JP2012191899A - Microorganism producing azasugar - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide foods, food additives, animal or fish feeds, medicines, cosmetics and the like containing azasugar.SOLUTION: There are provided microorganisms producing azasugar 1-deoxynojirimycin, especially, Bacillus amyloliquefaciens AS385 strain or Bacillus subtilis B4 strain of the genus Bacillus, wherein the azasugar is detected by analyzing a culture supernatant or fungus body milled product thereof.

Description

  The present invention relates to the provision of microorganisms of the genus Bacillus obtained from foods with experience as a fermented soybean food and products thereof.

  Azasugar is a general term for iminocyclitol in which a ring oxygen atom of a monosaccharide is substituted with an imino group. As typical azasugars, nojirimycin, mannojirimycin, galactstatin and their deoxy forms are known. These are all inhibitors of glucosidase, mannosidase and galactosidase.

  In particular, inhibitors of glucosidase are attracting attention as postprandial hyperglycemia improving agents in type 2 diabetes. They bind to α-glucosidase at the small intestinal mucosal brush border, and can inhibit postprandial hyperglycemia by inhibiting or delaying the degradation and absorption of carbohydrates in the small intestine. Several therapeutic drugs have been approved, and their positioning as postprandial hyperglycemia improving drugs has already been established.

  Azasugar is present in mulberry (Morus alba), camellia communis, Streptomyces subrutilus, S. lavenduae, and Bacillus subtilis, us. Yes.

  It is also said that foods such as beans (Touchi), Daitokuji natto, Hama natto, and Kiyokuni soy contain aza sugar as an active ingredient (participating ingredient) and lower blood sugar levels.

  What is currently being promoted vigorously is the use of mulberry. Mulberry contains 1-deoxynojirimycin, a potent α-glucosidase inhibitor. Products include dried mulberry leaves into tablets and mulberry leaves made into tea. In particular, the root bark has been used as a traditional Chinese medicine since ancient times. In addition, as a technique, there is a technique using an extract.

  It has also been reported that 1-deoxynojirimycin shows very potent α-glucosidase inhibition in in vitro experiments, but does not perform as well in vitro in human tests in humans. Although a significant difference is shown at a high concentration in terms of a decrease in postprandial blood glucose level, an effective result has not yet been obtained in the long-term administration.

  On the other hand, since effective results have been obtained in animal tests (rats), efficient production of azasugar including 1-deoxynojirimycin in an amount that can be expected to be effective when administered to humans A method is desired.

  The products based on the mulberry leaves mentioned above may be affected by external factors such as weather and temperature, so it takes time to grow and several processing steps are required to process the leaves. Become.

  In fermentation production with microorganisms, production can be artificially controlled, and particularly simple methods such as spray drying can be used for drying and concentration.

  There have been few reports on the production of azasugars by actinomycetes and bacteria belonging to the genus Bacillus (Patent Literature 1, Non-Patent Literature 1, Non-Patent Literature 2, and Non-Patent Literature 3).

JP 2008-222701 A JP 2004-294384 A

Hardick D. J. et al: Tetrahedron Vol 48, NO. 30, pp 6285-6296 (1992) Hardick D. J. et al: Tetrahedron Vol 49, No. 30, pp 6707-6716 (1993). Stein D.C. et al: Appl. Environ. Microbiol. Vol. 48, No. 2, pp. 280-284 (1984) Kojima M .: Journal of Fermentation and Bioengeneering Vol 79, No.4 pp391-394 (1995) Kimura T. et al: J. Agric. Food Chem., 52, 1415-1418 (2004) Kimura T. et al: J. Agric. Food Chem., 55, 8928-8933 (2007) Nakagawa K. et al: Anal. Biochem., 404, 217-222 (2010)

  In the reports of Patent Document 1 and Non-Patent Documents 1 to 4 mentioned above, the presence of azasugar including 1-deoxynojirimycin is not shown or quantitatively shown. Absent.

  The final object of the present invention is to provide foods, food additives, animal or fish feeds and pharmaceuticals, cosmetics and the like containing azasugar.

The invention described in claim 1
It is a microorganism that produces azasugar from which azasugar can be detected by analyzing the culture supernatant or cell disruption.

The invention according to claim 2
The microorganism for producing azasugar according to claim 1, which is classified into Bacillus and has the ability to produce azasugar.

The invention described in claim 3
The microorganism for producing an azasugar according to claim 1 or 2, wherein the azasugar is 1-deoxynojirimycin.

The invention according to claim 4
The microorganism producing a microbial azasugar according to any one of claims 1 to 3, wherein the 16sRNA gene is represented by the nucleotide sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing. It is.

The invention according to claim 5
The microorganism is a Bacillus amyloliquefaciens AS385 (FERM AP-22049) strain or a Bacillus subtilis B4 (FERM AP-22050) strain. A microorganism that produces azasugar according to any one of 1 to 4.

  ADVANTAGE OF THE INVENTION By this invention, the microorganisms which can produce azasugar including 1-deoxynojirimycin which can be utilized as a foodstuff, orally ingestible foodstuff, food additive, etc. can be provided.

2 is a separation pattern (chromatogram) of Example 1. The horizontal axis represents the analysis time, and the vertical axis represents the detected amount. 2 is a separation pattern (chromatogram) of Example 2. The horizontal axis represents the analysis time, and the vertical axis represents the detected amount.

  High safety is required for the purpose of the present invention to provide foods, food additives, animal or fish feeds and pharmaceuticals, cosmetics and the like containing azasugar including 1-deoxynojirimycin. Microorganisms having the ability to produce azasugar in the present invention are derived from soybean fermented foods such as bean, Daitokuji natto, Hama natto, and Kiyokuni soy, or soybean-derived ingredients. Since these foods have a long eating experience, there is no need to worry about their safety.

  In the present invention, microorganisms isolated from these foods are used to produce azasugar by fermentation, foods containing, additives, animal or fish feeds, pharmaceuticals, and cosmetics.

  The present invention relates to a Bacillus amyloliquefaciens AS385 strain (deposit number: FERM AP-22049) and a Bacillus subtilis B4 strain (deposit number: FERM AP) isolated from a fermented soybean food. -22050) to produce fermented soybean foods or fermented products using a medium mainly composed of soybean-derived materials, foods, animal or fish feeds, pharmaceuticals, cosmetics, and the like.

  As an isolation source of the microorganism of the present invention, fermented soybean foods such as bean jam, Daitokuji natto, Hama natto, and Kiyokuni soy can be used. Although it does not limit to these, the thing which has a food experience for a long time is good.

  These fermented foods are suspended in sterilized water, and a part of the suspension is smeared on an LB medium, a standard agar medium, etc., the medium is cultured at 37 ° C., and a fungus is collected from the resulting colony to obtain a candidate strain.

  The obtained candidate strain is cultured in a liquid medium, and the inhibitory activity of α-glucosidase is evaluated on the culture supernatant. The medium may be any medium as long as it can produce azasugar. Specifically, a medium using soy broth (Brix 2% pH 7.3), an LB medium, 2% soy peptone, etc. Can be mentioned.

  Cultivation is performed at 37 ° C. for 3 days, and after centrifugation at 13,000 rpm for 5 minutes, a part of the culture supernatant is collected, and the inhibitory activity of α-glucosidase is measured using the sample as a specimen.

  When evaluating the α-glucosidase inhibitory activity, a product derived from baker's yeast, rat small intestine, rabbit small intestine or the like is used as an enzyme, and p-nitrophenyl-α-D-glucopyranoside, sucrose, maltose or the like is used as a substrate. To stop the reaction, a Tris-HCl buffer or carbonate buffer is used.

  The α-glucosidase in the present invention is an enzyme that catalyzes a reaction that hydrolyzes an α-glycoside bond of a sugar. Therefore, α-glucosidase inhibitory activity refers to the amount of free glucose and fructose produced when sucrose and maltose are decomposed, and the amount of p-nitrophenol released in the synthetic substrate p-nitrophenyl-α-D-glucopyranoside. Using the amount as an index, the inhibition rate is the value obtained by dividing the increase in the amount of production or absorbance during the addition reaction of the sample solution by the increase in the amount of production or absorbance during the reaction without addition of the sample solution. The sample concentration at the time of the enzyme reaction when the inhibition rate is 50% is defined as an IC50 value, and this is used as a measure of the α-glucosidase inhibitory activity.

  The strain with good evaluation of α-glucosidase inhibitory activity was cultured again and centrifuged, and then the culture supernatant was lyophilized.

  The specimen obtained as described above is separated by a method described in Patent Literature 2, Non-Patent Literature 5, Non-Patent Literature 6, Non-Patent Literature 7, etc. (for example, a sample is separated by hydrophilic mutual liquid chromatography (HILIC)). The method for detecting 1-deoxynojirimycin with a light scattering detector) detected and quantified azasugar.

  Thus, the present inventors isolated the Bacillus amyloliquefaciens AS385 strain (Bacillus amyloliquefaciens AS385 strain) and the Bacillus subtilis B4 strain according to the examples described later. Deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology on the 16th. The strains have been deposited as FERM AP-22049 and FERM AP-22050, respectively.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to these.

Screening Commercially available natto, Hama natto, rice straw, beans imported from Japan, and Kiyokuni soy from a mass retailer in Korea were suspended in sterile physiological saline. 100 μl of the suspension was collected per medium, smeared on a standard agar medium, and cultured at 37 ° C. for 24 hours. After culturing, it was picked from some of the formed colonies and used as an acquired strain. Each was cultured with shaking in 5 ml of 2% soybean peptone at 37 ° C. for 3 days. The culture solution was centrifuged at 13,000 rpm for 5 minutes, and the supernatant was collected and used as a sample.

  The α-glucosidase inhibitory activity was evaluated using the culture supernatant as a sample. The method for evaluating α-glucosidase inhibitory activity is as follows.

  Intestine acetone powder 5 mL of 50 mM sodium phosphate buffer / 100 mM NaCl (pH 7.0) was added to 0.125 g of rat-derived (SIGMA) and allowed to stand at 4 ° C. for 2 hours or more. Centrifugation was performed at 4 ° C. and 13,000 rpm for 10 minutes, and the supernatant was used as an enzyme solution. 1.0 mM p-nitrophenyl-α-glucopyranoside (50 mM sodium phosphate buffer / 100 mM NaCl (pH 7.0)) was used as a substrate solution, and 0.3 M sodium carbonate solution was used as a reaction stop solution.

  50 μl of the enzyme solution was added to 50 μl of the sample solution. After preincubation for 10 minutes at room temperature, 100 μl of substrate solution was added. The reaction was performed for 120 minutes, and 100 μl of a reaction stop solution was added. A product obtained by adding a reaction stop solution in advance before the enzyme reaction was used as a blank, and the absorbance at 405 nm of the test solution was measured with a microplate reader (Bio-Rad: Model 550).

  The strain obtained from the above method was again subjected to a confirmation test in the same manner, and a strain having reproducibility was selected as a candidate strain. Of the candidate strains, the base sequence of the 16s RNA gene of a strain obtained from soybean chi was determined, and the result was SEQ ID NO: 2 in the sequence listing. It was concluded that this strain was classified as Bacillus subtilis, and this strain was named Bacillus subtilis B4 strain. And liquid culture was performed using this strain.

  Next, the candidate strain was cultured for 7 days in a 500 ml Sakaguchi flask in a medium containing 50 ml of 4% soy peptone (DIFCO Select Soytone: Becton, Dickinson and Company) and 2% carbon source. An appropriate amount of the culture solution was collected on the first day, the third day, and the seventh day, and after centrifugation at 13,000 rpm for 10 minutes, the supernatant was lyophilized to prepare a sample.

  To the sample, add the same amount of ethanol: distilled water (1: 1, V / V) as the supernatant before lyophilization, and filter through a PTFE membrane of 45 μm pore size. 10 μl of the filtrate is acetonitrile: distilled water: formic acid. A product obtained by adding 990 μl of (50: 49.9: 0.1, V / V / V) was used as a measurement sample solution.

  Quantification of 1-deoxynojirimycin and detection of azasugar were performed by HILIC-tandem mass spectrometry (HILIC-MS / MS). For HPLC, Shimadzu Corporation (pump: LC-20AD, column thermostatic chamber: CTO-20A, autosampler: SIL-20AC) was used. The column uses TSKgel Amide-80 (2.0 × 100 mm) manufactured by Tosoh Corporation, the column temperature is 40 ° C., the mobile phase is acetonitrile (including 0.1% formic acid), water (including 0.1% formic acid), and the flow rate. Elution was performed with a gradient development of 0.2 mL / min. The mass spectrometer used was API 3200 manufactured by ABC Scientific Co., Ltd. The amount of 1-deoxynojirimycin mulberry leaves was calculated from a calibration curve using a 1-deoxynojirimycin preparation.

As a result, the content of 1-deoxynojirimycin shown in Table 1 was confirmed in the culture supernatant. FIG. 1 shows an example of a chromatogram.

  Based on the above, it was concluded that the Bacillus subtilis B4 strain has the ability to produce aza sugars including deoxynojirimycin, and was deposited at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (deposit number: FERM AP). -22050).

  The rice straw collected from all over Japan and the Kiyokuni soy obtained at a mass retailer in Korea were suspended in sterile physiological saline. 100 μl of the suspension was collected per medium, smeared on a standard agar medium, and cultured at 37 ° C. for 24 hours. After culturing, it was picked from some of the formed colonies and used as an acquired strain. Each was cultured with shaking in 5 ml of 2% soybean peptone at 37 ° C. for 3 days. The culture solution was centrifuged at 13,000 rpm for 5 minutes, and the supernatant was collected and used as a sample.

  The α-glucosidase inhibitory activity was evaluated using the culture supernatant as a sample. The method for evaluating α-glucosidase inhibitory activity is the same as in Example 1.

  The obtained candidate strain was cultured in an LB liquid medium, and the obtained culture solution was diluted 1,000 times with sterilized water to obtain a seed solution. Soybean fermented food was made in the same process as natto. Next, a method for making a soybean fermented food is shown.

  The whole soybeans were washed thoroughly and immersed in water 3 times the weight of soybeans overnight. The absorbed soybean water was drained and autoclaved at 121 ° C. for 50 minutes to obtain boiled beans. After the boiled beans were filled, the seed solution was inoculated at a rate of 1 ml per 50 g and fermented at 40 ° C. for 17 hours. Thereafter, it was stored at 4 ° C. for 3 days and aged to obtain a soybean fermented food. The obtained soybean fermented product was freeze-dried to prepare a sample.

  The obtained sample was pulverized, 1 mL of ethanol: distilled water (1: 1, V / V) was added to 50 mg of the powder, extracted for 10 minutes with an ultrasonic cleaner, centrifuged at 3000 rpm, and the supernatant was reduced to 0.00. It filtered with the RC membrane filter of 45 micrometer pore size, and the 1-deoxynojirimycin content was measured like Example 1 by making a filtrate into a measurement sample solution.

  The quantification of 1-deoxynojirimycin and the detection of azasugar were performed by HILIC-tandem mass spectrometry as in Example 1.

As a result, the content of 1-deoxynojirimycin shown in Table 2 was confirmed in the fermented soybeans. FIG. 2 shows an example of a chromatogram.

  From these candidate strains, the base sequence of the 16sRNA gene of the AS385 strain with the highest production amount of 1-deoxynojirimycin was determined. It was concluded that this strain was classified as Bacillus amyloliquefaciens, and this strain was named Bacillus amyloliquefaciens AS385 and concluded that it has the ability to produce aza sugars including deoxynojirimycin. Deposited at the Research Center for Biological Biology (deposit number: FERM AP-22049).

  The present invention can provide a microorganism that produces azasugar (Azasugar-Producible Bacteria).

  By utilizing the microorganism of the present invention, fermentation production of azasugar becomes possible. The present invention can be widely used for fermented foods, raw materials for health foods, animal or fish feeds, pharmaceuticals, cosmetics, and the like.

FERM AP-22049
FERM AP-22050

Claims (5)

  A microorganism that produces azasugar from which azasugar is detected by analyzing the culture supernatant or crushed cells.   The microorganism that produces azasugar according to claim 1, which is classified into Bacillus and has the ability to produce azasugar.   The microorganism for producing an azasugar according to claim 1 or 2, wherein the azasugar is 1-deoxynojirimycin.   The microorganism producing a microbial azasugar according to any one of claims 1 to 3, wherein the 16sRNA gene is represented by the nucleotide sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing. .   The said microorganism is a Bacillus amyloliquefaciens AS385 (Bacillus amyloliquefaciens AS385) strain (FERM AP-22049) or a Bacillus subtilis B4 (Bacillus subtilis B4) (FERM AP-22050) strain, A microorganism producing the azasugar according to any one of 1 to 4.

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