JP6785324B2 - Bacillus licheniformis NY1505 strain that mass-produces α-glucosidase inhibitors - Google Patents

Description

The present invention relates to the Bacillus licheniformis NY1505 strain, which is a new strain that mass-produces α-glucosidase inhibitors.

α-Glucosidase inhibitors (α-glucosidase inhibitors) are substances that are distributed in some plants and microorganisms and have an action of suppressing the decomposition of oligosaccharides into monosaccharides.

Inhibition of α-glucosidase activity is expected to have ancillary effects such as treatment of obesity as well as suppression of postprandial blood glucose elevation by inhibiting smooth absorption of sugars in the intestine. The need for economical mass production is increasing.

Plant-derived α-glucosidase inhibitors include aza sugars such as 1-deoxynojirimycin contained in mulberry leaves, genistine contained in soybeans, and daizein. Isoflavone such as (daidzein), some flavon glycosides (flavone glycoside), etc. are known, and as an α-glucosidase inhibitor derived from a microorganism, a similarity derived from a strain of Actinoplanes is known. 1-Deoxynojirimycin, tris base, etc. produced by microorganisms such as oligosaccharide (pseudoglucosidase), Streptomyces or Bacillus are known, and acarbose (acrabose) is known as a pharmaceutical. ), Α-Glucosidase inhibitors such as boglibose are already on the market as therapeutic agents for diabetes.

Recently, research on edible natural products has been actively promoted in order to ingest an α-glucosidase inhibitor that is effective in lowering blood glucose as a food. Typical examples of α-glucosidase inhibitors derived from natural products used as foods are mulberry leaves in the case of plants and some natto in the case of microorganisms. Although these primary processed products or extracts are mass-produced, there is a limit to the diversification of products because they have a common odor and a factor that reduces the taste such as color. In addition, since these are cases where secondary metabolites produced by plants or microorganisms are used, there is a disadvantage that the intended purpose can be achieved only after taking a certain amount several times a day.

On the other hand, intestinal bacteria such as lactic acid bacteria have the property of growing while inhabiting the intestines. Therefore, gut flora that produce secondary metabolites such as α-glucosidase inhibitors are expected to produce and supply α-glucosidase inhibitors seamlessly into the intestinal environment while growing in the intestine. However, while some Bacillus subtilis, previously known, such as Bacillus subtilis, are known to produce α-glucosidase inhibitors, Bacillus subtilis is an aerobic bacterium in the intestine. It is difficult to supply α-glucosidase inhibitor in the intestine by the growth of Bacillus subtilis because it is difficult to inhabit, and in order to supply α-glucosidase inhibitor through ingestion of natto, it is necessary to take a large amount. There is a problem that it does not become.

Therefore, research and development of a new strain that can live in the intestine, can produce a large amount of α-glucosidase inhibitor, and can be effectively used for purposes such as weight loss and hypoglycemia. is required.

Republic of Korea Published Patent No. 10-2014-0123847 (2014.10.23) Republic of Korea Published Patent No. 10-2007-0028997 (2007.03.13)

Overseas paper (Zhu Y.P, Li X.T, Teng C, Sun B.G, "Enhanced production of α-glucosidase inhibitors by a newly isolated strain of Bacillus subtilis B2 using response surface methodology" FOOD AND BIOPRODUCTS PROCESSING Vol.91 No. 3 264p-270p) Republic of Korea paper (Kim Hyun-soo, Li Ze-yeon, Hwang Ky-yeol, Cho Young-suk, Park Young-sik, Kang Kyung-dong, Song Sui-il, Bacillus strain producing “α-glucosidase inhibitor 1-deoxynojirimycin” Separation and Identification of "Korea J. Microbiol. Biotechnol. Vol. 39, No. 1, 49-55 (2011)

As a result of diligent research to select intestinal microorganisms having strong α-glucosidase inhibitory activity, the present inventors are facultative anaerobic, form spores, and intestines from among microorganisms exhibiting α-glucosidase inhibitory activity. A novel Bacillus likeniformis NY1505 strain that produces a large amount of α-glucosidase inhibitor as a GRAS bacterium capable of growing within the strain was isolated and identified, and the vegetative cells containing the fermented product or spores of the microorganism were used for weight loss and blood glucose. It was confirmed that it can be usefully used in foods, medicines, feeds, etc. for the purpose of descent.

Accordingly, it is an object of the present invention to provide a technical content concerning a Bacillus licheniformis NY1505 strain, which is a new strain that mass-produces an α-glucosidase inhibitor.

In order to fulfill the above technical problems, the present invention provides a Bacillus licheniformis NY1505 strain (accession number KCTC13021BP) that highly produces an α-glucosidase inhibitor.

In addition, the strain is characterized by containing 16s RNA of SEQ ID NO: 1.

Also, the strain can be anaerobic or aerobic.

In addition, the strain forms spores and can be excreted from the organism within 2 weeks after the strain is administered.

In addition, the strain produces an α-glucosidase inhibitor in the intestine of an animal, and the α-glucosidase inhibitor produced in the intestine is excreted from the organism within 2 weeks after the administration of the strain. sell.

The present invention also provides a food composition containing the strain.

Moreover, the food composition, the strain is included in the form of spores can be included in ¹⁰ 6 ^{to 10 10} spores per 1 kg.

In addition, the food composition further contains a fermented product of the strain, and the fermented product may be a fermented soybean product.

The Bacillus licheniformis NY1505 strain according to the present invention is a new strain that can live even under anaerobic conditions, and since it produces and supplies an α-glucosidase inhibitor in the intestine, it is excellent in keeping the effective concentration constant. The strains administered as needed or the α-glucosidase inhibitors produced by the strains, which show the effects of weight loss and hypoglycemia, do not stick to the intestine, and show the property of temporarily inhabiting, are excreted. It is possible and can be used safely.

In addition, there is no need for separate fermentation costs and processing costs for the production of α-glucosidase inhibitors, which reduces production costs, does not cause problems such as fermented odors, and can be applied to various forms of food. Is.

It is a graph which shows the effect of administration of Bacillus licheniformis NY1505 strain spores on the body weight of a mouse when feeding a high-carbohydrate diet (control group, high-carbohydrate diet group and high-carbohydrate diet + spore group). It is a graph which shows the effect of administration of Bacillus licheniformis NY1505 spores on the body weight of a mouse when feeding a high-fat diet (control group, high-fat diet group and high-fat diet + spore group). It is a graph which shows the change of the number of strains contained in the feces of a mouse (high carbohydrate diet + spore group and high fat diet + spore group). It is a graph which shows the inhibitory activity of α-glucosidase contained in mouse feces (high-carbohydrate diet + spore group and high-fat diet + spore group). It is an electron micrograph of Bacillus licheniformis NY1505 strain. It is a schematic diagram which shows the taxonomic position of the Bacillus licheniformis NY1505 strain.

Hereinafter, the present invention will be described in detail.

The present invention provides a Bacillus licheniformis NY1505 strain (accession number KCTC13021BP) that produces a high α-glucosidase inhibitor.

The strain can be obtained by separating strains that form heat-resistant spores using rice straw and hay as samples, and selecting a strain that can grow in vivo and produces a high α-glucosidase inhibitor.

The strain is characterized by containing the 16s RNA of SEQ ID NO: 1 below.

SEQ ID NO: 1:
AGGGCTTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGAACAAAGGGCAGCGAAGCCGCGAGGCTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGTACAGGGAAAA

The strain may simultaneously have an anaerobic property capable of producing an α-glucosidase inhibitor in high yield in the intestine and an aerobic property capable of producing an α-glucosidase inhibitor in vitro.

The strain forms spores, does not adhere to the intestine, exhibits the property of temporarily inhabiting, and can remove the administered strain as needed, and can be used safely. Desirably, the strain can be excreted from the organism two weeks after being administered to the organism, and more preferably, it can be excreted one week later.

In addition, the strain can produce the α-glucosidase inhibitor in the intestine, keep the effective concentration of the α-glucosidase inhibitor constant, and the α-glucosidase inhibitor produced in the intestine can be used. It can be excreted and can be used safely. Desirably, the α-glucosidase inhibitor can be excreted from the organism two weeks after the strain is administered to the organism.

As a result, the α-glucosidase inhibitor can be produced in a high yield in the intestine by ingesting a food containing the strain, and can be excreted from the organism, resulting in weight loss. It can be effectively used for the purpose of lowering blood glucose.

The present invention provides a food composition containing the strain.

The food composition may include a strain, spores of the strain or a fermented product of the strain cultivated by a commonly used Bacillus licheniformis culture method.

As an example, the food composition comprises spores of the strain, the food composition is a 10 ⁶ to 10 ¹⁰ spores per 1 kg, the strain comprising said strain, in the intestines α- A large amount of glucosidase inhibitor can be produced.

As yet another example, the food composition may further comprise a fermented product of the strain, which may be a fermented soybean product fermented by inoculating soybean with the strain.

Hereinafter, the present invention will be described in more detail with reference to examples.

The examples presented are specific examples of the present invention and are not intended to limit the scope of the present invention.

<Example>
1. 1. Search for strains Microorganisms were isolated from about 500 rice straws and hay collected from all over the world such as Korea, China, Japan, and the United States. After adding a small amount of each sample to sterile saline and suspending it, the spore solution obtained by heat-treating in a constant temperature water bath at 80 ° C. for 20 minutes is LB plate medium (1% tryptone, 0.2% sugar) containing 2% agar. , 0.5% yeast extract and 0.5% NaCl, pH 7.0), and anaerobically cultured in a 55 ° C. incubator for 2 days. After culturing, the cells forming the bacterial colony were separated. In order to investigate the production of α-glucosidase inhibitor, the isolated strain was inoculated into a medium in which 5 ml of 5% soybean flour was suspended and cultured at 37 ° C. for 24 hours. The culture broth was centrifuged and the α-glucosidase inhibitor activity was measured from the supernatant (aerobic culture in Table 1). After primary selection of about 60 facultative anaerobic bacteria as α-glucosidase inhibitor-producing bacteria in the above process, a strain capable of anaerobic growth at 50 ° C. and magnetizing propionic acid. , Three species having high α-glucosidase inhibitory activity were secondarily selected, the culture solution was centrifuged, and the α-glucosidase inhibitor activity was measured from the supernatant (anaerobic culture in Table 1). The three strains are classified by the method of analyzing taxonomic characteristics according to the Bergey's Manual of Systematic Bacteriology (2002), and one of them is an edible bacterium, Bacillus licheniformis. ), Named Bacillus licheniformis NY1505, confirmed that the strain has the 16S RNA sequence of SEQ ID NO: 1 below, and transferred the sequence to the Biological Resources Center (KCTC) of the Korea Institute of Biotechnology in 2016. Deposited on May 3 (deposit number: KCTC13021BP).

SEQ ID NO: 1:
AGGGCTTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGAACAAAGGGCAGCGAAGCCGCGAGGCTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGTACAGGGAAAA

2. Production of natto Soybeans were soaked at room temperature for 12 hours and then boiled at 121 ° C. for 40 minutes. Before cooling the boiled soybean, following inoculation with g per 10 ^Three spores, 50g each placed in natto manufacturing styrofoam container, after covered with vinyl cover, and 20 hours at 37 ° C.. After refrigerating the cultured natto for 12 hours, the α-glucosidase inhibitory activity was measured and sensory evaluation was performed. (Natto in Table 1).

3. 3. Measurement of α-glucosidase inhibitory activity α-glucosidase was extracted from the small intestine of pigs and partially purified, and used as an enzyme source. The α-glucosidase enzyme solution is appropriately diluted with 0.5 M phosphate buffer (pH 7.0) so as to be 24 units / ml, and the substrate is p-nitrophenyl α-D. -Glucopyranoside (p-nitrophenyl α-D-glucosidase, pNPG) was used. 30 μl of the culture supernatant and 50 μl of the enzyme were mixed in a test tube and preheated at 37 ° C. for 10 minutes, then 50 μl of 3 mM pNPG was added, and the mixture was reacted at 37 ° C. for 20 minutes. 0.1 M Na ₂ CO ₃ was added to the reaction solution to stop the reaction, the absorbance was measured at 405 nm, the inhibitory activity was measured by the following formula 1, and the measurement results of the α-glucosidase inhibitory activity are shown in Table 1 below. Indicated. At this time, the α-glucosidase inhibitory unit (α-glucosidase inhibition unit, AGI unit) is the amount of the inhibitor when 100% of the α-glucosidase (α-glucosidase) used for the activity measurement in the following formula 1 is inhibited. Defined.

As shown in Table 1, the fact that Bacillus licheniformis is a facultative anaerobic bacterium, showing uniform AGI inhibitory activity with or without oxygen supply, can grow even under anaerobic conditions. I was able to confirm. Therefore, it was confirmed that Bacillus licheniformis can proliferate and produce useful bioactive substances in the intestinal tract of animals.

4. Dietary experiment of Bacillus licheniformis NY1505 strain In order to confirm whether the Bacillus licheniformis NY1505 strain produces useful substances in the intestinal tract of animals, the Bacillus licheniformis NY1505 spores are mixed and administered to the mouse feed. The presence or absence of intestinal growth of the Bacillus licheniformis NY1505 strain and the presence or absence of α-glucosidase inhibitor production in the intestine were examined.

First, alpha-feed by mixing spores of Bacillus licheniformis NY1505 producing strain glucosidase inhibitor (10 ⁸ spores / kg diet) and salary mice were observed changes in body weight. For this purpose, the standard feed and water to be sold were freely fed, and male mice (00 weeks old) were used after pre-breeding for 1 week. The light-dark cycle is from 08:00 to 20:00 in the light period and from 20:00 to 08:00 in the dark period, with a temperature of 22.5 ± 0.5 ° C and a humidity of 50 to 60%. Breeding in a retained environment. High carbohydrate diet and high-fat diet and west in the following Table 2 and Table 3 in obese inductive, feed spores of Bacillus licheniformis NY1505 strain is contained, produced by mixing the respective obesity inductive 1kg per 10 ⁸ spores did. Mice were divided into 5 groups (8 animals each) and divided into a control group, a high-carbohydrate diet group, a high-carbohydrate diet + spore group, a high-fat diet group, and a high-fat diet + spore group, and bred for 7 weeks each. did.

(1) Analysis of the effect of administration of Bacillus licheniformis NY1505 spores on the body weight of mice when fed a high-carbohydrate diet. Analysis of the body weight (body weight, unit gram) of Bacillus likeniformis NY1505 spores As a result of analyzing the effect of administration, as shown in FIG. 1, the high-carbohydrate feed group showed a clear weight gain as compared with the control group, but the spores of the Bacillus likeniformis NY1505 strain were mixed and high. It was confirmed that the carbohydrate feed group showed the same aspect of weight gain as the control group.

(2) Effect of administration of Bacillus licheniformis NY1505 spores on body weight of mice fed with high-fat diet Effect of administration of Bacillus licheniformis NY1505 spores on body weight of mice fed with high-fat diet As a result of the analysis, as shown in FIG. 2, the high-fat feed group showed a clear weight gain as compared with the control group, but in the high-fat feed group mixed with spores of the Bacillus licheniformis NY1505 strain. , It was confirmed that the body weight gain mode was similar to that of the control group.

(3) Changes in the number of Bacillus licheniformis NY1505 excreted in feces The number of spores contained in the spores of mice to which Bacillus licheniformis NY1505 spores were administered was measured to analyze changes in the strains excreted in the stool. as a result, as shown in FIG. 3, 10 ^five spores per feed 1g are mixed, since intake of feed of approximately 5g per day per animal, about 5x10 ^five spores per day Ingest. After spore administered three weeks, since about ¹⁰⁷ animals of Bacillus licheniformis NY1505 per feces 1g is detected, it was found that this strain in the intestine is discharged by vigorous proliferation. When spores are administered for 7 weeks, the number of bacteria decreases dramatically after 8 weeks, which is one week when spores are not administered, so it can be judged that they do not stick to the intestines and inhabit temporarily. did it. Therefore, if the administration of this strain is discontinued, changes in the intestinal flora are expected, and it was confirmed that there is an advantage that the strain can be removed in the intestine as needed.

(4) Changes in α-glucosidase inhibitory activity excreted in stool As a result of analyzing the inhibitory activity of α-glucosidase contained in the stool of mice to which Bacillus licheniformis NY1505 bacterial spores were administered, as shown in FIG. In addition, it was confirmed that the Bacillus licheniformis NY1505 strain proliferates vigorously in the intestine to the extent that it is excreted by stool and produces an α-glucosidase inhibitor. The Bacillus licheniformis NY1505 strain grown in the intestine was excreted in the stool after 2 weeks from the mice to which the spores were administered, and the activity was detected. From the 4th week onward, it can be confirmed that the amount of α-glucosidase inhibitor excreted during the period when the amount of Bacillus licheniformis NY1505 strain excreted until the 7th week is stabilized is also constant. did it. That is, since the α-glucosidase inhibitor is always produced in the intestine, it has the advantage that the concentration in the intestine is maintained in a constant amount and acts with high efficiency, and a certain amount of the produced inhibitor is discharged. You can see that. Looking at the activity of the α-glucosidase inhibitor excreted in feces, a considerable amount of the α-glucosidase inhibitor was produced in the intestine as compared with the activity of natto produced by the Bacillus licheniformis NY1505 strain (Table 1). We were able to confirm the fact that some of it was discharged.

Through the above results, the Bacillus licheniformis NY1505 strain according to the present invention does not use the physiologically active substance produced by fermentation, but the strain continuously produces and supplies the physiologically active substance in the body. There is no need for additional fermentation costs and processing costs for the production of glucosidase inhibitors, and there is the advantage that the effective concentration can be kept constant by reducing the production cost and continuously producing in the body. By using the spores of useful bacteria, it is possible to use it as an additive in various processed foods without causing problems such as fermented odor, so it can be applied to various forms of food. I was able to confirm the fact that it can be done.

5. Investigation of morphological and biochemical characteristics of the isolated strain (1) Morphological characteristics of the isolated strain The Bacillus licheniformis NY1505 strain isolated as described above is the central portion of the colony on the LB agar plate medium. Showed a light rust color, and the edges showed an irregular outline (Fig. 5). The temperature range in which the bacteria can grow is in the range of 25 to 50 ° C., and the growth is good, and above 60 ° C., cell growth has not been confirmed. As a result of microscopic observation in the logarithmic phase of cell growth using LB liquid medium, if a short gram-positive rod was shown, it was similar to the comparative strain Bacillus licheniformis. In addition, it should be confirmed that the isolated strains are Gram-positive and the cell size is approximately 0.5 to 0.7 × 1.5 to 1.8 μm, as shown in Table 4 below. The above results were combined, and the taxonomic position of the Bacillus licheniformis NY1505 strain is shown in FIG.

(2) Biochemical characteristics of the isolated strain The biochemical of the isolated strain using the analysis method of the taxonomic characteristics of the Bacillus strain according to the Verjay's Manual and the API50CHB kit (BioMeriaux, France). As a result of investigating the characteristics and the like, the isolated strain had a Gram-positive cylindrical shape, spores were formed in the center of the cell, the reducing power of nitrate was positive, and indole formation was negative. In addition, it was confirmed that casein and starch were decomposed, catalase-positive, and grown under aerobic and anaerobic conditions, and that it had biochemical properties (API test results) as shown in Table 5 below. We were able to.

The Bacillus licheniformis NY1505 strain according to the present invention is used in food compositions because it produces and supplies an α-glucosidase inhibitor in the intestine.

Depositary name: Korea Research Institute of Bioscience and Biotechnology Korea Bioresource Center (KCTC)
Access number: KCTC13021BP
Contract date: 20160503

(Certificate of acceptance)

Claims (12)

Bacillus licheniformis NY1505 strain containing 16s RNA of SEQ ID NO: 1 and highly producing an α-glucosidase inhibitor (accession number KCTC13021BP). The strain according to claim 1, wherein the strain is anaerobic. The strain according to claim 2 , wherein the strain is aerobic. The strain according to claim 1, wherein the strain forms spores. The strain according to claim 1, wherein the strain is excreted from an organism within 2 weeks after administration. The strain according to claim 1, wherein the strain produces an α-glucosidase inhibitor in the intestine. The strain according to claim 6 , wherein the α-glucosidase inhibitor produced in the intestine is excreted from the organism within 2 weeks after the strain is administered. A food composition containing the strain according to any one of claims 1 to 7 . The food composition according to claim 8 , wherein the strain is contained in the form of spores. The strain, the food composition according to claim 9, characterized in that contained in ¹⁰ 6 ^{to 10 10} spores per 1 kg. The food composition according to claim 8 , wherein the food composition further contains a fermented product of the strain. The food composition according to claim 11 , wherein the fermented product is a fermented soybean product.