JP5364352B2 - Novel fucoidan-utilizing microorganism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means/method for not only oligomerizing but also desulfating fucoidan, and to provide a desulfated and oligomerized fucoidan by such a means/method. <P>SOLUTION: The method for producing the desulfated and oligomerized fucoidan includes subjecting fucoidan to fucoidan-assimilative bacteria having the respective activities of desulfating and oligomerizing fucoidan, i.e. bacteria belonging to the genus Flavobacterium such as Flavobacterium limicola NITE AP-674 or Luteolibacter algae AP-675, AP-676, or extract thereof. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

  The present invention relates to a novel fucoidan-assimilating bacterium and a low-molecular-weight fucoidan obtained thereby. The fucoidan-assimilating bacterium of the present invention is a unique one having both the low molecular weight activity and the desulfation activity of fucoidan.

  Fucoidan, a natural substance, is a slime ingredient in brown seaweeds such as Okinawa mozuku, mozuku, wakame, and kelp, and has various biological activities such as killing cancer cells, regulating the immune system, and promoting tissue regeneration. It has become clear to have. In addition, fucoidan has an action of tightening the skin and has a moisturizing action, so that it is also used in cosmetics. Thus, the demand for fucoidan is increasing as a raw material or ingredient for health foods, functional foods, supplements, cosmetics and pharmaceuticals.

  These various physiological activities possessed by fucoidan are said to correlate with the degree of sulfation of fucoidan molecules, but there are cases where physiological activity is not recognized when there are many sulfate groups. Fucoidan with a molecular weight of 32,000 and a low sulfate group content promotes the proliferation of normal lymphocytes and enhances the production of cytokines such as TNF-a and IL-6, whereas it has a high sulfate group content at the same molecular weight. There is a report that fucoidan does not show these physiological activities (see Non-Patent Document 1).

  Thus, the molecular weight of fucoidan and the number of sulfate groups greatly influence the physiological activity of fucoidan. As for the low molecular weight of fucoidan, heat treatment, acid hydrolysis, decomposition by microorganisms or enzymes have been reported so far (see Patent Document 1, Non-Patent Documents 2 to 7). In these reports, there are cases in which enzymes and genes involved in lowering the molecular weight of fucoidan have been isolated. However, in either case, no mention is made of enzymatic desulfation of fucoidan. Furthermore, there are also examples of fucoidan molecular weight reduction utilizing hydrothermal reaction, and the elimination of sulfate groups is not observed when the molecular weight is reduced by hydrothermal reaction (see Patent Document 2).

From this situation, if a method or means for reducing the molecular weight of fucoidan, and further capable of desulfating (desulfating) sulfate groups, conventional heating, hydrothermal, acid treatment, Fucoidan with a low degree of sulfation that cannot be prepared by methods such as enzyme treatment can be obtained, elucidation of the physiological activity of fucoidan, a different activity from the original activity, or an increase in the original activity, a low sulfation degree and Low molecular fucoidan can be produced.
US Pat. No. 6,489,155 Japanese Patent Application No. 2008-50534 Japanese Journal of Nutrition and Food, 58, 273-280 (2005) Marine Biotechnology, 5, 70-78 (2003) Marine Biotechnology, 5, 536-544 (2003) Marine Biotechnology, 6, 335-346 (2004) Marine Biotechnology, 8, 27-39 (2006) Glycobiology, 16, 1021-1032 (2006) J. Microbiol. Biotechnol., 18, 616-623 (2008)

  An object of the present invention is to develop a means / method for desulfating as well as reducing the molecular weight of fucoidan, and to obtain a low molecular weight fucoidan having a low degree of sulfation by such means / method.

In order to complete the present invention, the present inventors have intensively studied to solve the above problems and succeeded in isolating fucoidan-utilizing microorganisms having desulfation activity and low molecular weight activity of fucoidan from seawater. It came.
That is, the present invention provides:
(1) A desulfated low-molecular-weight fucoidan characterized in that a fucoidan-utilizing bacterium Flavobacterium bacterium having an activity of desulfating and reducing the molecular weight of fucoidan or an extract thereof is allowed to act on fucoidan. Manufacturing method of
(2) Desulfated low-molecular-weight fucoidan characterized by allowing fucoidan to act on a fucoidan-utilizing bacterium, Luteolibacter genus bacterium having the desulfating activity and low-molecularizing activity, or its extract. Manufacturing method of
(3) The method according to (1), wherein the bacterium is Flavobacterium limicola F31 strain deposited as an accession number NITE AP-674 at the National Institute of Technology and Evaluation of the National Institute of Technology and Evaluation,
(4) The method according to (2), wherein the bacterium is Luteolibacter algae H18 strain deposited at the National Institute of Technology and Evaluation Microorganisms Deposit Center under the receipt number NITE AP-675;
(5) The method according to (2), wherein the bacterium is a Luteolibacter algae SWi-1-Y strain deposited as an accession number NITE AP-676 with the Patent Evaluation Microorganism Depositary, National Institute for Product Evaluation and Technology.
(6) Desulfated low molecular weight fucoidan obtained by the method according to any one of (1) to (5);
(7) Desulfated low molecular weight fucoidan having a molecular weight of 140,000 or less obtained by the method according to (5);
(8) The fucoidan-assimilating bacterium Flavobacterium limicola F31 strain having the desulfation activity and the low molecular weight activity of fucoidan deposited at the National Institute of Technology and Evaluation of Microorganisms of the National Institute of Technology and Evaluation under the receipt number NITE AP-674;
(9) Fucoidan-assimilating bacterium Luteolibacter algae H18 strain having the desulfation activity and the low-molecular-weight activity of fucoidan deposited at the National Institute of Technology and Evaluation of Microorganisms under the accession number NITE AP-675;
(10) Fucoidan-utilizing bacterium Luteolibacter algae SWi-1--having fucoidan desulfation activity and low-molecular-weight activity deposited at the National Institute of Technology and Evaluation of Microorganisms under the accession number NITE AP-676. Y shares are provided.

  ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of the desulfurized low molecular-weight fucoidan by the fucoidan utilization bacterium which has the desulfation activity of a fucoidan and a low molecular-weight activity is provided. By using the production method of the present invention, low molecular weight fucoidans having various molecular weights and different degrees of sulfation can be prepared. By examining their physiological activities, the correlation between the structure and function of fucoidan is clarified. can do. The low molecular weight fucoidan obtained by the present invention can be used as a desulfated low molecular weight fucoidan having an activity different from the original activity or enhanced original activity. Furthermore, according to the present invention, a bacterial strain suitable for the method for producing the desulfated low molecular weight fucoidan of the present invention is also provided.

  In one aspect, the present invention is a desulfated low molecule characterized by allowing fucoidan-utilizing bacteria having fucoidan desulfating activity and low molecular weight reducing activity or an extract thereof to act on fucoidan. A method for producing a fucoidan is provided. Any microorganism cell or extract thereof used in the method for producing a desulfated low molecular weight fucoidan of the present invention may be used as long as it is a fucoidan-utilizing microorganism having fucoidan desulfating activity and low molecular weight activity. But preferably a bacterium, more preferably a bacterium of the genus Flavobacterium (more preferably a Flavobacterium limicola species), or a bacterium of a bacterium of the genus Luteolibacterium (more preferably a species of Luteolibacter algae) or its extraction Can be used.

  Accordingly, the present invention is directed to a desulfated low-activator characterized in that a cell of a bacterium belonging to the genus Flavobacterium having an activity of desulfating fucoidan and a molecule-lowering activity, or an extract thereof, acts on fucoidan. A method for producing molecular fucoidan is provided. Furthermore, the present invention, in another aspect, is characterized in that a fucoidan-utilizing bacterium Luteolibacter bacterium having an activity of desulfating and reducing the molecular weight of fucoidan or an extract thereof is allowed to act on fucoidan. A method for producing a desulfated low molecular weight fucoidan is provided.

The most preferred microbial cells or extracts thereof used in the method for producing a desulfated low molecular weight fucoidan of the present invention are the following three bacterial strains or extracts thereof:
Flavobacterium limicola F31 strain deposited with the National Institute of Technology and Evaluation Patent Microorganisms Deposit Center as receipt number NITE AP-674,
Luteolibacter algae H18 strain deposited at the National Institute of Technology and Evaluation Technology Patent Microorganisms Deposit Center as the receipt number NITE AP-675, or deposited at the National Institute of Technology and Evaluation Microorganisms Deposit Center as the patent number NITE AP-676 The fucoidan-utilizing bacterium Luteolibacter algae SWi-1-Y strain having fucoidan desulfation activity and low molecular weight activity.

  The fucoidan-assimilating microorganism used in the present invention is different from conventional ones in that it has both a low molecular weight activity and a desulfation activity of fucoidan. By using the fucoidan-assimilating bacterium of the present invention, fucoidan can be reduced in molecular weight while releasing sulfate groups. Moreover, since the desulfation pattern of fucoidan of these bacteria is different from the demolecularization pattern of fucoidan, the molecular weight and the degree of desulfation of fucoidan can be adjusted by appropriately using these bacteria. In the present specification, fucoidan molecular weight-reducing activity refers to the ability to cleave fucoidan sugar chains to reduce molecular weight, and fucoidan desulfation activity refers to the release of sulfate groups bound to fucoidan sugar residues. Refers to the ability to Desulfation includes not only the case where all of the sulfate groups of fucoidan are eliminated, but also the state where a portion is eliminated. Furthermore, other terms used herein have meanings that are commonly understood by those of ordinary skill in the art.

  The most preferred three strains of the fucoidan-assimilating bacterium of the present invention are a medium containing fucoidan as a single carbon source, a medium containing fucoidan as a single carbon source, and a microbe in seawater, seaweed and soil collected at the Shirakaba coast of Tottori City, and a single carbon source. And obtained by culturing and screening in a medium containing a single sulfur source. DNA was extracted from the isolated strain, 16s rRNA gene of each strain was PCR amplified, and the nucleotide sequence was determined to identify and name Flavobacterium limicola F31 strain, Luteolibacter algae strain H18, and Luteolibacter algae SWi-1-Y strain. . These strains were deposited at the National Institute of Technology and Evaluation Patent Microorganisms Deposit Center, and were given receipt numbers NITE AP-674, NITE AP-675, and NITE AP-676, respectively. November 14, In the following, Flavobacterium limicola is referred to as F. limicola and Luteolibacter algae Sometimes abbreviated as algae.

  Fucoidan can be desulfated and reduced in molecular weight using the fucoidan-assimilating bacterium of the present invention or an extract thereof. The fucoidan used in the method for producing the desulfated low molecular weight fucoidan of the present invention may be a purified product, a crude product or a partially purified product, and may be a protein, lipid, other saccharide, etc. It may contain impurities. For example, seaweeds such as mozuku, kombu, seaweed and hijiki may be used in the method of the present invention as they are, or a crude extract thereof as fucoidan. Preferably fucoidan is treated as an aqueous solution. In consideration of the characteristics of the microorganism or its enzyme, substances such as salts and buffers may be added to the aqueous solution.

  You may make the fucoidan utilization microorganisms of this invention act on a fucoidan directly. In this case, you may culture | cultivate as usual using instruments, such as a test tube and a flask. In order to increase the scale, culture may be performed using a bioreactor or a tank. The bacterium of the present invention is preferably cultured aerobically, and shaking culture or aeration culture is suitable. In addition to fucoidan, for example, salts, buffers, vitamins, nutrient sources other than fucoidan, and the like may be appropriately added to the medium in order to promote or control the growth and enzyme activity of microorganisms. The culture temperature of the fucoidan-assimilating bacterium of the present invention is usually about 15 ° C. to about 40 ° C., preferably about 20 ° C. to about 37 ° C., more preferably about 25 ° C. to about 33 ° C., and the culture pH is usually about 6 to about 10, preferably about 7 to about 9, more preferably a pH range of seawater, such as a pH of about 7.5 to about 8.5. These conditions can be appropriately determined by a person skilled in the art through simple tests. The ratio of the amount of fungus body and the amount of fucoidan can also be determined appropriately. One type of bacterial cell of the present invention that acts on fucoidan may be used, or two or more types thereof.

  As the culture time elapses, fucoidan is reduced in molecular weight and desulfated. The molecular weight of fucoidan and the sulfation degree (desulfation degree) of fucoidan can be measured by known methods. The molecular weight of fucoidan can be measured using, for example, gel filtration HPLC (high performance liquid chromatography). To measure the degree of sulfation, sulfate groups can be quantified by, for example, a barium sulfate precipitation method.

  In the method for desulfating and reducing the molecular weight of fucoidan according to the present invention, the cell extract of fucoidan-assimilating bacteria of the present invention may be allowed to act on fucoidan. The extract of fucoidan-assimilating bacteria can be obtained by a known method. For example, what disrupted the microbial cell using the ultrasonic wave or the crusher can be used as an extract as it is. Alternatively, the supernatant of the crushed material can be used as an extract. Further, the extract of the fucoidan-assimilating bacterium of the present invention may be purified by a known method to obtain a fraction having a strong desulfation activity and a low molecular weight activity, and this may be used as the extract. Thus, the extract may be a crude enzyme or an enzyme purified to a single substance. Such purification can be performed by a known method, for example, ammonium sulfate fractionation, or various chromatography such as ion exchange chromatography or gel filtration chromatography. The shapes of these extracts, crude enzymes, and purified fractions are not particularly limited, and may be liquids or powders or solids such as lyophilized products.

  The extract obtained as described above can be added to fucoidan, preferably fucoidan aqueous solution, to perform desulfation and molecular reduction. In order to promote or control the reaction, a buffer, salts and the like may be appropriately added to the reaction system. The reaction temperature is usually about 15 ° C. to about 40 ° C., preferably about 20 ° C. to about 37 ° C., more preferably about 25 ° C. to about 33 ° C., and the pH of the reaction is usually about 6 to about 10, preferably about 7 To about 9, more preferably from about 7.5 to about 8.5. The reaction is preferably carried out with stirring. These conditions can be appropriately determined by a person skilled in the art through simple tests. The ratio between the amount of extract and the amount of fucoidan can also be determined as appropriate. As the culture time elapses, fucoidan is reduced in molecular weight and desulfated. The molecular weight of fucoidan and the sulfation degree (desulfation degree) of fucoidan can be measured by known methods (see above). The extract of the microbial cell of the present invention that acts on fucoidan may be one type, or two or three types.

  As described above, all of the fucoidan-utilizing bacteria of the present invention have both fucoidan low-molecularization activity and desulfation activity, but there are differences between these strains in the desulfation pattern and the low-molecularization pattern. F. Release of sulfate groups by the extract of limicola strain F31 begins after the molecular weight of fucoidan reaches about 20,000, whereas L. When an extract of algae SWi-1-Y strain is used, the release of sulfate groups can be seen at the stage where the molecular weight of fucoidan is about 140,000. L. in the case of algae H18 strain. Similar results are obtained with the limicola F31 strain.

  Therefore, F.R. limicola F31 strain or L. When the cells of algae H18 strain or an extract thereof are used, a desulfated relatively low-molecular fucoidan can be obtained. When the cells of algae SWi-1-Y strain or an extract thereof are used, a desulfated relatively high molecular fucoidan can be obtained. L. The cells of the algae SWi-1-Y strain or its extract have a higher desulfation activity than the other two strains.

  Using the microorganism of the present invention or an extract thereof, low molecular weight fucoidan having various molecular weights and different degrees of sulfation can be prepared. They can be used as materials for investigating the correlation between the structure and function of fucoidan. The low molecular weight fucoidan obtained by the present invention can be used as a low sulfated and low molecular weight fucoidan having an activity different from the original activity or having an enhanced original activity. Examples of the intrinsic activity of fucoidan include, but are not limited to, immunostimulation, antitumor, cholesterol and neutral fat reduction, gastric mucosa protection, liver function improvement, and antioxidant activity. Therefore, the low molecular weight fucoidan obtained by the present invention can be used as a raw material or ingredient for health foods, functional foods, supplements, cosmetics, and pharmaceuticals.

  EXAMPLES The present invention will be described more specifically and in detail below with reference to examples, but the examples are not intended to limit the present invention.

Isolation and identification of strains The following F medium was used for isolation of microorganisms capable of growing using fucoidan as a single carbon source. F medium is 2.5 g of fucoidan (manufactured by Marine Products Kimuraya Co., Ltd.), 4.0 g of dipotassium hydrogen phosphate, 0.5 g of potassium dihydrogen phosphate, 2.0 g of ammonium sulfate, magnesium sulfate heptahydrate per liter of distilled water. 0.41 g of the product, 10 ml of the metal mixed solution-F, and 1 ml of the vitamin mixed solution-F, and the pH is adjusted to 7.4. Metal mixture-F is 1 g of sodium chloride, 2 g of calcium chloride, 0.5 g of iron (II) sulfate, 0.5 g of zinc sulfate, 0.5 g of manganese chloride tetrahydrate, and 0 of copper (II) sulfate per liter of distilled water. .05 g, a solution containing 0.1 g of sodium molybdate dihydrate and 0.05 g of sodium tungstate dihydrate. Vitamin mixture-F contains 100 mg of biotin, 100 mg of thiamine hydrochloride, 100 mg of riboflavin, 100 mg of calcium pantothenate, 100 mg of pyridoxal phosphate, 100 mg of nicotinamide, 20 mg of sodium parabenzoate, 10 mg of cyanocobalamin and 10 mg of lipoic acid per liter of distilled water. It is a solution.

  The following AF medium was used for isolation of microorganisms capable of growing fucoidan as a single carbon source and a single sulfur source. The AF medium is 2.5 g of fucoidan (manufactured by Kimuraya Co., Ltd.), 1 g of dipotassium hydrogen phosphate, 0.5 g of potassium dihydrogen phosphate, 1.0 g of ammonium chloride, 0.1 g of magnesium chloride per liter of distilled water. 2 g, metal mixture-AF 10 ml, vitamin mixture-AF 1 ml, pH adjusted to 7.4. Metal mixture-AF is 1 g of sodium chloride, 2 g of calcium chloride, 0.5 g of iron (II) chloride, 0.5 g of zinc chloride, 0.5 g of manganese chloride tetrahydrate, and 0 of copper (II) chloride per liter of distilled water. .05 g, a solution containing 0.1 g of sodium molybdate dihydrate and 0.05 g of sodium tungstate dihydrate. Vitamin mixed solution-AF is a solution containing 400 mg of calcium pantothenate, 200 mg of inositol, 200 mg of pyridoxal phosphate, 400 mg of nicotinamide, 200 mg of sodium parabenzoate, and 0.5 mg of cyanocobalamin per liter of distilled water.

  Samples such as soil, mozuku algae, and seawater concentrate were suspended in autoclaved F medium and AF medium, shaken at 30 ° C., and samples with increased turbidity of the medium were transplanted to a new medium. . If a sample in which turbidity increase is observed even if this operation is repeated 10 times or more is applied to a plate medium having the same composition as the liquid medium and containing 1.5% of agar, once a single colony is obtained, the liquid is again removed from the colony. Inoculated into the medium. By repeating these operations, fucoidan-assimilating microorganisms were purified and named F31 strain, H18 strain from the system using F medium, and SWi-1-Y from the system using AF medium. One strain of target microorganism was isolated. The F31 strain was deposited at the Patent Evaluation Microorganism Deposit Center of the National Institute of Technology and Evaluation, and was given a receipt number NITE AP-674 as of November 14, 2008. The H18 strain was deposited with the National Institute of Technology and Evaluation Patent Microorganisms Deposit Center, and was given the receipt number NITE AP-675 as of November 14, 2008. The SWi-1-Y strain was deposited with the Patent Microorganism Deposit Center, National Institute of Technology and Evaluation, and was given a receipt number NITE AP-676 as of November 14, 2008.

  DNA was extracted from the isolates according to a conventional method, and the 16s rRNA gene of each strain was PCR amplified to determine the base sequence. As a result, the F31 and H18 strains were 97% homologous to the Flavobacterium limicola and Luteolibacter algae 16s rRNA genes, indicating that these strains were identified as the Flavobacterium limicola F31 strain, the Luteolibacterial strain H Named. In addition, the same experiment was performed on the SWi-1-Y strain, and it was identified and named as a Luteolibacter algae SWi-1-Y strain.

Preparation of crude enzyme and enzyme reaction (1) Preparation method and enzyme reaction method of crude enzyme The above strains were cultured in 500 ml of the medium used for screening, and the cells were collected by centrifugation when the growth stopped. After washing the cells with physiological saline, the cells were suspended in 20 mM Tris-HCl buffer (pH 8.0), and the cells were disrupted by ultrasonic treatment, and the centrifuged supernatant was used as a crude enzyme.

  The enzyme reaction was performed at 30 ° C. by adding the crude enzyme to a reaction solution containing 0.25% fucoidan and 100 mM Tris-HCl buffer (pH 8.0) so that the protein concentration was 2 mg / ml. After the enzyme was inactivated by heat treatment (80 ° C., 5 minutes) to stop the reaction, the molecular weight of fucoidan and free sulfate groups in the reaction solution were quantified.

The molecular weight of fucoidan was calculated based on the peak top retention time in gel filtration HPLC. Pullulan was used as the molecular weight standard. The HPLC conditions were as follows: Column: TSK-Gel GMPW _XL (φ7.8 mm x 300 mm) (TOSOH), detection method: RI, mobile phase: 0.1 M NaNO ₃ (pH 4.9), flow rate: 0.6 ml / min went.

The sulfate group was quantified according to Dodson's method (Biochem. J., 84, 350-356 (1962)). The amount of barium sulfate precipitated by adding a barium chloride-gelatin solution was measured for turbidity at 500 nm. Was done.
The enzyme activity was also measured by quantifying the reducing end of the sugar produced by the progress of the enzyme reaction. The amount of reducing terminal was quantified by measuring the absorbance at 420 nm after adding potassium ferricyanide solution to the enzyme reaction solution and heating for 15 minutes.

(2) F.E. Reduction of molecular weight and desulfation of fucoidan using crude enzyme of limicola F31 strain The low molecular weight desulfation reaction of fucoidan by the crude enzyme of limicola F31 strain proceeded as shown in FIG. As a result of this reaction, release of the reducing end of the sugar was observed as fucoidan was reduced in molecular weight. It was found that the desulfation reaction started after the molecular weight of fucoidan reached about 20,000.

上の四角で囲ったデータは脱硫酸化反応が確認された時点のデータ、下の四角で囲ったデータは反応２４時間後のデータであることを示す。 When the enzyme reaction was carried out for 24 hours using the crude enzymes of three types of isolates, the molecular weight and free sulfate group amount of fucoidan changed as shown in Table 1 and FIG. F. Release of sulfate groups by the crude enzyme of limicola F31 strain starts after the molecular weight of fucoidan reaches about 20,000, whereas L. When the crude enzyme of the algae SWi-1-Y strain was used, the release of sulfate groups was observed at the stage where the molecular weight of fucoidan was about 140,000. And L. When the crude enzyme of the algae SWi-1-Y strain was used, there was a tendency that the amount of released sulfate groups was larger than when the other two strains of crude enzyme were used. L. in the case of algae H18 strain. The result was similar to that of limicola F31 strain. L. The crude enzyme of the algae SWi-1-Y strain had higher desulfation activity than the other two strains of the crude enzyme.

The data enclosed in the upper square indicates data at the time when the desulfation reaction was confirmed, and the data enclosed in the lower square indicates data after 24 hours of reaction.

(3) F.E. Acquiring low molecular weight and desulfated fucoidan using crude enzyme of limicola F31 strain 400 mg fucoidan (manufactured by Kimuraya Co., Ltd., molecular weight 200,000, sulfate group content 19%) The enzyme reaction was carried out using a 100 mM Tris-HCl buffer (pH 8.0) containing 320 mg of the crude enzyme of the limicola F31 strain as a protein weight at 30 ° C. for 3.5 hours while mixing with a stir bar.

  After stopping the enzymatic reaction by heat treatment at 80 ° C. for 15 minutes, the high molecular compound and the salt are separated from the centrifuged supernatant by ultrafiltration membranes with a molecular weight cut off of 50,000 and 10,000, and the molecular weight is 8,500. A low molecular weight fucoidan was obtained. This solution was freeze-dried to obtain 84 mg of a powder sample. This sample was heat-treated in the presence of 1.7 M hydrochloric acid at 100 ° C. for 5 hours to remove sulfate groups and quantify the released sulfate groups. The resulting low molecular weight fucoidan contained sulfate groups. The rate was determined to be 16%.

(4) L. Acquisition of low molecular weight / desulfated fucoidan using crude enzyme of S. algae SWi-1-Y Fucoidan 500 mg (manufactured by Kimuraya Co., Ltd., molecular weight 200,000, sulfate group content 19%) The enzyme reaction was carried out using a 100 mM Tris-HCl buffer (pH 8.0) containing 400 mg of the crude enzyme of the algae SWi-1-Y strain as a protein weight, while mixing with a stir bar at 30 ° C. for 28 hours.

  After stopping the enzymatic reaction by heat treatment at 80 ° C. for 15 minutes, the polymer compound was separated from the centrifuged supernatant by an ultrafiltration membrane having a molecular weight cut off of 50,000. Further, desalting was performed by Sepharose CL-4B gel filtration chromatography (φ 2 cm × 90 cm, mobile phase was distilled water) to obtain fucoidan having a molecular weight of 4,000. This solution was lyophilized to obtain a 32 mg powder sample. This sample was heat-treated in the presence of 1.7 M hydrochloric acid at 100 ° C. for 5 hours to remove sulfate groups and quantify the released sulfate groups. The resulting low molecular weight fucoidan contained sulfate groups. The rate was determined to be 8.3%.

  As shown in the above experimental examples, low molecular weight fucoidans having different degrees of sulfation could be generated by using the enzyme of the novel microorganism of the present invention. L. When the algae SWi-1-Y strain was used, a desulfation reaction was observed even with a relatively high molecular weight fucoidan. Therefore, a higher molecular weight desulfated fucoidan was obtained than when the other two strains were used. It turns out that it is obtained. It was also found that the molecular weight and degree of sulfation of fucoidan can be controlled by appropriately selecting the amount of microorganisms or the amount of extract (enzyme), reaction time, and other reaction conditions.

  The present invention provides a research material for investigating the relationship among fucoidan activity, molecular weight and degree of sulfation. Furthermore, the low molecular weight fucoidan obtained by the present invention can be used as a raw material or ingredient for health foods, functional foods, supplements, cosmetics and pharmaceuticals.

F. It is a graph which shows the time-dependent change of the low molecular weight (fucoidan molecular weight), desulfation reaction (free sulfate group amount), and reducing terminal amount of fucoidan by the crude enzyme of limicola F31 strain. L. algae SWi-1-Y, L. algae F31 strain and L. It is a graph which shows the time-dependent change of the low molecular weight (fucoidan molecular weight) and desulfation reaction (free sulfate group amount) of fucoidan by the crude enzyme of algae H18 strain. The progress of the reaction up to 3 hours in the upper graph is shown in the lower graph.

Claims (5)

A method for producing a desulfated low-molecular-weight fucoidan, comprising causing a fucoidan-utilizing bacterium, Luteolibacter algae , having fucoidan-desulfating activity and low-molecular-weight activity to act on fucoidan. Bacteria, National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center entrusted number NITE The method of claim 1 wherein the Luteolibacter algae H18 strain deposited as P-675. Bacteria, National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center entrusted number NITE Luteolibacter algae SWi-1-Y strain The method of claim 1, wherein deposited as P-676. National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center entrusted number NITE The Fucoidan-utilizing bacterium Luteolibacter algae H18 strain deposited with P-675 and having fucoidan desulfation activity and low molecular weight activity. National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center entrusted number NITE Fucoidan-utilizing bacterium Luteolibacter algae SWi-1-Y strain deposited with P-676 and having fucoidan desulfation activity and low molecular weight activity.

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