JP2007028917A - Method for producing aldonic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing an aldonic acid capable of being safely used as a pharmaceutical, food, and feed, by using a bacterial cell of which the safety is guaranteed. <P>SOLUTION: This method for producing the aldonic acid comprises bringing the cell body of a microorganism belonging acetic acid bacteria (for example, Gluconobacter cerinus NBRC 3267 and Gluconobacter oxydans NBRC 3285) into contact with a saccharide having a hemiacetal hydroxy group other than glucose, oxidizing the hemiacetal hydroxy group, and forming the aldonic acid corresponding to the saccharide having the hemiacetal hydroxy group used as a substrate. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for producing aldonic acid using a microorganism belonging to acetic acid bacteria.

  In recent years, mineral supplements such as calcium, iron, and zinc have attracted attention. Among them, aldonic acid is considered to have a high mineral-reinforcing effect when it is in a salt form with a metal such as calcium or iron, and it is considered to have a high mineral reinforcing effect. Yes. In addition, aldonic acid obtained by oxidizing oligosaccharide is less susceptible to degradation by an in vivo hydrolase such as schuclase or β-galactosidase because the hemiacetal hydroxyl group of oligosaccharide is oxidized. For this reason, when ingested by humans, it can reach the intestine without being decomposed, and can be used as a factor that induces the growth of bifidobacteria. Because of such characteristics, methods for producing aldonic acid from various sugars have been searched.

  As a specific method for obtaining aldonic acid, a method is known in which a sugar having a hemiacetal hydroxyl group is oxidized by applying electricity together with sodium bromide. In addition, as a method obtained by microbial conversion / fermentation method, a method is known in which microorganisms such as Acinetobacter genus and Burkholderia genus are obtained by acting on a sugar having a hemiacetal hydroxyl group and oxidizing (eg, patent document). 1).

  Among aldonic acids, lactobionic acid (O-β-D-galactopyranosyl- (1-4) -D-gluconic acid) obtained by oxidizing the hemiacetal hydroxyl group of lactose has already been approved by the FDA in the United States. It is added to the pudding premix as a coagulant or used in cosmetics as a moisturizer. In terms of functionality, lactobionic acid is known to have bifidobacterial selective growth activity (for example, see Patent Document 2) and to have a mineral absorption promoting effect (for example, see Patent Document 3). Further, the present inventors have found that lactobionic acid has an effect of strengthening eggshell, and have already filed a patent application (Japanese Patent Application No. 2004-030898). Since it has such characteristics, it has been particularly desired to produce lactobionic acid easily and in a manner that ensures safety.

On the other hand, the genus Acetobacter known as acetic acid bacteria has been used for brewing acetic acid by oxidizing ethanol to acetic acid (see, for example, Non-Patent Document 1). Moreover, Gluconobacter genus oxidizes glucose to gluconic acid and 2 and 5-ketogluconic acid (for example, refer nonpatent literature 2). Gluconic acid bacteria belonging to the genus Gluconobacter are used for the production of L-sorbose from D-sorbitol (see, for example, Non-Patent Document 3). However, it has not been known to produce aldonic acid from a sugar having a hemiacetal hydroxyl group other than glucose.
JP 2001-245657 A Japanese Patent No. 3559063 Japanese Patent No. 3501237 Supervised by Toshiro Tochikura, Fermentation Handbook, Kyoritsu Shuppan, p.189-192 (2001) Supervised by Toshiro Tochikura, Fermentation Handbook, Kyoritsu Shuppan, p.163-164 (2001) Supervised by Toshiro Tochikura et al., Fermentation Handbook Kyoritsu Shuppan, p.257-258 (2001)

  As mentioned above, it was particularly desirable to produce lactobionic acid in an easy and safe manner. However, chemical synthesis of aldonic acid should be used for food and feed applications in accordance with domestic laws and regulations. I can't. In addition, microorganisms such as Acinetobacter and Burkholderia are not sufficiently safe, and there are problems in using aldonic acid produced using these microorganisms for food and feed applications. It was.

  An object of the present invention is to provide a method for efficiently producing aldonic acid using a microorganism whose safety is ensured.

  In order to achieve the above-mentioned problems, the present inventors have comprehensively examined bacteria with guaranteed safety, and as a result, microorganisms selected from acetic acid bacteria oxidize the hydroxyl groups of sugars having hemiacetal hydroxyl groups. The headline and the present invention were reached.

  That is, according to the first aspect of the present invention, an aldonic acid is characterized in that a microbial cell belonging to acetic acid bacteria is brought into contact with a sugar having a hemiacetal hydroxyl group other than glucose to oxidize the hemiacetal hydroxyl group to produce aldonic acid. The microbial cells belonging to acetic acid bacteria are preferably obtained by culturing in a medium supplemented with a sugar having a hemiacetal hydroxyl group and / or an alcohol as an inducer. The method is a microbial cell, and preferably the sugar having a hemiacetal hydroxyl group is D-mannose, D-galactose, D-ribose, D-xylose, L-arabinose, cellobiose, maltose, maltotriose, maltotetra At least one selected from the group consisting of ose, maltopentaose, melibiose and lactose Is one sugar.

  In the second aspect of the present invention, microorganisms belonging to acetic acid bacteria are cultured in a medium containing a sugar having a hemiacetal hydroxyl group other than glucose, whereby aldonic acid is accumulated in the culture solution, and aldonic acid is obtained from the culture solution. A method for producing an aldonic acid characterized by separation, wherein a sugar having a hemiacetal hydroxyl group is preferably D-mannose, D-galactose, D-ribose, D-xylose, L-arabinose. And at least one sugar selected from the group consisting of cellobiose, maltose, maltotriose, maltotetraose, maltopentaose, melibiose and lactose.

  ADVANTAGE OF THE INVENTION According to this invention, aldonic acid can be efficiently manufactured with the microorganisms used for food manufacture from a long time, and the safety | security was ensured, Moreover, it is not restrict | limited to the kind of sugar which has hemiacetal hydroxyl group, Corresponding aldonic acid Can be manufactured.

  Hereinafter, the present invention will be described in detail.

  The sugar having a hemiacetal hydroxyl group used in the present invention refers to a sugar whose reducing end is an aldehyde. Specific examples of such compounds include monosaccharides such as D-glucose, D-mannose, D-galactose, D-ribose, D-xylose, L-arabinose, cellobiose, maltose, maltotriose, maltotetraose, malto Examples thereof include oligosaccharides such as pentaose, melibiose, and lactose. Among these, D-mannose, D-galactose, D-ribose, D-xylose, L-arabinose, cellobiose, maltose, maltotriose, maltotetraose, maltopentaose, melibiose and lactose are particularly preferable. Lactose.

  In the present invention, aldonic acid includes not only those in which the hemiacetal hydroxyl group of aldose, which is a monosaccharide, is oxidized, but also those in which the hemiacetal hydroxyl group of a sugar having a aldose structure at the reducing end is oxidized, and these The salt form is also included.

  Accordingly, the present invention is a method for converting a sugar having a hemiacetal hydroxyl group into the corresponding aldonic acid. Specifically, D-mannose is mannonic acid, D-galactose is galactonic acid, D-ribose is ribbon acid, D-xylose is xylonic acid, L-arabinose is alabonic acid, and cellobiose is cellobionic acid. To convert maltose to maltobionic acid, maltotriose to maltotrionic acid, maltotetraose to maltotetraonic acid, maltopentaose to maltopentanoic acid, melibiose to melbionic acid, and lactose to lactobionic acid It is.

  The microorganism used in the present invention is an acetic acid bacterium, and specifically refers to a microorganism belonging to the genus Acetobacter or Gluconobacter. Since acetic acid bacteria not described above have been used for fermentation of foods for a long time, the safety of microorganisms is ensured.

  The acetic acid bacterium used in the method of the present invention can be an acetic acid bacterium isolated from the natural world. However, as in the case of the Independent Administrative Institution Product Evaluation Technology Infrastructure, it can be distributed to any person's request. It is also possible to use acetic acid bacteria stored by various depository institutions. These include, for example, Acetobacter aceti NBRC 3281, Acetobacter syzygii NBRC 16604, Acetobacter cibinongensis NBRC 16605, Acetobacter indonesiens , Gluconacetobacter xylinus NBRC 16670, Gluconacetobacter liquefaciens NBRC 12388, Gluconacetobacter sp. NBRC 3261, Gluconobuser cerinas NBRC 3267, Gluconobacter fraturii NBRC 3260, Gluconobacter oxydans NBRC 3285, and the like. However, it is not restricted to these strains.

  In the first production method of the present invention, a microbial cell belonging to acetic acid bacteria is brought into contact with a sugar having a hemiacetal hydroxyl group other than glucose, and an aldonic acid is generated by oxidizing the hemiacetal hydroxyl group with an enzyme in the microbial cell. Is the method. The microbial cell belonging to the acetic acid bacterium used in the present invention refers to a living microbial cell of the acetic acid bacterium as it is, or a processed microbial cell product, or those immobilized on a carrier.

  First, in order to obtain bacterial cells of acetic acid bacteria, it is necessary to perform culture. The medium can be cultured in the same manner as normal microorganisms. What mixed suitably components, such as a carbon source, a nitrogen source, a vitamin, and a mineral which can be normally utilized by microorganisms, is used. Examples of carbon sources include monosaccharides such as glucose and fructose, oligosaccharides such as sucrose and maltose, polysaccharides such as starch, sugar alcohols, and glycerol. Common carbohydrates include corn starch and malt. In addition, vegetable oils such as olive oil and corn oil are also included in the carbon source. CSL (corn steep liquor) or soybean flakes may be used. In addition, carbon compounds such as alcohol, organic acid, and alkane may be used.

  As the nitrogen source, both inorganic and organic nitrogen can be used. As the inorganic nitrogen, ammonia gas, ammonia salt, nitric acid or the like is used. Organic nitrogen is provided in the form of amino acids, proteins or urea. As natural organic nitrogen complex, CSL, soybean, soybean flake, peanut meal, cotton meal, Distillers' solubles, casein hydrolyzate, slaughterhouse waste, fish meal, yeast extract and the like are used.

  As vitamins, vitamins necessary for the growth of microorganisms can be sufficiently supplemented by using natural carbon sources and nitrogen sources, but calcium pantothenate, biotin, vitamin B1 and the like are added as necessary.

  As minerals, magnesium, phosphoric acid, potassium, sulfuric acid, calcium, and chlorine are required, but cobalt, copper, iron, manganese, molybdenum, zinc, and the like are also required in a trace amount.

  As other components, for controlling pH, calcium carbonate may be added to the culture solution, or phosphate may be added to provide a buffering action. In order to control the pH of the culture system, ammonia, caustic soda, hydrochloric acid, sulfuric acid or the like may be added.

  Cultivation is preferably carried out under the optimum culture conditions for the strain to be used, but the approximate temperature is preferably 20 to 30 ° C., and the pH is adjusted to 5 to 8 with hydrochloric acid, sodium hydroxide aqueous solution, calcium carbonate or the like. It is preferable to maintain.

  Examples of the culture method include stationary culture, shaking culture, and deep aeration and agitation culture. In the case of mass culture, deep aeration and agitation culture using a batch method, a sequential addition culture method or a continuous culture method is preferable. When culturing is performed under such conditions, a sufficient amount of microorganisms can be obtained in 15 to 72 hours after culturing.

  In the present invention, it is preferable to add a saccharide having a hemiacetal hydroxyl group as an enzyme inducer to the medium during the above-described culture because the conversion efficiency to aldonic acid can be increased. Examples of the sugar having a hemiacetal hydroxyl group used herein include monosaccharides such as D-glucose, D-mannose, D-galactose, D-ribose, D-xylose, and L-arabinose, cellobiose, maltose, maltotriose, malto Examples thereof include oligosaccharides such as tetraose, maltopentaose, melibiose, and lactose. In addition to these sugars, alcohols such as ethanol and isopropanol may be added as inducers.

  The addition amount of the derivatizing agent is 0.1 to 100 g / L, more preferably 0.1 to 10 g / L.

  In this case, when acetic acid bacteria cannot grow only with the sugar, it is necessary to add and cultivate a carbon source that can be assimilated. Examples of carbon sources that can be generally assimilated by acetic acid bacteria include monosaccharides such as glucose and fructose, oligosaccharides such as sucrose and maltose, polysaccharides such as starch, sugar alcohol, and glycerol. The amount of carbon source added is generally 10 to 200 g / L, more preferably 10 to 50 g / L. As other medium components, the aforementioned nitrogen source, minerals, vitamins, buffers and the like may be added in order to optimize the culture conditions.

  In the case where the live cells of acetic acid bacteria obtained as described above are used as they are, the collected cells can be used as they are.

  When used as a cell-treated product of acetic acid bacteria, cells treated with drugs with acetone, quaternary ammonium compounds, lauryl soda sulfate, Tween, or an enzyme that breaks down the specific binding of the cell walls of microorganisms, frozen cells Freeze-dried microbial cells obtained by sublimating water under reduced pressure, homogenizers and glass beads, physically crushed microbial cells, and cell-free extract that is the supernatant of microbial cell crushed materials, A crude enzyme solution obtained by extracting an enzyme from these can be used.

  In order to immobilize acetic acid bacterial cells or treated products on a carrier, a method of adsorbing to a substance such as a cellulose carrier, a ceramic carrier, a glass bead carrier, a gel-like substance having a lattice structure, such as agar, calcium alginate, Examples thereof include carrageenan and a method included in known polymers. This method allows the microorganism to be used repeatedly.

  In the first production method of the present invention, a sugar having a hemiacetal hydroxyl group serving as a substrate is dissolved in a reaction solvent, and a culture solution, bacterial cells, or treated bacterial cells prepared as described above are added. If necessary, the reaction is carried out while controlling the reaction temperature and the pH of the reaction solution. As the reaction solvent, an aqueous solvent such as ion-exchanged water or a buffer solution can be used. The substrate concentration of the reaction solution is not particularly limited, but it is preferably 10 to 50% by mass in consideration of the solubility and productivity of the sugar as a substrate.

  Although there is no restriction | limiting in particular in reaction time, It is preferable to select the conditions which reaction completes normally in 6 to 24 hours, preferably 6 to 12 hours. The reaction pH depends on the optimum pH of the microbial enzyme to be used, but is generally in the range of pH 4 to 8, particularly 5 to 7. Moreover, the reaction temperature should just be the conditions which a microbial enzyme does not deactivate, 20-60 degreeC is preferable, but 30-45 degreeC is more preferable.

  Examples of the reaction method include standing, shaking, and deep aeration stirring. When culture is performed under such conditions, a sufficient amount of product is obtained in 15 to 72 hours.

  In order to isolate and purify the target aldonic acid from the reaction solution obtained as described above, a general separation method such as extraction or column separation can be used. For example, ethanol is added to the reaction solution, and the target product is precipitated and collected, and is isolated using activated carbon, adsorption chromatography using porous organic resin particles, gel filtration, or ion exchange resin chromatography. be able to.

  The second production method of the present invention comprises culturing a microorganism belonging to acetic acid bacteria in a medium containing a sugar having a hemiacetal hydroxyl group other than glucose, thereby accumulating aldonic acid in the culture solution, The acid is separated. As the medium component used for the culture, the above-described medium components used in the first production method of the present invention are similarly used except that the medium contains a sugar having a hemiacetal hydroxyl group. As the sugar having a hemiacetal hydroxyl group, the same sugars as described above can be used. As these content, it is 10-500 g / L, More preferably, it is 10-100 g / L.

  The culture conditions can be the same as the culture conditions described above except that the culture period is usually 1 to 10 days.

  Aldonic acid accumulated in the culture solution can be separated and purified in the same manner as described above.

  The aldonic acid produced by the present invention exhibits an intestinal regulating action, a lipid metabolism improving action, a mineral absorption promoting action, and an eggshell strengthening action, and is useful as a pharmaceutical product, food or feed. When the aldonic acid is used as a pharmaceutical product, tablets using excipients, lubricants, diluents, pH adjusters, preservatives, sweeteners, fragrances, emulsifying powders and the like conventionally used in the field are used. Can be administered orally or parenterally as powders, wettable powders, emulsions. Moreover, when using the said aldonic acid as a foodstuff or a feed, it can be used as it is, or it can be added or mixed with another foodstuff or a feed.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited only to these examples. In the examples,% represents mass%.

In the examples, the aldonic acid in the reaction solution or the culture solution was quantified using high performance liquid chromatography (HPLC) under the following conditions.
HPLC analysis condition column Asahipak NH2P-50 (manufactured by Shodex)
Eluent Acetonitrile: 40 mM citrate-NaH2PO4 buffer (pH 5.0) = 60: 40 (volume ratio)
Condition Temperature: 40 ℃
Flow rate: 0.8mL / mL
Detector: differential refractometer Example 1
[Preparation of bacterial cells] (Lactose added as inducer)
In a test tube (18 mm × 200 mm), dispense 3 mL of medium containing 0.5% lactose, 0.5% glucose, 0.5% yeast extract, 0.5% polypeptone, and 0.1% magnesium sulfate (pH 7.0). Sterilized for a minute. One platinum loop of various gluconobacters (obtained from the National Institute for Product Evaluation and Technology) shown in Table 1 was inoculated into the test tube, and cultured with shaking (220 rpm) at 30 ° C. for 3 days. Thereafter, the culture broth was centrifuged to collect the cells.
[Production of lactobionic acid]
The cells obtained by the above method were resuspended in 1 mL of 2% lactose and incubated at 40 degrees for 6 hours, and lactobionic acid in the reaction solution was measured. The results are shown in Table 1.

Example 2
[Preparation of bacterial cells] (No inducer added)
A test tube (18 mm × 200 mm) was dispensed with 3 mL of medium containing 0.5% glucose, 0.5% yeast extract, 0.5% polypeptone, and 0.1% magnesium sulfate (pH 7.0), and sterilized at 121 ° C. for 20 minutes. One platinum loop of various gluconobacters (obtained from the National Institute for Product Evaluation and Technology) shown in Table 1 was inoculated into the test tube, and cultured with shaking (220 rpm) at 30 ° C. for 3 days.
[Production of lactobionic acid]
The same method as in Example 1 was used. The lactobionic acid contained in the reaction solution is shown in Table 2.

As shown in Table 2, compared to Example 1, Example 2 has a low lactobionic acid concentration in any strain. Therefore, when preparing bacterial cells, lactose may be added as an inducer. It was shown to be good.

Example 3 [Production of various aldonic acids]
In the same manner as in Example 1, cells of Gluconobacter oxydans NBRC 3285 were prepared, and 12 types of sugars having a hemiacetal hydroxyl group (D-mannose, D-galactose, D-ribose, D-xylose, L- (Arabinose, cellobiose, maltose, maltotriose, maltotetraose, maltopentaose, melibiose, lactose) were resuspended in 1 mL of a 2% aqueous solution and incubated at 40 ° C. for 6 hours. Table 3 shows the quantitative results of various aldonic acids contained in the reaction solution.

Example 4 [Fermentative production of lactobionic acid]
[Pre-culture]
A test tube (18 mm × 200 mm) was dispensed with 3 mL of medium containing 0.5% glucose, 0.5% yeast extract, 0.5% polypeptone, and 0.1% magnesium sulfate (pH 7.0), and sterilized at 121 ° C. for 20 minutes. One platinum loop of Gluconobacter cerinas NBRC 3267 was inoculated into the test tube, and cultured with shaking (220 rpm) at 30 ° C. overnight. Next, 1 L of the above composition medium was dispensed and the test tube culture solution was inoculated into a 3 L Erlenmeyer flask sterilized at 121 ° C. for 20 minutes, and cultured with shaking (220 rpm) at 30 ° C. for 3 days.
[Main culture]
Cultivation was performed using a jar fermenter. 20 L of a medium (pH 7.0) containing 1.5% glucose, 15% lactose, 0.5% yeast extract, 0.5% polypeptone, 0.1% magnesium sulfate and 7.5% calcium carbonate was prepared and sterilized at 121 ° C. for 20 minutes. This was inoculated with 1 L of the above pre-cultured solution and subjected to deep agitation culture (300 rpm, 1 vvm) at 30 ° C. The amount and conversion rate of lactobionic acid accumulated in the culture medium are shown in FIG.

It is a graph which shows the result of having measured the production | generation of the lactobionic acid by Gluconobacter cerinas NBRC 3267 with time.

Claims (4)

  A method for producing aldonic acid, which comprises contacting a microbial cell belonging to acetic acid bacteria with a sugar having a hemiacetal hydroxyl group other than glucose to oxidize the hemiacetal hydroxyl group to produce aldonic acid.   The method for producing aldonic acid according to claim 1, wherein the microbial cell belonging to the acetic acid bacterium is a microbial cell obtained by culturing in a medium to which sugar and / or alcohol having a hemiacetal hydroxyl group is added as an inducer. .   By culturing a microorganism belonging to acetic acid bacteria in a medium containing a sugar having a hemiacetal hydroxyl group other than glucose, aldonic acid is accumulated in the culture solution, and aldonic acid is separated from the culture solution. A method for producing aldonic acid. A group in which the sugar having a hemiacetal hydroxyl group is composed of D-mannose, D-galactose, D-ribose, D-xylose, L-arabinose, cellobiose, maltose, maltotriose, maltotetraose, maltopentaose, melibiose and lactose The method for producing aldonic acid according to any one of claims 1 to 3, wherein the aldonic acid is at least one sugar selected from the group consisting of: