JP5100987B2 - Method for producing aldonic acid - Google Patents

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 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.

The present inventors have found that in order to achieve the above object, comprehensively considering the results for bacteria backed safety, Gluconobacter, Gluconobacter Acetobacter, or microorganisms belonging to the genus Acetobacter, hemi It has been found that the hydroxyl group of an oligosaccharide having an acetal hydroxyl group is oxidized, and the present invention has been achieved.

Specifically, according to the first aspect of the present invention , an oligosaccharide that is a substrate having a hemiacetal hydroxyl group is brought into contact with a microbial cell belonging to the genus Gluconobacter, Gluconoacetobacter, or Acetobacter , and the hemiacetal hydroxyl group is contacted. And producing an aldonic acid corresponding to the oligosaccharide that is the substrate , preferably a aldonic acid production method, preferably, Gluconobacter, Gluconoacetobacter, Alternatively , the microbial cell belonging to the genus Acetobacter is a microbial cell obtained by culturing in a medium supplemented with the same oligosaccharide as the substrate oligosaccharide as the inducer , and preferably, , Oligosaccharides are cellobiose, maltose, maltotriose, maltotetraose, maltopentaose, melibiose and lacto It is at least one method is a sugar selected from the group consisting of scan.

In the second aspect of the present invention, by culturing a microorganism belonging to the genus Gluconobacter, Gluconoacetobacter, or Acetobacter in a medium containing an oligosaccharide having a hemiacetal hydroxyl group , to accumulate aldonic acid corresponding to oligosaccharide is intended to subject matter a method for manufacturing a aldonic acid and separating the aldonic acid from the culture broth, preferably, oligosaccharide, cellobiose, maltose, The method is at least one sugar selected from the group consisting of maltotriose, maltotetraose, maltopentaose, melibiose and lactose.

ADVANTAGE OF THE INVENTION According to this invention, the aldonic acid corresponding to the oligosaccharide which is a substrate can be efficiently manufactured with the microorganisms which were used for food manufacture from a long time, and the safety | security was ensured, and also the oligosaccharide which has a hemiacetal hydroxyl group The corresponding aldonic acid can be produced without being limited by the type.

  Hereinafter, the present invention will be described in detail.

The oligosaccharide having a hemiacetal hydroxyl group used in the present invention refers to a sugar whose reducing end is an aldehyde. Specific examples of such, cell Robiosu, maltose, maltotriose, maltotetraose, maltopentaose, melibiose, lactose and the like. Preferably Among these is the La Kutosu.

In the present invention, aldonic acid refers to an oligosaccharide having a hemiacetal hydroxyl group oxidized by the oxidation of a hemiacetal hydroxyl group , and further including these salt forms.

Therefore, the present invention is a method for converting an oligosaccharide having the above-described hemiacetal hydroxyl group into the corresponding aldonic acid. Specifically, the cell Robiosu to Serobion acid, maltose maltobionic acid, maltotriose malto-trione acid, maltotetraose to maltotetraose propionic acid, maltopentaose into maltopentaose propionic acid, Meribion melibiose In this method, lactose is converted to lactobionic acid.

The microorganism used in the present invention refers A Setobakuta genus Gluconobacter, or a microorganism belonging to the Gluconobacter genus Acetobacter. Since acetic acid bacterium as described above are used in such fermentation foods since ancient times, the safety of the microorganisms is ensured.

Microorganisms belonging to the genus Gluconobacter, Gluconoacetobacter, or Acetobacter used in the method of the present invention can be those isolated from the natural world, but like the Incorporated Administrative Agency Product Evaluation Technology Infrastructure, It is also possible to use one stored by a public depository that can be distributed for any person's request. These include, for example, Acetobacter aceti NBRC3281, Acetobacter syzygii NBRC16604, Acetobacter cibinongensis NBRC16605, Acetobacter indonesiens 471 Gluconacetobacterxylinus NBRC16670, Gluconacetobacter liquefaciens NBRC12388, Gluconacetobacter rsp. NBRC3261, Gluconobactercerinus 267 Examples thereof include fragrant obliter (Gluconobacter fraturii) NBRC3260, gluconobacter oxydans NBRC3285, 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 the genus Gluconobacter, Gluconoacetobacter, or Acetobacter is brought into contact with an oligosaccharide which is a substrate having a hemiacetal hydroxyl group, In this method, the hemiacetal hydroxyl group is oxidized by the above enzyme to produce aldonic acid corresponding to the oligosaccharide that is the substrate . The microbial cell belonging to the genus Gluconobacter, gluconoacetobacter, or acetobacter used in the present invention is gluconobacter, gluconoacetobacter, or a living cell of acetobacter as it is, or This refers to a treated product of cells or those immobilized on a carrier.

First, in order to obtain microbial cells belonging to the genus Gluconobacter, Gluconoacetobacter, or Acetobacter , 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, during the above culture, adding the same oligosaccharide as the substrate oligosaccharide to the medium as an enzyme inducer may increase the conversion efficiency to aldonic acid. It is preferable in that it can be performed. The sugar having a hemiacetal hydroxyl group as used herein, Se Robiosu, maltose, maltotriose, maltotetraose, maltopentaose, melibiose, oligosaccharides such as lactose and the like.

  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 the microbial cells belonging to the genus Gluconobacter, Gluconoacetobacter, or Acetobacter cannot grow on the sugar alone, it is necessary to add and cultivate a carbon source that can be assimilated. It is. Carbon sources that can be generally assimilated by microorganisms belonging to the genus Gluconobacter, Gluconoacetobacter, or Acetobacter include monosaccharides such as glucose and fructose, oligosaccharides such as sucrose and maltose, starch, etc. Polysaccharides, sugar alcohols, glycerol and the like. 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.

When the living microbial cells of microorganisms belonging to the genus Gluconobacter, Gluconoacetobacter, or Acetobacter obtained as described above are used as they are, the recovered cells can be used as they are.

Specific binding of acetone, quaternary ammonium compounds, lauryl sulfate, Tween, or the cell wall of microorganisms when used as a treated product of microorganisms belonging to the genus Gluconobacter, Gluconoacetobacter, or Acetobacter Microbial cells treated with drugs such as enzymes that degrade lyophilized cells, freeze-dried microbial cells obtained by sublimating moisture under reduced pressure, crushed microbial cells physically crushed using a homogenizer or glass beads, Furthermore, a cell-free extract that is a supernatant of a crushed cell body, a crude enzyme solution obtained by extracting an enzyme from these, and the like can be used.

In order to immobilize microbial cells or processed products of microorganisms belonging to the genus Gluconobacter, Gluconoacetobacter, or Acetobacter to a carrier, a method of adsorbing to a substance such as a cellulose carrier, a ceramic carrier, or a glass bead carrier And a gel-like substance having a lattice structure such as agar, calcium alginate, carrageenan, and a method encompassed by known polymers. This method allows the microorganism to be used repeatedly.

In the first production method of the present invention, an oligosaccharide having a hemiacetal hydroxyl group serving as a substrate is dissolved in a reaction solvent, and the gluconobacter genus, gluconoacetobacter genus, or acetobacter genus prepared as described above is used. A culture solution, fungus body, or treated cell product of a microorganism belonging to the microorganism is added, and the reaction is performed while controlling the reaction temperature and the pH of the reaction solution as necessary. 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.

In the second production method of the present invention, a microorganism belonging to the genus Gluconobacter, Gluconoacetobacter, or Acetobacter is cultured in a culture medium containing an oligosaccharide having a hemiacetal hydroxyl group. In addition, aldonic acid corresponding to the oligosaccharide as a substrate is accumulated, and the aldonic acid is separated from the culture solution. As the medium components 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 the same oligosaccharide as the substrate oligosaccharide . The same oligosaccharides as those described above can be used as the same oligosaccharide as the substrate oligosaccharide . 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 3 [Production of various aldonic acids]
In the same manner as in Example 1, cells of Gluconobacter oxydans NBRC 3285 were prepared, oligosaccharides seven having hemiacetal hydroxyl group (Se Robiosu, maltose, maltotriose, maltotetraose, maltopentaose, Resuspended in 1 mL of a 2% aqueous solution of melibiose and lactose) and incubated at 40 degrees for 6 hours. Table 3 shows the quantitative results of various aldonic acids contained in the reaction solution.

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 (5)

Oligosaccharide, which is a substrate having a hemiacetal hydroxyl group , is contacted with a microbial cell belonging to the genus Gluconobacter, Gluconoacetobacter, or Acetobacter , and the hemiacetal hydroxyl group is oxidized, whereby the oligo which is the substrate A method for producing aldonic acid, comprising producing aldonic acid corresponding to a sugar . The microbial cell belonging to the genus Gluconobacter, Gluconoacetobacter, or Acetobacter was obtained by culturing the same oligosaccharide as the substrate oligosaccharide as a inducer in a medium added with the same . The method for producing aldonic acid according to claim 1, which is a microbial cell. The gluconobacter genus, gluconoacetobacter genus, or acetobacter genus is cultured without using the same oligosaccharide as the substrate oligosaccharide as the inducing agent. The method for producing aldonic acid according to 1. By culturing a microorganism belonging to the genus Gluconobacter, Gluconoacetobacter, or Acetobacter in a medium containing an oligosaccharide having a hemiacetal hydroxyl group, an aldonic acid corresponding to the oligosaccharide is cultured in the culture solution. Is accumulated , and the accumulated aldonic acid is separated from the culture solution. Oligosaccharide which is a substrate having a hemiacetal hydroxyl group, cellobiose, maltose, maltotriose, maltotetraose, maltopentaose, according to claim 1-4 is at least one sugar selected from the group consisting of melibiose and lactose The method for producing aldonic acid according to any one of the above.