JP2006034166A - Method for producing degraded product of mannan - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a highly safe method for producing a degraded product of a mannan by converting the mannan to a low-molecular product by using microorganisms associated to human beings. <P>SOLUTION: The mannan is degraded by culturing Bacteroides vulgatus having ability for producing β-mannanase in a medium containing the mannan. The Bacteroides vulgatus is highly safe because of being derived from the human beings, and can produce the degraded product of the mannan in the culture solution when the producing microorganisms are cultured by using the mannan as a carbon source. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a novel β-mannanase, a β-mannanase producing bacterium, a method of producing a mannan degradation product by culturing a β-mannanase producing bacterium and mannan.

  Mannan is a water-soluble indigestible polysaccharide, and typical examples include galactomannan present in guar and locust bean, glucomannan present in konjac potato, iris, aloe and the like. For example, mannan derived from konjac potato is solidified by adding slaked lime and used as edible konjac. In addition to being used as food, mannan is also used as a raw material for pharmaceuticals as a water-soluble dietary fiber that is not degraded by human digestive enzymes. As the action of dietary fiber, constipation is improved, blood cholesterol is lowered, and the number of Bifidobacterium in the intestine is increased.

  On the other hand, mannan has various actions as described above, but its aqueous solution has a high viscosity, so it is limited to applications such as konjac and jelly, and if it has a blockage in the digestive tract due to its high viscosity, it will pass through the digestive tract. May cause trouble. In some cases, calcium, oily vitamins, and the like are incorporated into mannan and absorption into humans is inhibited. For this reason, attempts have been made to degrade the polymer mannan to lower the viscosity, and various cellulases and mannanases have been used.

  For example, a method for producing a partial degradation product of a polysaccharide contained in konjac flour, which is decomposed by cellulase derived from a microorganism belonging to the genus Aspergillus to obtain water-soluble dietary fibers having an average molecular weight of 2,000 to 15,000 Is disclosed (Patent Document 1). The aqueous solution of konjac flour is highly viscous and gels, and has the property of adsorbing minerals necessary for human nutrition. Therefore, in order to overcome the above difficulties while maintaining the function of dietary fiber of konjac flour, Among enzymes that hydrolyze β-glucoside bonds, in particular, a cellulase derived from a microorganism belonging to the genus Aspergillus is used. In the examples, commercially available cellulase is added to konjac flour for decomposition.

  There is also a method for producing mannose-containing saccharides using β-mannanase (Patent Document 2). Using β-mannanase obtained by producing and accumulating any of the alkaliphilic microorganisms belonging to the genus Bacillus and having an alkalinity optimum pH in the culture medium, any one of the deposits No. 8856 to No. 8860 The β-mannanase is allowed to act on a saccharide substrate having a β-1,4-mannopyranoside bond as a main skeleton. Note that β-mannanase is used after being produced outside the cells by culturing enzyme-producing microorganisms and then separated from the cells.

  Also disclosed is a method using an enzyme produced by Bacillus subtilis M-1 deposited under the accession number FERM P-14139 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology as β-mannanase (Patent Document 3). ). The producing bacterium is isolated from soil and is characterized by producing only β-mannanase. In the examples, β-mannanase obtained by culturing the producing bacterium is separated from the microbial cells by centrifugation and then hydrolyzed by acting on the konjac mannan solution.

Furthermore, a method using an enzyme produced by Bacillus polymixer strain KT551 deposited as FERM P-18001 as β-mannanase is also disclosed (Patent Document 4). The producing bacteria are isolated from soil, and β-mannanase can be efficiently produced extracellularly by culturing in a medium containing the copula oil residue of coconut oil. Mannanase is not described in the literature. In the examples, β-mannanase obtained by culturing the producing bacterium by supplying copra oil residue as a carbon source was separated from the microbial cells by centrifugation, and then was allowed to act on the konjac mannan solution to cause hydrolysis. .
JP-A-2-222659 JP-A 63-49093 JP 7-274960 A JP 2002-65257 A

  However, all of the methods described in the above documents use the enzyme after isolating the mannan-degrading enzyme, which requires an enzyme isolation step. As a result, the manufacturing process of the mannan-decomposed product becomes complicated. ing. In addition, many enzyme-producing bacteria are collected from soil, and their safety to humans is unknown.

  In addition, conventional enzymes may be degraded to oligosaccharides due to their own properties or because of the difficulty in controlling enzyme action, and the function of dietary fiber by mannan may be lost.

  In view of the above situation, the present invention has an object to provide a method for reducing the molecular weight of mannan using a microorganism having high safety and relevance to humans.

  The present invention also provides a method for producing a mannan degradation product having a low molecular weight without decomposing oligosaccharides so that functions such as dietary fiber can be exhibited.

  The present inventor paid attention to human intestinal bacteria from the viewpoint of safety and investigated in detail microorganisms that can act as mannan-degrading enzymes. As a result, Bacteroides bulgatus can produce β-mannanase. It has been found that when Bacteroides bulgatus is cultured in a mannan solution, mannan can be reduced in molecular weight in the same manner as in the case where the isolated β-mannanase is allowed to act. Moreover, since the β-mannanase is an endo type, mannan is not decomposed into monosaccharides.

  According to the present invention, it is possible to easily produce a mannan degradation product having a reduced molecular weight. Since β-mannanase produced by Bacteroides bulgatus is an endo-type, when this enzyme is used, it is possible to produce a mannan degradation product that retains the function of dietary fiber and the like without degradation to oligosaccharides. Moreover, since Bacteroides bulgatus can be cultured in a mannan solution to produce a mannan degradation product, it is not necessary to isolate β-mannanase, and the production process and production time can be simplified. In addition, Bacteroides bulgatus that produces β-mannanase is an obligate anaerobic bacterium derived from the human intestine, and is quickly killed when exposed to air, so there is little possibility of contamination to the product or the environment. Excellent safety.

  The first of the present invention is β-mannanase produced by Bacteroides bulgatus.

Bacteroides bulgatus is not particularly limited, but can be obtained by culturing Bacteroides bulgatus GM4 (FERM P-20107) in a medium containing mannan as a carbon source, and has the following properties.
(1) Action and substrate specificity: this enzyme is an endo-type β-mannanase that acts on β-mannans such as glucomannan and galactomannan and hydrolyzes its β-1,4-D-mannopyranosyl bond, It does not act on α-mannan.

  (2) Optimal pH: The optimal pH of the enzyme is 6.5 to 7.5.

  (3) Molecular weight: It is considered that there are a plurality of isozymes having different molecular weights, and typical molecular weights by SDS-polyacrylamide electrophoresis are about 36 kDa and 59 kDa.

The mycological properties of this bacterium are shown below. However, + shows positive and-shows negative.
1. Cell morphology (a) Neisseria gonorrhoeae (size: 0.5-0.8 μm × 1.5-8.0 μm)
(B) Motility −
(C) Gram staining −
(D) Endospores −
2. Physiological properties (a) Indole production −
(B) Formation of nitrite from nitrate-
(C) Catalase −
(D) Starch hydrolysis +
(E) Hydrolysis of esculin +
3. Sugar decomposition Acid generation (a) Arabinose +
(B) Cellobiose −
(C) Esculin −
(D) Fructose +
(E) Glucose +
(F) Inositol −
(G) Maltose +
(H) Mannitol −
(I) Raffinose +
(J) Salicin −
(K) Starch +
(L) Sucrose +
(M) Trehalose −
(N) Xylose +
4). 4. Attitude toward oxygen Optimal ph 7.2
6). Major acids produced from glucose Acetic acid, formic acid, propionic acid, succinic acid End-type β-mannanase +
8). Exo-type β-mannanase −
From the above properties, it is clear that this strain belongs to the genus Bacteroides, which is described in Berge's Manual of Systematic Bacterilogy Vol. As a result of searching based on 1,1984, it was identified as Bacteroides bulgatas. None of the known strains are similar to or similar to this strain, and it was recognized as a new strain, and this strain was designated as Bacteroides bulgatus GM4 strain. This strain has been deposited with the National Institute of Advanced Industrial Science and Technology, and its deposit number is FERM P-20107. That is, the second of the present invention is Bacteroides bulgatus GM4 (FERM P-20107) having the β-mannanase producing ability. In the present invention, even a mutant obtained by mutating this strain naturally or by artificial means is included as long as it has the ability to produce the target β-mannanase.

  The β-mannanase of the present invention is produced by centrifuging the culture solution because β-mannanase is released outside the cells when the Bacteroides bulgartus GM4 (FERM P-20107) bacterium is cultured in a mannan-containing liquid medium. The enzyme can be purified by separating from the cells and subjecting the supernatant to salting out, gel filtration, ultrafiltration, chromatography using an ion exchange resin, and the like. In particular, Bacteroides bulgatus GM4 (FERM P-20107) of the present invention is an extremely safe bacterium isolated from the intestines of healthy humans, and is an endo type and excellent in β-mannanase activity. It is extremely suitable for lowering the molecular weight of mannan. Conventionally, various β-mannanases have been used for the degradation of mannan, but there are no end-type ones derived from obligate anaerobes such as the present invention. Unlike other β-mannanase-producing bacteria, it is an obligate anaerobic bacterium, so it can be easily killed when exposed to air after the target operation is completed, and it has no spores like Bacillus genus. There is no worry about pollution to the environment and it is extremely safe.

  A third aspect of the present invention is a method for producing a mannan degradation product, which comprises culturing Bacteroides bulgatus having β-mannanase producing ability in a medium containing mannan and decomposing the mannan.

  In the present invention, Bacteroides bulgatus having a β-mannanase producing ability can be widely used. Bacteroides bulgatus GM4 (FERM P-20107) is preferable. Since it is human-derived, highly safe, and produced β-mannanase is an endo-type and does not decompose mannan into oligosaccharides, mannan should be included within the range that retains functions such as dietary fiber with easy control. Low molecular weight can be achieved.

  The mannan used in the present invention broadly means a polysaccharide mainly composed of mannose, and may be a galactomannan containing galactose or a glucomannan containing glucose. Mannan is widely present in wood and plants, but its origin is not limited. Therefore, konjac potatoes, arum roots of the taro family, jack pine of the genus pine, bulbs of the orchid family, spruce plants such as spruce and spruce, elephant palms, huenix canariensis, okis maquilata, copra, locust bean gum , Guar gum, spino gum, soybeans, coffee beans, purple palm, red clover, fenugreek. Examples of other mannan-containing plants include mannan obtained from Asparagus officinalis, Elemlus Huscus, Elemras reggery, Elemras Spectabilis, Huaceoles aureus, and the like. These are generally obtained by an alkali extraction method or the like. Furthermore, commercially available konjac flour (milling, medium content, coarse content, etc.), refined mannan, and various β-D-mannans conventionally used as a paste or thickener in the food industry or textile industry may be used. In addition, these mannans may contain starch. This is because Bacteroides bulgatus used does not affect the isolation of mannan from the culture solution because it also degrades starch. The mannan is added as a carbon source for Bacteroides bulgatus. In the present invention, konjac mannan is preferably used because it is easily available.

  The culture of this production bacterium may be basically carried out according to the medium and culture conditions used for cultivation of Bacteroides bulgatus, but a medium containing the above mannan as a carbon source is used. As the nitrogen source, peptone, polypeptone, meat extract, yeast extract and the like can be used alone or in combination. In addition, buffering agents such as monopotassium hydrogen phosphate, inorganic salts such as magnesium sulfate, and trace amounts of heavy metals can be added.

The culture is performed under anaerobic conditions. In the case of batch culture, the gas phase is replaced with nitrogen gas, carbon gas, or the like. Generally, the mannan concentration in the culture solution is 0.01 to 5% by mass, more preferably 0.5 to 2% by mass. When the concentration is higher than 5% by mass, the viscosity of mannan is too high and it may be difficult to culture and stir. On the other hand, when it is less than 0.01% by mass, productivity is lowered. The Bacteroides bulgatus is generally cultured at a rate of 1 × 10 ² to 1 × 10 ⁸ cells / ml in the medium. The culture temperature is 30 to 40 ° C., preferably 35 to 38 ° C., and the culture period is 2 to 7 days, preferably 3 to 5 days. During culture, the pH of the medium is preferably maintained at 6-8. In addition, completion | finish of reaction can be judged by the viscosity of a culture solution.

  A feature of the present invention is that mannan is contained as a carbon source in a medium of Bacteroides bulgatus that produces β-mannanase, instead of degrading mannan using β-mannanase isolated from Bacteroides bulgatus. This is in the point of lowering the molecular weight. Since β-mannanase is produced outside the cells, mannan is reduced in molecular weight by β-mannanase in the medium, but the difference is that there is no step to isolate β-mannanase from the producing bacteria. To do. Conventionally, since β-mannanase is superior in heat resistance to its producing bacteria, mannan is often reduced in molecular weight using the isolated β-mannanase. Therefore, the isolation of β-mannanase is an essential process. It was. In addition, when mannan degradation products are used for food, medical use, etc., the safety of the product is important. If the origin of the β-mannanase-producing bacterium is not clear, it is difficult to ensure safety, and for this reason, mannan was not decomposed by the microbial cells. However, in the present invention, since Bacteroides bulgatus derived from humans is used, safety is secured, mannan is supplied as a carbon source in the medium without isolating β-mannanase, and mannan is easily reduced in molecular weight. can do. In addition, the molecular weight of the target mannan can be adjusted by changing the culture time, and a complicated method for adjusting the enzymatic reaction with pH or temperature is not required. In the present invention, a mannan decomposition product having an average molecular weight of 2,000 to 1,000,000, more preferably 5,000 to 500,000, particularly 10,000 to 200,000 is preferable. Within this range, the viscosity is lower than that of mannan before decomposition, so that the handling is easy and the function as dietary fiber can be maintained.

  After completion of the culture, the culture contains microbial cells, mannan degradation product and β-mannanase. The cells can be separated by centrifuging the culture solution. The supernatant after cell separation is an aqueous solution containing a mannan degradation product.

  When inorganic salts such as potassium chloride and ammonium sulfate are added to the mannan decomposed product-containing aqueous solution at a saturation degree of 50 to 80%, more preferably 60 to 70%, β-mannanase is mainly precipitated. The precipitate is dialyzed with 50 mM phosphate buffer (pH 7.2) or the like, and β-mannanase can be recovered by performing anion exchange chromatography, gel filtration or the like. The obtained β-mannanase can be powdered by spray drying or freeze drying.

  When an organic solvent such as ethanol is added to the mannan degradation product-containing aqueous solution, the mannan degradation product is preferentially precipitated over β-mannanase. When the precipitate is collected and stirred with water, the mannan decomposition product dissolves in water. Therefore, the mannan decomposition product can be purified by repeating the above precipitation operation and water dissolution operation. The obtained mannan decomposition product can be powdered by spray drying or freeze drying.

  Meanwhile, an inorganic salt such as potassium chloride, ammonium sulfate or the like is added to the solution containing the mannan decomposition product-containing aqueous solution, the ethanol precipitate in water, or the purified mannan decomposition product in water. More preferably, when it is added at 15 to 35%, a mannan decomposition product mainly precipitates. This mannan decomposition product is insoluble and does not dissolve even when water is added again. Therefore, when purified water is added to prepare a suspension, and centrifugation and suspension are repeated, an insoluble mannan degradation product can be obtained. Such an insoluble mannan degradation product can be suitably used as an insoluble dietary fiber as a constipation therapeutic agent, an adsorbent, an excipient and the like.

  EXAMPLES Next, although an Example is given and this invention is demonstrated concretely, these Examples do not restrict | limit this invention at all.

(Example 1)
A 40-liter aqueous solution containing 200 g of potassium phosphate-potassium was added with 800 g of peptone (Mikuni Chemical) and 400 g of konjac mannan (Wako Pure Chemical) to prepare a medium, which was sterilized at 121 ° C. for 30 minutes.

The gas phase part of the medium was filled with nitrogen gas and cooled to 37 ° C. Next, 800 ml of Bacteroides bulgatus GM4 (FERM P-20107) culture solution (1 × 10 ⁸ / ml) pre-cultured in the same medium was inoculated into the medium and cultured at 37 ° C. for 4 days with stirring.

  The cells were removed by continuous centrifugation after culturing. An equal amount of ethanol was added to the supernatant, and the generated precipitate was removed by centrifugation. Next, ammonium sulfate was added to the supernatant so that the saturation was 60%, and the mixture was allowed to stand at 4 ° C. overnight, and then centrifuged at 10,000 g for 30 minutes to collect the precipitate. Dialyzed overnight against 50 mM phosphate buffer (pH 7.2) while cooling. Next, ion exchange chromatography was performed with an anion exchange resin (SOURCE 30Q; Amersham Bioscience), and then gel filtration was performed with Sephacryl 1 S-200HR (Amersham Bioscience) to collect fractions having β-mannanase activity. After concentration with an ultrafilter (Millipore), freeze drying was performed to obtain 800 mg of enzyme powder. The molecular weight by SDS PAGE was about 36 kDa and 59 kDa. The results are shown in FIG.

(Example 2)
A medium was prepared by adding 800 g of peptone (Mikuni Chemical) and 400 g of commercially available konjac powder to 40 liters of an aqueous solution containing 200 g of potassium phosphate-potassium, and sterilized at 121 ° C. for 30 minutes.

The gas phase part of the medium was filled with nitrogen gas and cooled to 37 ° C. Next, 800 ml of Bacteroides bulgatus GM4 (FERM P-20107) culture solution (1 × 10 ⁸ / ml) pre-cultured in the same medium was inoculated into the medium and cultured at 37 ° C. for 4 days with stirring.

  The cells were removed by continuous centrifugation after culturing. An equal amount of ethanol was added to the supernatant, and the generated precipitate was collected by centrifugation, and the precipitate was redissolved in purified water. An equal amount of ethanol was again added thereto, and the generated precipitate was collected by centrifugation. The operations of dissolving the precipitate in purified water and ethanol precipitation were repeated three times to remove impurities. When the purified precipitate was spray-dried, 345 g of partially decomposed mannan was obtained. When the molecular weight was measured by gel filtration, it was 10,000 to 30,000.

Example 3
The procedure was the same as in Example 2 except that the culture time was changed to 3 days, and 368 g of partially decomposed mannan was obtained by lyophilization. The molecular weight of this product was 100,000 to 280,000.

Example 4
After culturing in the same manner as in Example 2, the cells were removed by centrifugation. Ammonium sulfate was added to the supernatant so that the saturation was 20%, and the mixture was allowed to stand at 4 ° C. for 4 hours. The precipitate was collected by centrifugation at 10,000 g for 30 minutes. The precipitate was suspended in purified water and centrifuged at 10,000 g for 30 minutes. This centrifugation and suspension operation was repeated three times to remove impurities. When the purified precipitate was lyophilized, 330 g of partially decomposed mannan was obtained. This mannan decomposition product was insoluble in water and stable to acids and alkalis.

  According to the β-mannanase of the present invention, for example, konjac mannan can be easily reduced in molecular weight. Bacteroides bulgatus GM4 (FERM P-20107) that produces the β-mannanase is highly safe because it is derived from humans, and the mannan degradation product obtained using this is useful as a food or medicine. is there.

FIG. 1 shows SDS-PAGE of a novel β-mannanase. Lane 1 is a molecular weight marker, and lanes 2 and 3 are samples during the purification process of β-mannanase. The arrow indicates the band of novel β-mannanase.

Claims (8)

  Β-mannanase produced by Bacteroides bulgatus.   The β-mannanase according to claim 1, which is an endo type.   Bacteroides bulgatus GM4 (FERM P-20107) having the β-mannanase producing ability according to claim 1 or 2.   A method for producing a mannan degradation product, comprising culturing Bacteroides bulgatus having β-mannanase producing ability in a medium containing mannan and decomposing the mannan.   A mannan degradation product-containing aqueous solution comprising a step of charging Bacteroides bulgatus having β-mannanase-producing ability into a mannan solution having a concentration of 0.01 to 5% by mass and culturing at 30 to 40 ° C. Production method.   The manufacturing method of a mannan decomposition product including the process of adding alcohol to the said mannan decomposition product containing aqueous solution, and precipitating a mannan decomposition product.   An insoluble mannan decomposition product characterized by adding an inorganic salt at a saturation degree of 10 to 50% to an aqueous solution in which a mannan decomposition product having a molecular weight of 2,000 to 1,000,000 is dissolved to precipitate the mannan decomposition product. Production method.   The method for producing a mannan degradation product according to any one of claims 4 to 6, wherein the Bacteroides bulgatus is Bacteroides bulgatus GM4 (FERM P-20107).