JP2008245536A - Method for producing aldonic acid by lactic acid bacterium - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing aldonic acid by using a lactic acid bacterium. <P>SOLUTION: A saccharide having a hemiacetal hydroxyl group is brought into contact with a bacterial cell of a lactic acid bacterium [e.g. Lactococcus lactis subsp. cremoris KYG-33 (FERM P-21207)] to oxidize the hemiacetal hydroxy group. Thus, the aldonic acid corresponding to saccharide having the hemiacetal hydroxyl group as a substrate is produced. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for producing aldonic acid using lactic acid bacteria.

  Lactobionic acid, one of the aldonic acids, has already been approved by the FDA in the United States and is added to purine premixes as a coagulant or used in cosmetics as a moisturizer. In terms of function, it is known that lactobionic acid has bifidobacterial selective growth activity (for example, see Patent Document 1) and has a mineral absorption promoting effect (for example, see Patent Document 2). 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.

As a specific method for obtaining aldonic acid, a method of oxidizing a sugar having a hemiacetal hydroxyl group by applying electricity together with sodium bromine is known. 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). 3).
Japanese Patent No. 3559063 Japanese Patent No. 3501237 JP 2001-245657 A

  However, chemically synthesized products of aldonic acid cannot be used for food and feed applications due to domestic laws and regulations. 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. Moreover, the food / beverage products containing an aldonic acid are not known.

  An object of this invention is to provide the method of manufacturing aldonic acid using lactic acid bacteria.

  As a result of exhaustive studies on microorganisms in order to achieve the above-mentioned problems, the present inventors have found that lactic acid bacteria produce aldonic acid from a sugar having a hemiacetal hydroxyl group, and have reached the present invention.

  That is, the first of the present invention is a method for producing an aldonic acid characterized by contacting a microbial cell belonging to a lactic acid bacterium with a sugar having a hemiacetal hydroxyl group, and oxidizing the hemiacetal hydroxyl group to produce aldonic acid. Preferably, the microorganism belonging to the lactic acid bacterium is a microorganism belonging to the genus Lactococcus, and more preferably, the microorganism belonging to the genus Lactococcus is Lactococcus lactis subspices cremolith KYG-33 ( FERM P-21207) is a method for producing aldonic acid.

  In the second aspect of the present invention, a microorganism belonging to a lactic acid bacterium is cultured in a medium containing a sugar having a hemiacetal hydroxyl group, whereby aldonic acid is accumulated in the culture solution and the aldonic acid is separated from the culture solution. The microorganism belonging to the genus Lactococcus is preferably a microorganism belonging to the genus Lactococcus, more preferably the microorganism belonging to the genus Lactococcus is a lactococcus. -It is a manufacturing method of aldonic acid which is Lactis subspecies Cremolis KYG-33 (FERM P-21207).

  The third aspect of the present invention is summarized as Lactococcus lactis subspecies Cremolis KYG-33 (FERM P-21207) having the ability to produce aldonic acid from a sugar having a hemiacetal hydroxyl group.

  Furthermore, a fourth aspect of the present invention is a method for producing lactobionic acid in fermented milk by fermenting Lactococcus lactis subspecies Cremolis KYG-33 (FERM P-21207) in milk. The gist of the production method of the acid-rich fermented milk.

  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 a hemiacetal hydroxyl group, Corresponding aldonic acid Can be manufactured. Moreover, if fermented milk is produced using the lactic acid bacteria which produce aldonic acid, fermented milk containing aldonic acid can be obtained.

  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 include oligosaccharides such as pentaose and lactose. Among these, D-mannose, D-galactose, D-ribose, D-xylose, L-arabinose, cellobiose, maltose, maltotriose, maltotetraose, maltopentaose, and lactose are particularly preferable. It is.

  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. In addition, lactobionic acid among aldonic acids includes not only free lactobionic acid but also lactobionic acid salt or lactobionolactone.

  The lactic acid bacteria used in the present invention include, for example, Lactobacillus genus, Lactococcus genus, Tetragenococcus genus, Pediococcus genus, Bifidobacterium genus, Lactobacillus genus・ Brugaricus (Lactobacillus delbrueckii subsp.Bulgaricus), Lactococcus lactis, Lactococcus lactis subsp. Preferably, it is a genus Lactococcus. Furthermore, among these lactic acid bacteria, Lactococcus lactis subspecies Cremolis KYG-33 (FERM P-21207) can be used. Lactococcus lactis sub-species Cremolis KYG-33 is a cell body newly isolated by the present inventors from dairy products, and has the accession number FERM at the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary. It is deposited at P-21207 (date of deposit: February 9, 2007).

The mycological characteristics of Lactococcus lactis subspecies Cremolis KYG-33 (FERM P-21207) are as shown below.
(Morphological characteristics)
Culture temperature: 30 ° C
Shape: Cocci Size: 0.9-1.0μm
Existence of motility: None Spore formation: None (culture characteristics)
Color: Cream colony Morphology: Circular ridged state: Lenticular rim: Full edge surface: Smooth transparency: Opaque consistency: Butter-like (physiological properties)
Gram staining: +
Catalase:-
Oxidase:-
O / F test: +
Growth at 40 ° C:-
Growth with 4% NaCl: −
(Fermentation test)
Glycerol:-
Erythritol:-
D-arabinose:-
L-arabinose:-
Ribose:-
D-xylose:-
L-xylose:-
Adonitol:-
β-methyl-D-xyloside: −
Galactose: +
Glucose: +
Fructose: +
Mannose: +
Sorbose:-
Rhamnose:-
Dulcitol:-
Inositol:-
Mannitol:-
α-methyl-D-mannoside: −
α-methyl-D-glucoside: −
N-acetylglucosamine: +
Amygdalin:-
Arbutin:-
Esclin: +
Salicin: +
Cellobiose: +
Maltose: +
Lactose: +
Melibiose:-
Sucrose:-
Trehalose: +
Inulin:-
Meletetose:-
Raffinose:-
Starch:-
Glycogen:-
Xylitol:-
Gentiobiose: +
D-Tulanose:-
D-lyxose:-
D-Tagatose:-
D-Fucose:-
L-Fucose:-
D-arabitol:-
L-arabitol:-
Gluconate:-
2-ketogluconate:-
5-ketogluconate:-
From the bacteriological properties of the strain and the results of homology search of 16S rDNA base sequence by GenBank / DDBJ / EMBL, the strain belongs to L. lactis, and at the subspecies level, L. lactis subsp. The most likely attributed to cremoris. However, as described later, since it was different from the known L. lactis subsp. Cremoris in terms of the ability to produce lactobionic acid, it was deposited as a new strain.

  Lactic acid bacteria isolated from the natural world can be used in this way, but public institutions that can be distributed to any person's demand, such as the Institute for Molecular Cell Biology and the National Institute for Product Evaluation Technology, the University of Tokyo. It is also possible to use strains conserved by traditional depository institutions. These include, for example, Lactococcus lactis subspices cremolith NBRC100676 (Lactococcus lactis subsp. Examples include Lactobacillus lactis subsp. Lactis IAM10070.

  The first production method of the present invention is a method for producing an aldonic acid by contacting a lactic acid bacterial cell with a sugar having a hemiacetal hydroxyl group, and oxidizing the sugar having a hemiacetal hydroxyl group by an enzyme in the bacterial cell or on a cell membrane. It is. Examples of the lactic acid bacteria used in the present invention include live lactic acid bacteria as they are, processed microbial cells, or those immobilized on a carrier.

  First, in order to obtain bacterial cells of lactic 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, sugar alcohols, and glycerol. Common carbohydrates include corn starch and malt. Vegetable oils such as olive oil and corn oil are also included in the carbon source. Also, 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, but as the inorganic nitrogen, ammonia salt, nitric acid or the like is used. Organic nitrogen is provided in the form of amino acids, proteins or urea. Natural organic nitrogen complexes include CSL, soybeans, soybean flakes, peanut meal, cotton meal, Distillers'solubles, casein hydrolyzate, slaughterhouse waste, fish meal, yeast extract, etc. .

  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, potassium, and calcium 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 for buffering. In order to control the pH of the culture system, ammonia, caustic soda, hydrochloric acid, sulfuric acid or the like may be added.

  The culture is preferably performed under the optimal culture conditions for the strain to be used. In general, the temperature is preferably 20 to 35 ° C, and the pH is 4 to 8 with hydrochloric acid, aqueous sodium hydroxide, calcium carbonate, or the like. Is preferably maintained.

  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 the culture is performed under such conditions, a sufficient amount of microorganisms can be obtained in 15 to 168 hours after the culture.

  When the live microbial cells obtained as described above are used as they are, the recovered and washed cells can be used as they are.

  When used as a treated product of lactic acid bacteria, the cells treated with acetone, quaternary ammonium compounds, lauryl sulfate, Tween, or an enzyme that breaks down the specific binding of the cell walls of microorganisms, or frozen cells are decompressed. Freeze-dried microbial cells obtained by sublimation of moisture under the water, homogenizers and glass beads, and crushed microbial cells and cell membrane fractions, as well as the supernatant of microbial cell lysates Cell extracts, crude enzyme solutions obtained by extracting enzymes from these, and the like can be used.

  In order to immobilize lactic acid bacterial cells or treated products on a carrier, a method of adsorbing to a substance such as a cellulose carrier, a ceramic carrier or a glass bead carrier, or a gel-like substance having a lattice structure such as agar, calcium alginate, carrageenan, etc. And a method encompassed by known polymers. These methods make it possible to repeatedly use lactic acid bacteria having a oxidization activity and treated cells.

  In the first production method of the present invention, a saccharide having a hemiacetal hydroxyl group serving as a substrate is dissolved in a reaction solvent, and a lactic acid bacterium culture solution, a microbial cell, a processed microbial cell, or a microbial cell prepared as described above A product obtained by immobilizing a cell-treated product on a carrier is added, and the reaction is carried out while controlling the reaction temperature and 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 enzyme of the lactic acid bacterium to be used, but is generally in the range of pH 4 to 8, and 5 to 7 is particularly preferable. Moreover, the reaction temperature should just be the conditions which a microbial enzyme does not deactivate, 20-60 degreeC is preferable, but 25-45 degreeC is more preferable.

  Examples of the reaction method include standing, shaking, and deep aeration stirring, but it is convenient to aerobically react to increase the yield of aldonic acid. When culture is performed under such conditions, a sufficient amount of product can be obtained in 6 to 24 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, lactic acid bacteria are cultured in a medium containing a sugar having a hemiacetal hydroxyl group, whereby aldonic acid is accumulated in the culture solution and aldonic acid is separated from the culture solution. It is. As the medium components used for the culture, the above-mentioned medium components used for the culture for obtaining the cells by the first production method of the present invention are used in the same manner except that the medium contains a sugar having a hemiacetal hydroxyl group. It is done. The content of the sugar having a hemiacetal hydroxyl group is 0.1 to 500 g / L, preferably 0.5 to 200 g / L, and more preferably 0.5 to 150 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.

  A fourth aspect of the present invention is a method for producing fermented milk having a high lactobionic acid content from milk using Lactococcus lactis subspecies Cremolis KYG-33 (FERM P-21207).

  Examples of the milk used in the present invention include cow's milk and goat milk, and fresh ones are preferably sterilized in advance. Moreover, as milk used by this invention, the foodstuffs containing lactose, such as skim milk powder and whey, may be sufficient. If necessary, sugars such as sucrose, fructose, invert sugar and glucose, water, pulp, fruit juice, flavor, acidulant and the like can be appropriately added to these milks.

  Lactic acid bacteria to be added to milk is Lactococcus lactis subspecies Cremolis KYG-33 (FERM P-21207), and the cells are added as they are to the nonfat dry milk medium and then freeze-dried. Powdered inoculum, so-called yogurt inoculum, may be added.

  As conditions for fermentation, 20 to 50 ° C., preferably 25 to 40 ° C., and more preferably 25 to 30 ° C. can get.

  The fermented milk produced by the above method is not limited to yogurt (including hard yogurt, soft yogurt, drink yogurt, frozen yogurt, sterilized yogurt, plain yogurt, and sweetened yogurt). You may be made into the form related to various food-drinks. Examples of the food and drink include frozen confectionery such as lactic acid beverages and popsicles, raw confectionery such as pudding, jelly, cream puff and cake, confectionery such as ramune, candy, chocolate and biscuits, cheese, butter, bread, stew, dressing and others Food and drink or health food.

  The aldonic acid produced by the production method of the present invention, especially lactobionic acid, 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 medicine, food and drink, and feed. When using the above-mentioned aldonic acid, especially lactobionic acid as a pharmaceutical, excipients, lubricants, diluents, pH regulators, preservatives, sweeteners, fragrances, emulsifying dispersions conventionally used in the field It can be administered orally or parenterally as tablets, powders, wettable powders and emulsions. Moreover, when using the said aldonic acid, especially lactobionic acid as food / beverage products and feed, it can be used as it is, or it can be added or mixed with other food / beverage products and 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 Examples, lactobionic acid in a reaction solution or a culture solution was quantified using high performance liquid chromatography (HPLC) under the following conditions.
HPLC analysis condition column Asahipak NH2P-50 (Showa Denko)
Eluent Acetonitrile: 40 mM citrate-NaH ₂ PO ₄ buffer (pH 5.0) = 60: 40 (volume ratio)
Condition Temperature: 40 ℃
Flow rate: 0.8mL / mL
Detector: Differential refractometer

Examples 1 to 4 [Production of lactobionic acid by contacting cells]
[Preparation of bacterial cells]
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. In the test tube, Lactococcus lactis sub-species Cremolis KYG-33 (FERM P-21207) (Example 1) and lactic acid bacteria obtained from the Institute for Molecular Cell Biology (National Institute for Molecular and Cell Biology) One platinum loop of Examples 2, 3, and 4) was inoculated, and cultured with shaking (220 rpm) at 27 ° C. for 3 days. Thereafter, the culture broth was centrifuged to collect the cells.
[Production of lactobionic acid]
The whole amount of the cells obtained by the above method was resuspended in 1 mL of 2% lactose, shaken at 27 degrees for 3 days, and lactobionic acid in the reaction solution was measured. The results are shown in Table 1.

Example 5 [Production of various aldonic acids]
In the same manner as in Example 1, cells of Lactococcus lactis subspecies Cremolis KYG-33 (FERM P-21207) 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, lactose) were resuspended in 1 mL of a 2% aqueous solution and shaken at 27 degrees for 3 days. Table 2 shows the quantitative results of various aldonic acids contained in the reaction solution.

Example 6 [Production of lactobionic acid by a medium containing lactose]
[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 Lactococcus lactis subspecies Cremolis KYG-33 (FERM P-21207) was inoculated into the test tube, and cultured overnight at 27 ° C. with shaking (220 rpm). Next, 1 L of the above-mentioned 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, followed by shaking culture (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 precultured solution and subjected to deep stirring culture (300 rpm, 1 vvm) at 30 ° C. The concentration of lactobionic acid accumulated in the culture solution is shown in FIG.

Example 7, Comparative Examples 1 and 2 [Production of Fermented Milk High in Lactobionic Acid]
[Cultivation of lactic acid bacteria]
A 500 mL Erlenmeyer flask was charged with 100 mL of Lactobacilli MRS Broth (Defico) and sterilized at 121 ° C. for 20 minutes. There is one platinum ear Lactococcus lactis subspecies cremolith KYG-33 (FERM P-21207) (Example 7), Lactococcus lactis subspecies cremolith NBRC100676 (comparative example 1) Lactococcus lactis Subspecies lactis IAM10070 (Comparative Example 2) was inoculated and cultured at 27 ° C. for 2 days.

[Preparation of yogurt inoculum]
The cultured lactic acid bacteria were added to the skim milk medium and powdered by lyophilization to obtain a powdered inoculum.
[Production of fermented milk with high lactobionic acid content]
In a sterilized container, 100 ml of fresh sterilized milk was placed. 10 g of the prepared yogurt inoculum was added thereto. The lid was closed and left at 27 ° C. for 24 hours. The fermented milk thus prepared was sampled and the lactobionic acid content was measured. The lactobionic acid content in the fermented milk is shown in Table 3.

It is a figure which shows the result of having measured the production | generation of the lactobionic acid by a Lactococcus lactis subspecies Cremolis KYG-33 with time.

Claims (6)

  A method for producing aldonic acid, comprising contacting a microbial cell belonging to a lactic acid bacterium with a sugar having a hemiacetal hydroxyl group to oxidize the hemiacetal hydroxyl group to produce aldonic acid.   Production of aldonic acid characterized by accumulating aldonic acid in a culture solution by culturing a microorganism belonging to lactic acid bacteria in a medium containing a sugar having a hemiacetal hydroxyl group, and separating the aldonic acid from the culture solution Method.   The method for producing aldonic acid according to claim 1 or 2, wherein the microorganism belonging to the lactic acid bacterium is a microorganism belonging to the genus Lactococcus.   The method for producing aldonic acid according to claim 3, wherein the microorganism belonging to the genus Lactococcus is Lactococcus lactis subspecies Cremolis KYG-33 (FERM P-21207).   Lactococcus lactis subspecies cremolith KYG-33 (FERM P-21207) having the ability to produce aldonic acid from a sugar having a hemiacetal hydroxyl group.   Lactococcus lactis subspecies cremolith KYG-33 (FERM P-21207) is fermented in milk to produce lactobionic acid in fermented milk, and a method for producing fermented milk containing high lactobionic acid .