature and high pressure extraction is preferably carried out for 1 to 3 hours at 100 ~ 135 ℃, 1.0 ~ 2.0 kgf / cm2, whereby low molecular fucoidan can be obtained. The longer the extraction time, the lower molecular weight fucoidan can be obtained. In particular, when extracted for 2 hours or more, a crude fucoidan solution containing a low molecular weight fucoidan having an average molecular weight of about 7000 can be obtained. This is a safe decomposition method corresponding to the ultra-low molecular weight fucoidan technology that has been tried in Japan.

(d) Fucoidan  Obtaining Step

This step is a step of purifying the crude fucoidan solution to obtain the fucoidan, desalination, concentration of the crude fucoidan solution by ultrafiltration, eluting with a gradient of NaCl concentration in an ion exchange column to obtain a fraction, sulfuric acid groups and constituent sugar of the fraction The composition is measured to collect a high content fraction to obtain fucoidan.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

<Example 1>

Fucoidan Generation ability  Exploration of Having Microorganisms

<1-1> Isolation and Culture of Microorganisms

Since 1999, more than 200 samples have been taken from the coast, soil, plants, animals and livestock feces near Yeosu, Jeonnam. Each sample was subcultured for one week using each medium of Table 1 below, followed by seed culture. Each of the media in Table 1 is A, bacteria, B is fungi, C is a medium composition for the separation of marine bacteria in consideration of the microbial phase to be separated. Each microbial sample cultivated above was incubated for 48 hours in medium A, 96 hours in medium B, and 48 hours in medium C. Bacteria and marine bacteria were gram-stained after pure separation and stored after microscopic examination. After the separation was separated and stored in culture.

Composition A medium (for bacterial separation) B medium (for fungal isolation) C medium (for marine bacteria separation) Glucose 1 g 0.5g 1 g peptone 5 g 3 g 3 g Yeast extract 1 g 3 g 1 g Malt extract 0.5g 2 g 1 g Seaweed Degradation Algae with Alginic Acid and Salts Removed 50 mL (solid 5 Brix) 50 mL (solid 5 Brix) 50 mL (solid 5 Brix) Algae and salt-free kelp seaweeds 20 mL (solid 5 Brix) 20 mL (solid 5 Brix) 20 mL (solid 5 Brix) NaCl 20% final concentration Potato Powder 5 g

<1-2> Alginic Acid and Salt-Free Algae Degradation

Seaweed ear ( Undaria pinnatifida 200 g of Sporophyll ) was washed twice with 2 kg of distilled water to remove mannitol and salt from the raw materials. 2 kg of distilled water at 90 ° C. was added thereto, and then 1.6 kg of distilled water at 4 ° C. was further added to adjust to about 50 ° C., and then adjusted to pH 4.8 by adding citric acid-sodium carbonate buffer. This algae is inventors micro tumefaciens in the deposit as sugars released (Microbacterium sp.) A2203 (KACC 91096) is isolated from the microorganism algae sugars released (Republic of Korea Patent Application No. 2004-21662 call) the addition of 30g and 8 Decomposition over time to liquefy the water-soluble components. This was again treated at 121 ° C. for 10 minutes to lower the viscosity below 60 cP (25 ° C.). 1.7 kg of ethyl alcohol was added to the seaweed digested product, 25 g of calcium chloride was added, stirred at 30 ° C. for 2 hours, centrifuged (17,000 g, 20 minutes) to obtain a supernatant, and filtered in a centrifugal dehydrator equipped with a 25 μm filter cloth. (First filtrate obtained). The unfiltered residue was washed with distilled water again, and the supernatant obtained by centrifugation (17,000 g, 20 minutes) was filtered in a centrifugal dehydrator equipped with a 25 μm filter cloth as described above to obtain a filtrate. The obtained filtrate was combined with the primary filtrate and then desalted and concentrated four times with an ultrafilter (Millipore Co. Ltd, USA) MWCO 5,000. Distilled water was added thereto so that the solid content was adjusted to 5 Brix (60 cP viscosity; 25 ° C.) or less or 10 Brix (80 cP viscosity; 25 ° C.) or less to prepare alginic acid and salt-free seaweed decomposition products.

Meanwhile, kelp ( Laminarea japonica ) was prepared in the same manner as above.

<1-3> Strain Isolation with Excellent Fucoidan Formation

In order to identify microorganisms capable of producing fucoidan from seaweeds, the sample was collected in Example <1-1> using a seaweed decomposition product having a solid content of 10 Brix and prepared in Example <1-2> as a substrate. Fucoidan production ability of each microorganism isolated was investigated. That is, the strains collected and separated in Example <1-1> were again seeded and cultured, and inoculated in an amount of 1% by volume with respect to the seaweed digested product and left for 7 hours. Since the sulfuric acid group content was measured according to the following method and the content of fucose in the constituent sugar.

The determination of sulfuric acid group content is known in the art (Dogson, KS et. al . , A Note on the Determination of the Ester Sulphate Content of Sulphated Polysaccharides. Biochem. J., vol 84, 106, 1962). That is, 4% TCA solution (Trichloroacetic acid, Sigma Co. Ltd, USA> 3.8 mL and barium chloride-gelatin reagent 1mL were added and stirred to 0.3 mL of the digested solution, and left for 20 minutes to measure absorbance at 360 nm. The calibration curve was K2SO4 (Sigma Co. Ltd., USA), in which barium chloride-gelatin reagent was dissolved in 400 ml of distilled water, 2 g of gelatin was completely dissolved at 65 ° C, left overnight at 4 ° C, and 2 g of barium chloride was dissolved for 2 hours. It was used after standing.

The measurement of the fucose content was carried out according to a known method (Somogyi-Nelson method). That is, 1 ml of the prepared sample solution and 1 ml of the copper reagent were respectively placed in a test tube, and heated for 20 minutes in a water bath to produce cuprous oxide (Cu 2 O). After 1 ml of molybdenum solution was added and developed, absorbance was measured at 520 nm, and the fucose-reducing sugar was quantified from the calibration curve. However, it was measured using fucose as a standard product when preparing the calibration curve.

Analysis of the composition per composition was performed using ion chromatography. 50 ml of the sample was centrifuged and the supernatant was collected in a reaction vial, 5 ml of 1N HCl was added, hydrolyzed at 110 ° C. for 5 hours, neutralized with NaOH, diluted with 20 ml of distilled water, concentrated under reduced pressure, and lyophilized. It was. After dissolving 1 g of the analytical sample in 10 ml of distilled water and passing through Amberite IR 200 to remove metal ions first, it was then desalted with a Sep-pak cartridge (Waters Co., LTD, USA) to obtain ion chromatography (Table 2). IC) Measured under the analysis conditions. L-fucose, D-galactose, D-xylose, L-ribose, D-glucose, L-rhamnose, and mannitol were used as standard sugars.

Device Dionex 600 ion chromatography (Dionex Co., Ltd, USA) column Cabopack-PA10 from Dionex Co., Ltd, USA Eluate 16 mM NaOH Flow rate 1.0 mL / min Injection volume 20 μl detection Amms-ice Analysis time 60 minutes / 1 sample

As a result, four kinds of sulfuric acid group content and fucose content among microorganisms isolated from about 200 samples were selected and named as A2104 strain, B3321 strain, B2203 strain and B2206 strain, respectively. The sulfuric acid group content and the fucose content in the constituent sugars of the four strains are shown in FIGS. 1 and 2, respectively. As can be seen in Figures 1 and 2, the sulfate group content and the fucose content of the B2203 strain and B2206 strain of the four strains were relatively higher than the A2104 strain and B3321 strain. From the above results, it can be seen that the fucoidan production ability of the B2203 strain and the B2206 strain is the best.

<1-4> Identification of B2203 strain with excellent fucoidan production

The B2203 strain identified above with high fucoidan production capacity was identified using a classification based on homology of the nucleotide sequence of 16S ribosomal DNA (rDNA; DNA encoding ribosomal RNA). As a result of the identification, the B2203 strain was identified as a new strain of Caulobacter sp.

Biochemical and culture characteristics of the microorganisms were analyzed according to the Burgy's Manual (Bergey's Manual of Systematic Bacteriology) as shown in Table 3 below.

 Shape  rod  Color  colorless  Gram stain  negative  Morphology  Stalk invariably central NaCl   ≥ 0.05% required for growth  +   4% tolerated  - Sugar Utilization   Glucose  +   Galactose  +   Mannose  +   Fructose  -   Sucrose  + Starch hydrolysis  + Amino acid utilization  Alanine  +  Aspartate  +  Glutamate  +  Proline  - Utilization of primary alcohols  Methanol  -  Ethanol  -  Propanol  -  Butaol  - Orgnic growth factors needed  Vitamin b2  -  Biotin  -

The new strain Caulobacter sp.) To determine whether the fucoidan-producing ability of B2203 is a general property of the Kaulobacterium microorganisms, the new strain Caulobacter from KCCM. sp.) B2203 and the homology of 95% or more bakteo cowl segment varnish (Caulobacter segnis (AB023427.1)), Caulrobacter vibrioides (ATCC11764)), a cowl bakteo Crescent sekeun tooth (Caulobacter cresecntus (AJ227757.1) in bakteo accept the pre-sale) to receive the results from the pre-sale cowl Korea Gene Bank (KCCM) performed the same experiment with the (Caulobacter sp.) Sulfate absorbance of the strains was significantly lower than 0.325, and no peak corresponding to fucose among the constituent sugars was observed. From the results, it was confirmed that the fucoidan-producing ability of the new strain Caulobacter sp. B2203 was the unique mycological properties of the strain.

The Caulobacter sp.) B2203 was deposited with the Korea Agricultural and Microbiological Conservation Center (KACC) on March 24, 2004, and deposited as accession number KACC 91095.

<Example 2>

Kaulobacter ( Caulobacter sp From B2203 Fucoidan  Purification of Producing Enzymes

<2-1> Caulobacter sp.) Isolation of enzyme fractions from B2203

The strain Caulobacter sp.) The intracellular and extracellular coenzyme solutions were separated from B2203, and the molecular weight distribution was confirmed by SDS-PAGE electrophoresis, and fractions were obtained by molecular weight through gel-colum chromatography in the effective molecular weight range.

That is, the strain Kaulobacter sp.) B2203 contains 2.0 g of Peptone (Difco, USA), 1.0 g of Yeast extract (Difco, USA), 0.2 g of MgSO4-7H2O (Sigma Co Ltd, USA), 10.0 g of Agar, 1.0 L of distilled water, 3 g of Wakame powder The inoculated medium was maintained at 30 ° C. in a 10 L fermenter (Korea fermenter Co., Ltd, Korea) and aerated for 72 hours, followed by separation of extracellular coenzyme solution and intracellular coenzyme solution, respectively.

The extracellular coenzyme solution was centrifuged to 17,000 g of the cultured strain, and the supernatant was first filtered through 9 sheets of a multi-filter press (KHS Processtechnik, Germany), followed by secondary filtration with a 0.1 μm microfilter (millipore). Cut off with an ultrafilter (millipore) MWCO (molecular weight cut off) 5000, and concentrated repeated desalting three times. After taking 1 mL of the coenzyme solution, gel permeation chromatography (GPC, gel permeation chromatography) using OHpak SB-803, 804, 805, and HQ columns with a flow rate of 0.5 ml / min and mobile phase PBS (phosphate buffered saline, pH 7.2). Molecular weight distribution at 280 nm with SDS-PAGE, and then gel-column chromatography using Biogel p-100 (Biorad Co Ltd) with a flow rate of 1 ml / min and a mobile phase (phosphate buffered saline, pH 7.2). Gel-column chromatography; Biorad Co. Ltd, USA) collected and fractionated the corresponding molecular weight range. As a result, five fractions were obtained: CFX-1 (average molecular weight 85kD), CFX-2 (average molecular weight 73kD), CFX-3 (average molecular weight 52kD), CFX-4 (average molecular weight 13kD) and CFX-5 ( Average molecular weight 5.7kD). After each addition of a pH 7.0 phosphate buffer solution in each of the fractions and then desalted by ultrafiltration MWCO 5000.

Intracellular coenzyme solution was centrifuged at 17,000g after culturing the strain, the supernatant was removed, the cells were recovered, and phosphate buffer solution of pH 7.0 was added. After crushing the cells with an ultrasonic crusher, the fractions were purified using the same method as the fractionation method of the enzyme in the cells. As a result, three enzyme liquid fractions were obtained through gel-column chromatography (Biorad Co. Ltd), which were CFN-1 (average molecular weight 71kD), CFN-2 (average molecular weight 54kD), and CFN- It was named 3 (average molecular weight 27kD).

<2-2> Confirmation of Fucoidan-producing Enzyme

Fucoidan decomposition experiments were performed using the seaweed digested seaweeds prepared in Example <1-2> as a substrate for each of the enzyme liquid fractions obtained in Example <2-1>. In the present embodiment, the content of fucose in the sulfate group and the constituent sugar which can be detected only by liberation with fucoidan type was measured.

The enzyme solution was added to 1% by volume of the algae and algae seaweeds from which the alginic acid and salts were removed, followed by reaction for 24 hours. The sulfuric acid group content was then measured and the composition sugar composition was analyzed. Specific sulfuric acid group content measurement and determination of the content of fucose in the sugar composition using the same method as used in Example <1-3>, various pH (pH 4, pH 5, pH 6, pH 7, pH 8 and Algae acid and salts of pH 9) were removed from seaweed algae digests. In addition, the measurement process of each said condition was measured by reaction temperature of 30 degreeC and 50 degreeC.

The sulfuric acid group content and the fucose content of the fractions of CFX-1, CFX-2, CFX-3, CFX-4, CFX-5, CFN-1, CFN-2 and CFN-3 performed at 50 ° C are shown in FIG. 3, FIG. 4 and Table 4, respectively. In the case of Figure 4 and Table 4, the pH of the algae seaweed degradation products from which alginic acid and salts were removed is 5.0. As shown in FIG. 3 and Table 4, the sulfuric acid group content was highest in CFN-2, followed by CFX-2, highest in the substrate at pH 5.0, and next highest at pH 6.0. In addition, as shown in FIG. 4 and Table 4, the fucose content was highest in CFN-2, followed by CFN-3. On the other hand, the temperature condition is preferably 50 ℃, but did not show a significant difference with 30 ℃.

Fraction Sulfuric Acid Content (ABS 360nm) Fucose Content of Degraded Products (ABS 520nm) Fractional average molecular weight (Dalton) CFX-1 0.402 0.020 85,000 CFX-2 0.821 0.040 73,000 CFX-3 0.556 0.025 52,000 CFX-4 0.454 0.035 13,000 CFX-5 0.354 0.012 5,700 CFN-1 0.604 0.015 71,000 CFN-2 1.210 0.099 54,000 CFN-3 0.304 0.065 27,000

From these results, the caulobacter of the present invention In sp .) B2203) (KACC 91095), it was found that CFN-2 had the best fucoidan production ability, and CFX-2 and CFN-3 were the next excellent.

As described above, the genus Caulobacter of the present invention sp .) Microorganisms can be used very effectively to produce fucoidan from seaweeds. In the case of using the microorganism of the present invention, since it can lead to an improvement in safety, a reduction in production cost, and an improvement in production yield, compared to the conventional acid decomposition extraction method, there is an advantage of mass production of fucoidan, which is attracting attention in the food, cosmetic, and pharmaceutical fields.

Claims (14)

Caulobacter sp . B2203 (KACC 91095) microorganism producing fucoidan from seaweed. delete A method of culturing a microorganism of Kaulobacter sp . B2203 (KACC 91095), wherein the microorganism of claim 1 is cultured in a medium containing a carbon source and a nitrogen source. The culture method according to claim 3, further comprising algae in the medium. A culture of Caulobacter sp . B2203 (KACC 91095) microorganisms cultured by the method of claim 3 or 4. delete delete A method of producing fucoidan from seaweeds, comprising treating the microorganisms or a culture of the microorganisms of claim 1, wherein the present invention comprises a step of Caulobacter sp . B2203 (KACC 91095). The method of claim 8, (a) breaking down algae; (b) removing alginic acid and salt from the seaweed degradation products; (c) treating the algaic acid and salt-free algae digestion with Caulobacter sp . B2203 (KACC 91095) microorganism or a culture of the microorganism in seaweed to obtain a crude fucoidan solution; And        (d) purifying the crude fucoidan solution, the method of producing fucoidan from seaweed, characterized in that it comprises the step of recovering the fucoidan. 10. The method of claim 9, wherein the step (a) is performed by adding an algae polysaccharide degrading agent to the algae. 10. The method according to claim 9, further comprising a seaweed pretreatment step of washing the seaweed with desalination followed by washing with hot water. 10. The method of claim 9, further comprising the step of extracting the crude fucoidan solution of step (c) at 100 to 135 ° C and 1.0 to 2.0 kgf / cm ² . The method according to any one of claims 8 to 12, wherein the seaweed is Laminaria japonica , Undaria pinnatifida Sporophyll ), Padina arborescens , Fucus evanescence evanescens , Himanthulia , Bifurcaria , and Fucus Bezikulusus vesiculosus and Phaeophyceae Chordariales nemacystus ), characterized in that at least one selected from the group consisting of. The method of claim 10, wherein the algae polysaccharide is separated from Microbacterium sp. A2203 (KACC 91096).

Images