Bacillus licheniformis strain with high polyamine productivity

Abstract

The present invention relates to a Bacillus licheniformis strain producing a large amount of polyamines or α-glucosidase inhibitors, and to intestinal production and in vivo supply of polyamines or α-glucosidase inhibitors using same. Since the Bacillus licheniformis strain of the present invention can proliferate under anaerobic conditions as well as aerobic conditions, a large amount of physiologically active substances such as polyamines or α-glucosidase inhibitors can be continuously produced in the intestine and supplied to the human body. Therefore, the strain, spores thereof, cultures thereof, or fermented products thereof of the present invention can be applied and utilized for various purposes such as food and feed capable of producing polyamines, particularly spermidine, or α-glucosidase inhibitors with high efficiency.

Description

Bacillus licheniformis strain with high polyamine production

[0001] The present invention relates to a Bacillus licheniformis strain produced by mutation breeding from Bacillus licheniformis 203 (accession number KCTC 13021BP) strain, which produces polyamines and α-glucosidase inhibitors (AGI) in high quantities, and to a use thereof.

[0002] Polyamines are a general term for small polycationic aliphatic hydrocarbons with amino groups at both ends of their molecular structure. The major polyamines include putrescine, spermidine, and spermine. In both eukaryotic and prokaryotic cells, polyamines are known to be essential for life, possessing a wide range of biological functions, including gene regulation, stress resistance, cell proliferation, and differentiation. Because polyamines are essential for cell proliferation, they are found in high concentrations in actively proliferating cells. At the individual level, tissue polyamine concentrations are reported to be highest in young adults and decrease with age.

[0003] Polyamines are supplied through three routes: oral intake through food, intestinal microflora, and intracellular biosynthesis. The body's ability to biosynthesize polyamines is known to decline with age, and this is currently difficult to control. Therefore, to maintain polyamine concentrations in the body, methods for efficiently supplying polyamines derived from food and intestinal bacteria are needed.

[0004] Natto is known as a food containing a large amount of polyamines, but various supply methods from food sources have been proposed, such as manufacturing natto with increased polyamine content by changing the fermentation conditions of natto or fermenting it with a natto fermentation bacteria with significantly increased polyamine biosynthetic ability (Korean Patent No. 10-1794772, Japanese Patent No. 6054052), or extracting and supplying polyamines from food raw materials such as soybean or rice germ (Japanese Patent Laid-Open No. 10-101624, Japanese Patent Laid-Open No. 2007-291027) or fish testes (Japanese Patent Laid-Open No. 8-238094).

[0005] A promising non-food source of polyamines is intestinal bacteria. This occurs when polyamines produced by various intestinal microorganisms are absorbed in the intestines. However, there have been no examples of artificially modulating the intestinal microbiota to supply polyamines to the body through intestinal production. While indirect methods using Bifidobacteria, which promote polyamine production by intestinal microorganisms, have been proposed, direct intestinal production by polyamine-producing microorganisms could maximize the efficiency of polyamine supply to the body.

[0006] Meanwhile, α-glucosidase inhibitors are distributed in some plants and microorganisms, and by inhibiting the breakdown of oligosaccharides into monosaccharides in the intestines, they inhibit the smooth absorption of sugars in the intestines, thereby suppressing the rise in blood sugar after a meal and having secondary effects such as treating obesity.

[0007] Recently, research on edible natural products that can be consumed as food as α-glucosidase inhibitors that are effective in lowering postprandial blood sugar levels is actively being conducted.

[0008] Known plant-derived α-glucosidase inhibitors include aza sugars such as 1-deoxynojirimycin contained in mulberry leaves, isoflavones such as genistein and daidzein contained in soybeans, and some flavone glycosides. Known microbial-derived α-glucosidase inhibitors include pseudooligosaccharides from Actinoplanes strains, 1-deoxynojirimycin and tris base produced by microorganisms such as Streptomyces or Bacillus, and pharmaceuticals such as acarbose and voglibose are already commercially available as diabetes treatments.

[0009] An example of a microbial α-glucosidase inhibitor used as a food is natto, which is produced using the Bacillus subtilis DC-15 strain (Japanese Patent No. 4465337). Natto or extracts made with the Bacillus subtilis DC-15 strain for the purpose of consuming α-glucosidase inhibitors are mass-produced, but they commonly have factors that reduce palatability, such as odor and color, which limits the diversification of related products. In addition, the effective concentration of the α-glucosidase inhibitor in natto itself is low, so it must be consumed in large quantities or concentrated and purified, which requires a lot of time and money for processing.

[0010] Recently, it has been reported that some facultative anaerobic bacteria proliferate in the anaerobic intestinal environment and continuously produce and supply physiologically active substances such as α-glucosidase inhibitors to the intestines.

[0011] The intestines of animals are anaerobic spaces, primarily inhabited by anaerobic bacteria. Intestinal microorganisms reside within the intestines and provide the host with various physiologically active substances, such as vitamins and enzymes, produced by them. Therefore, the inventors of the present invention have studied strains capable of proliferating in the intestines and mass-producing physiologically active substances, such as polyamines and α-glucosidase inhibitors. As a result, they developed a Bacillus licheniformis strain with dramatically increased polyamine and α-glucosidase inhibitor production capacity, thereby completing the present invention.

[0012]

[0013] Prior art literature

[0014] Patent documents

[0015] Korean Patent No. 10-1794772

[0016] Japanese Patent No. JP6454052

[0017] Japanese Patent No. JP4465337

[0018] Japanese Patent No. JP6785324

[0019] The purpose of the present invention is to provide a novel strain having the ability to produce large amounts of polyamines and α-glucosidase inhibitors in the intestines of animals and supply them to the body.

[0020] In order to achieve the above object, the present invention provides a Bacillus licheniformis strain produced by mutation breeding from a Bacillus licheniformis 203 (accession number KCTC 13021BP) strain, which highly produces at least one selected from the group consisting of polyamines and α-glucosidase inhibitors.

[0021] In addition, the present invention provides a culture or spore comprising at least one selected from the group consisting of polyamines and α-glucosidase inhibitors, obtained by fermentation or culturing using the strain.

[0022] Furthermore, the present invention provides a food composition, a health functional food composition and a feed composition comprising the strain, a spore thereof, a culture thereof or a fermented product thereof.

[0023] Furthermore, the present invention provides a method for producing at least one selected from the group consisting of polyamines and α-glucosidase inhibitors, comprising a fermentation step using the strain.

[0024] The Bacillus licheniformis strain according to the present invention can survive even under anaerobic conditions, and thus produces and supplies physiologically active substances such as polyamines and α-glucosidase inhibitors in the intestines, thereby maintaining a constant effective concentration and exhibiting an appropriate effect. In addition, since it exhibits the characteristic of not remaining in the intestines but temporarily residing therein, the strain administered or the α-glucosidase inhibitor produced by the strain can be excreted as needed, and thus can be used safely.

[0025] In addition, by administering the spores of these strains in the form of food or feed, separate fermentation and processing costs for the production of physiologically active substances such as α-glucosidase inhibitors including polyamines are not required, so production costs can be reduced, and problems such as fermentation odor do not occur, so they can be applied to various forms of food and feed.

[0026] Figure 1 shows the results of thin-layer chromatography (TLC) analysis of polyamine production under aerobic (left) and anaerobic (right) conditions in Bacillus licheniformis BO825 and Bacillus licheniformis BO104 strains. Lane 1: Bacillus subtilis EE5, lane 2: Bacillus licheniformis BO825, lane 3: Bacillus licheniformis BO104. Standards used for comparison were putrescine, spermidine, and spermine.

[0027] The polyamine produced in all 1, 2, and 3 was mainly spermidine. The aerobic strain Bacillus subtilis EE5 produced spermidine under aerobic conditions, but no spermidine production was confirmed under anaerobic conditions. However, the Bacillus licheniformis BO825 and Bacillus licheniformis BO104 strains were confirmed to produce spermidine under both aerobic and anaerobic conditions.

[0028] Hereinafter, the present invention will be described in detail.

[0029]

[0030] Since substances such as polyamines and α-glucosidase inhibitors exhibit physiological activities such as anti-aging and weight loss in humans and animals, it is very important to provide a method for efficiently supplying these ingredients. In the process of pursuing a method for efficiently supplying physiologically active substances, the inventors of the present invention have confirmed the possibility of intestinal production of physiologically active substances by anaerobic microorganisms. Accordingly, the purpose of the present invention is to provide technical contents regarding Bacillus licheniformis BO825 (KCTC 15645BP), a strain that produces a large amount of polyamines, and Bacillus licheniformis BO104 (KCTC 15644BP), a strain that produces a large amount of α-glucosidase inhibitors and polyamines.

[0031] The polyamine content of cultures of strains secured in the inventor's laboratory and strains isolated from nature was examined. As a result, Bacillus licheniformis 203 (accession number KCTC 13021BP), a strain deposited at the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology, which is an α-glucosidase inhibitor-producing strain, exhibited the highest polyamine productivity. The Bacillus licheniformis strain with the accession number KCTC 13021BP showed the highest polyamine productivity among the screened microorganisms, but its industrial usability is low. Therefore, through mutant breeding using the Bacillus licheniformis 203 (accession number KCTC 13021BP) strain as the starting strain, Bacillus licheniformis BO104 (KCTC 15644BP), a strain that produces a large amount of α-glucosidase inhibitor and polyamine, and Bacillus licheniformis BO825 (KCTC 15645BP), a strain that produces a large amount of polyamine, were bred.

[0032]

[0033] In one aspect, the present invention provides a Bacillus licheniformis strain produced by mutation breeding through chemical mutagenesis from a Bacillus licheniformis 203 (accession number KCTC 13021BP) strain, which is highly productive of at least one selected from the group consisting of polyamines and α-glucosidase inhibitors.

[0034] The strain may have a 16s rRNA sequence represented by sequence number 1, and may be, for example, a Bacillus licheniformis BO104 (accession number KCTC 15644BP) strain or a Bacillus licheniformis BO825 (accession number KCTC 15645BP) strain.

[0035] 상기 서열번호 1로 표시되는 16s rRNA서열은 tctcccaggcgggagtgcttaatgcgtttgctgcagcactaaagggcggaaaccctctaacacttagcactcatcgtttacggcgtggactaccagggtatctaatcctgttcgctccccacgctttcgcgcctcagcgtcagttacagaccagagagtcgccttcgccactggtgttcctccacatctctacgcatttcaccgctacacgtggaattccactctcctcttctgcactcaagttccccagtttccaatgaccctccccggttgagccgggggctttcacatcagacttaagaaaccgcctgcgcgcgctttacgcccaataattccggacaacgcttgccacctacgtattaccgcggctgctggcacgtagttagccgtggctttctggttaggtaccgtcaaggtaccgccctattcgaacggtacttgttcttccctaacaacagagttttacgatccgaaaaccttcatcactcacgcggcgttgctccgtcagactttcgtccattgcggaagattccctactgctgcctcccgtaggagtctgggccgtgtctcagtcccagtgtggccgatcaccctctcaggtcggctacgcatcgtcgccttggtgagccgttacctcaccaactagctaatgcgccgcgggtccatctgtaagtggtagctgaaagccaccttttatgattgaaccatgcggttcaatcaagcatccggtattagccccggtttcccggagttatcccagtcttacaggcaggttacccacgtgttactcacccgtccgccgctgacctaagggagcaagctcccgtcggtccgctcgacttgcatgtattaggcacgccgccagcgttcgtcctga이다.

[0036] The above Bacillus licheniformis BO104 (accession number KCTC 15644BP) strain can be produced by mutagenesis breeding in which the above Bacillus licheniformis 203 (accession number KCTC 13021BP) strain is treated with a chemical mutagen selected from the group consisting of nitrosoguanidine, ethyl methanesulfonate (EMS), and a combination thereof.

[0037] The Bacillus licheniformis BO825 (Accession No. KCTC 15645BP) strain can be produced by mutagenic breeding in which the Bacillus licheniformis BO104 (Accession No. KCTC 15644BP) strain is treated with a chemical mutagen selected from the group consisting of nitrosoguanidine, ethylmethanesulfonic acid, and a combination thereof.

[0038] The above Bacillus licheniformis BO104 can produce high levels of polyamines as well as α-glucosidase inhibitors in the intestines. The α-glucosidase inhibitor produced in the intestines by the above strain can be excreted from the organism within two weeks after administration of the strain.

[0039] The above polyamine may be spermidine.

[0040] The above two strains can mass-produce polyamines and / or α-glucosidase inhibitors under both anaerobic and aerobic conditions. Anaerobic conditions, as used herein, refer to conditions with limited oxygen supply, such as animal intestines, while aerobic conditions refer to conditions with a smooth oxygen supply, such as shaking culture.

[0041] The above strain forms spores, so oral administration in a spore state can increase the rate of intestinal penetration. Furthermore, it exhibits the characteristic of temporarily residing in the intestines without settling, allowing for the removal of the administered strain as needed, making it safe for use. Preferably, the strain can be excreted from the organism 2 to 4 weeks after administration.

[0042] In addition, among the above-mentioned strains, the Bacillus licheniformis BO104 (KCTC 15644BP) strain has the characteristic of producing large amounts of polyamines and α-glucosidase inhibitors, while the Bacillus licheniformis BO825 (KCTC 15645BP) strain has the characteristic of producing large amounts of only polyamines without producing α-glucosidase inhibitors, so they can be used for different purposes.

[0043] The strain of the present invention can be provided in various forms. For example, it can be provided in the form of a culture or spore containing at least one selected from the group consisting of polyamines and α-glucosidase inhibitors obtained by fermentation or culture using the strain.

[0044] Accordingly, by administering feed or food containing the Bacillus licheniformis BO104 (KCTC 15644BP) strain, the α-glucosidase inhibitor produced in the intestine can be expected to have weight loss and blood sugar lowering effects, and the polyamine can be expected to have beauty and life extension effects.

[0045] In addition, by administering feed or food containing the Bacillus licheniformis BO825 (KCTC 15645BP) strain, polyamines can be produced in large quantities in the intestines, and thus, by administering a small amount of the strain, the physiological activity effects of polyamines other than beauty and life extension can be expected.

[0046]

[0047] In one aspect, the present invention provides a food composition, a health functional food composition and a feed composition comprising the Bacillus licheniformis strain, a spore thereof, a culture thereof or a fermented product thereof.

[0048] In the present invention, the term "spore" refers to a resting cell of a bacterium, and for the purpose of the present invention, the spore refers to a resting cell of Bacillus licheniformis.

[0049] The Bacillus licheniformis strain provided by the present invention is useful in that spores germinate in the intestines of an animal, proliferate into vegetative cells, and directly produce polyamines, thereby continuously supplying them to the host.

[0050] The above food, health functional food and feed composition may contain spores of the above strain, and the above food, health functional food and feed composition may contain 10 per 1 g. 6 10 inland 10 Since the strain is contained in the form of spores of a dog, the strain can proliferate in the intestine and produce large amounts of polyamine and α-glucosidase inhibitor.

[0051] As another example, the food, health functional food and feed composition may further include a fermented product of the strain, and the fermented product may be a plant-based fermented product obtained by inoculating the strain into a plant-based raw material such as soybeans or soybeans and fermenting it.

[0052] In the present invention, the term "culture" refers to a product obtained after culturing the strain, and may be a culture solution including cells, or may be cells obtained by removing or concentrating the cultured strain or culture supernatant. The composition of the culture may additionally include components that act synergistically on the growth of Bacillus licheniformis in addition to components necessary for the typical Bacillus licheniformis culture, and the composition accordingly may be easily selected by a person skilled in the art.

[0053] The novel isolated Bacillus licheniformis strain of the present invention can be cultured using a conventional culture method for Bacillus licheniformis strains, but is not limited thereto. A natural medium or a synthetic medium can be used as a medium. For example, glucose, sucrose, dextrin, glycerol, starch, etc. can be used as a carbon source of the medium, and a soybean hot water extract, peptone, meat extract, yeast extract, defatted soybean meal, ammonium salts, nitrates, and other organic or inorganic nitrogen-containing compounds can be used as a nitrogen source, but are not limited to these components. Inorganic salts included in the medium can be magnesium, manganese, calcium, iron, phosphorus, etc., but are not limited to these. In addition to the carbon source, nitrogen source, and inorganic salt components, amino acids, vitamins, nucleic acids, and related compounds can be added to the medium.

[0054] In the present invention, the term "fermented product" means a medium obtained by inoculating and culturing (fermenting) a Bacillus licheniformis BO825 or Bacillus licheniformis BO104 strain into a medium containing a main raw material of food, or a culture containing a microorganism cultured together with the medium. In addition, the fermented product may be a culture obtained by inoculating and culturing a food raw material with a Bacillus licheniformis strain. The food raw material may be a grain, a grain-derived food, etc., and the grain may be soybeans, rice, barley, wheat, corn, etc., and preferably may be soy milk or green tea, but is not limited thereto.

[0055] The above fermented product may be comprehensively interpreted to include, but is not limited to, all of the filtrate, dilution, concentrate thereof, adjusted product thereof, purified product, dried product, pulverized product, etc. of the above medium or culture.

[0056]

[0057] In the present invention, the term "food" refers to a natural product or processed product containing one or more nutrients, preferably a product that has gone through a certain degree of processing to become directly edible, and in its conventional sense, may include all of food, food additives, health functional foods, and beverages.

[0058] The above "health functional food" refers to a food group or food composition that has been given added value to perform and express the function of the food for a specific purpose, or a food that has been designed and processed to sufficiently express the body's internal regulatory function related to regulating the biological defense rhythm, disease prevention, and recovery. The above functional food may further include food science-acceptable food supplement additives, appropriate carriers, excipients, and diluents.

[0059] In the present invention, the "feed composition" may include both animal feed and animal feed additives. The feed composition of the present invention may be in the form of a dry or liquid formulation, and may further include other non-pathogenic microorganisms or enzyme preparations in addition to the strain, its spores, its culture, or its fermented product. Various grains and soy protein, as well as peanuts, peas, sugar beets, pulp, grain by-products, animal viscera powder, and fish meal powder, may be used as feed raw materials, and these may be used without limitation in an unprocessed or processed form.

[0060]

[0061] In one aspect, the present invention provides a method for producing a polyamine or α-glucosidase inhibitor, comprising a step of fermenting using the strain.

[0062] In the above method for producing polyamine, the fermentation may be fermentation of soybeans or soybeans, and the polyamine may be spermidine. In this case, the Bacillus licheniformis strain may be added to the soybeans or soybeans at a concentration of 10 1 ~10 9It can be fermented by inoculating cells / mL, preferably 10 4 ~10 8 It may be fermented by inoculating cells / mL.

[0063] In the above method for producing polyamine, the fermentation may be carried out at 30 to 50°C for 20 to 80 hours, and preferably, after strain inoculation, the fermentation may be carried out at 37 to 47°C for 24 to 72 hours.

[0064] The present invention will be described in more detail below through examples. However, the following examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0065]

[0066] Example 1. Search and selection of strains producing polyamines.

[0067] Strain exploration

[0068] Microorganisms were isolated from approximately 400 samples of rice straw and hay collected from various locations in Korea. Each sample was suspended in a small amount of sterile saline solution and heat-treated in a constant-temperature water bath at 80°C for 20 minutes. The spore solution was spread on LB plate medium (1% tryptone, 0.2% sucrose, 0.5% yeast extract, and 0.5% NaCl, pH 7.0) containing 2% agar and incubated anaerobically in an incubator at 55°C for 2 days. After incubation, the cells forming colonies were isolated. To investigate the polyamine production, the isolated strain was inoculated into a medium containing 5 ml of 5% soybean powder and incubated with shaking at 37°C for 24 hours. The culture solution was centrifuged and the polyamine content of the supernatant was measured (Table 1). After selecting about 200 facultative anaerobic bacteria through the above process, strains that can grow anaerobically at 50℃ and autogenize propionic acid were selected, and 7 strains with high polyamine contents in the culture medium were selected for the second time. Separately, 12 strains, including 5 strains of Bacillus licheniformis that were isolated from nature for various purposes, identified, and preserved in the inventor's laboratory, and 7 strains selected for the second time, were inoculated into a 5% soybean powder suspension medium and cultured with shaking at 37℃ for 24 hours, and the polyamine production of the culture medium was compared. As a result, Bacillus licheniformis strain 203 showed the highest polyamine productivity, and this strain is an α-glucosidase inhibitor-producing strain deposited at the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology (accession number KCTC 13021BP).

[0069]

[0070] Quantification of polyamines

[0071] After culturing, 1 ml of 5% soybean medium was added with 1 ml of 5% HClO4, shaken for 6 hours, and centrifuged. 1 ml of the supernatant was taken, 2 ml of saturated Na2HCO3 and 2 ml of 1% dansyl chloride were added, and the mixture was reacted at 70°C for 1 hour. 2 ml of 1% proline was added, and the mixture was reacted at 70°C for 30 minutes. 0.5 ml of toluene was added to 0.5 ml of the reaction mixture, vortexed, and centrifuged. The supernatant was used as a TLC analysis sample. The TLC analysis used cyclohexane: ethylacetate = 3:2 as a developing solvent, and 1 to 3 μl of the dansyl-treated sample and 1 to 3 μl of the spermidine standard were loaded, developed, photographed under ultraviolet light, and the intensity of the spot was measured with a densitometer to determine the amount of spermidine from the standard curve.

[0072]

[0073] Analysis using HPLC (Shiseido SP-LC [Shiseido, Japan]) was performed by eluting the dansyl derivative with a mobile phase of H2O:MeOH composition at 25°C at a flow rate of 0.2 ml / min using an HPLC column (ImtaktUnison UK-C18 Column, 75 X 2 mm, particle size 3 μm) with a gradient elution of 50:50 → 0:100, and performing HPLC under the conditions shown in Table 2 below.

[0074]

[0075] Example 2. Preparation of a high-polyamine-producing mutant strain

[0076] Bacillus licheniformis KCTC 13021BP strain, selected as a polyamine-producing strain, is a strain reported to be an α-glucosidase inhibitor-producing strain, but mutation breeding was performed to further increase the α-glucosidase inhibitor and polyamine productivity.

[0077] Bacillus licheniformis KCTC 13021BP strain in the logarithmic growth phase cultured in LB liquid medium at 37℃ for 3 hours was centrifuged to collect the cells and suspended in 0.8% saline solution, pH 7.0. 100μg / ml of nitrosoguanidine (NTG) was added, and the treatment time was adjusted to achieve a 99.9% mortality rate to induce mutations. Approximately 200 cells were plated per Spizizen minimal medium and cultured at 37℃. The surviving cells were individually cultured in 5% soybean medium with shaking to measure the α-glucosidase inhibitor activity. This process was repeated several times to secure strains with increased α-glucosidase inhibitor activity. The sporulation ability of mutant strains with increased α-glucosidase inhibitor activity was tested, and one strain with high sporulation rate and the highest α-glucosidase inhibitor activity and polyamine production ability was named Bacillus licheniformis BO104. In order to further increase polyamine production ability, Bacillus licheniformis BO104 strain was mutated using the above method, and then 10 per sheet of Spizaizen minimal medium containing 5 mM dicyclohexylamine 5 The cells were smeared and cultured at 37℃ for 5 days, and the surviving bacteria were cultured on 5% soybean medium and the amount of polyamine was measured. Bacillus licheniformis BO104 strain was mutated using the same process as above, and one strain with no α-glucosidase inhibitor activity and high polyamine productivity was selected and named Bacillus licheniformis BO825. These strains were deposited with the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology on October 16, 2023 as Bacillus licheniformis BO104 (Accession No.: KCTC 15644BP) and Bacillus licheniformis BO825 (Accession No.: KCTC 15645BP), respectively.

[0078]

[0079] Example 3. Evaluation of Bacillus licheniformis strains BO104 and Bacillus licheniformis strains BO825

[0080] Bacillus licheniformis KCTC 13021BP strain was a facultative anaerobic bacterium that produced α-glucosidase inhibitors and showed the highest polyamine productivity among the screened strains. Using this strain as a starting strain, mutations were performed to obtain Bacillus licheniformis BO104 strain and Bacillus licheniformis BO825 strain. To determine how oxygen supply affected the production of polyamines and α-glucosidase inhibitors in the culture of these two strains, 10 per mL of 5% soybean medium was used. 3After inoculating each spore, the cultures were shaken at 37℃ for 24 hours or statically cultured in an anaerobic chamber. Although there were slight differences depending on the oxygen supply during culture, the Bacillus licheniformis BO104 strain showed a 210 to 230% increase in α-glucosidase inhibitor activity and a 130 to 150% increase in polyamine content compared to the starting strain, Bacillus licheniformis KCTC 13021BP, and was evaluated as a strain with multiple activities that simultaneously produce large amounts of α-glucosidase inhibitor and polyamine. The Bacillus licheniformis BO825 strain showed a 220 to 250% increase in polyamine production compared to the starting strain, but did not produce any α-glucosidase inhibitors, indicating different physiologically active substance production characteristics from the starting strain Bacillus licheniformis KCTC 13021BP and the mutant Bacillus licheniformis BO104 strain. Therefore, the Bacillus licheniformis BO104 strain and the Bacillus licheniformis BO825 strain can be expected to have different industrial applications. Bacillus subtilis EE5 strain is a natto bacterium that produces a large amount of polyamines, and Bacillus subtilis DC-15 strain is a natto bacterium that produces an α-glucosidase inhibitor. Under aerobic conditions, they produce a large amount of polyamines or α-glucosidase inhibitors, but under anaerobic conditions, they produce almost no polyamines or α-glucosidase inhibitors. Therefore, unlike Bacillus subtilis strains that are obligately aerobic, both Bacillus licheniformis BO104 strain and Bacillus licheniformis BO825 strain have the characteristic of producing a large amount of polyamines or α-glucosidase inhibitors (AGI) regardless of the supply of oxygen, so they can be very usefully applied in terms of material production in the intestines.

[0081] In addition, as can be seen in Fig. 1, the polyamine produced in all 1, 2, and 3 was mainly spermidine, and the aerobic strain Bacillus subtilis EE5 produced spermidine under aerobic conditions, but no spermidine production was confirmed under anaerobic conditions. This suggests that the Bacillus licheniformis BO825 strain and the Bacillus licheniformis BO104 strain produce spermidine under both aerobic and anaerobic conditions.

[0082]

[0083] Example 4. Dietary experiment of Bacillus licheniformis strain BO104

[0084] To determine whether the α-glucosidase inhibitor produced in the intestine of animals by Bacillus licheniformis BO104 strain exhibits a weight-loss effect, it was administered to dogs in the form of feed containing spores.

[0085] For the purpose of effectively administering Bacillus licheniformis BO104 strain to dogs, 10 spores of Bacillus licheniformis BO104 strain were added per 1 g to chicken breast cubes for dog treats (approximately 0.5 g each). 9 After spraying, it was manufactured by drying so that the moisture content is less than 5%. Dogs whose weight changes were to be observed were maintained under the same breeding conditions as usual and fed one chicken breast cube containing spores (approximately 0.5 g) per 5 kg of body weight as a snack once a day. Twelve dogs weighing 3.5 to 33.1 kg participated, and their weights were measured twice a week and observed for 4 weeks. As a result of administering spores of the Bacillus licheniformis BO104 strain for 4 weeks, a weight loss effect of approximately 5% compared to the starting body weight was observed (Table 4). It was confirmed that the spores of the Bacillus licheniformis BO104 strain processed and administered in the form of feed proliferate in the intestine and efficiently produce α-glucosidase inhibitors, thereby having the function of weight loss.

[0086]

[0087] Example 5. Changes in blood polyamine concentrations following administration of Bacillus licheniformis strain.

[0088] In order to measure the change in blood polyamine concentration according to the administration of spores of Bacillus licheniformis strains BO104 and BO825 that produce large amounts of polyamines and Bacillus licheniformis general strains that do not produce large amounts of polyamines, 10 male mice were administered spore samples of each strain at 10 weeks of age. The microorganism administration group was administered spores of Bacillus licheniformis strains BO104 and BO825 and Bacillus licheniformis KCTC 1732 as general strains in 10 doses each. 7 The cells were administered three times a week, and the polyamine administration group was administered 30 μg and 60 μg of spermidine three times a week, respectively. The microbial administration group and the polyamine administration group were administered orally three times a week after diluting each of the above dosages in 200 μL of sterile water. As a control group, 200 μL of saline solution was administered orally three times a week instead of the strain spore sample. After four weeks of administration, blood samples were collected to measure the concentration of polyamine (spermidine) in the blood, which is shown in Table 5 below. Since the polyamine produced by the Bacillus licheniformis strains BO104 and BO825 is mainly spermidine, the amount of polyamine in the blood was expressed as the amount of spermidine.

[0089]

[0090] As shown in Table 5 above, when the spores of the Bacillus licheniformis KCTC 1732 strain were administered, there was no significant difference from the control group in the change in the blood polyamine concentration, whereas when the spores of the Bacillus licheniformis BO104 and BO825 strains of the present invention were administered, polyamines were produced about 2 to 4 times more than the Bacillus licheniformis KCTC 1732 strain, confirming that they are effective in proliferating in the intestines of animals, producing polyamines, and continuously supplying them to the host.

[0091]

[0092] As a result of the above, it can be confirmed that the Bacillus licheniformis BO104 and BO825 strains according to the present invention proliferate in the intestines and continuously produce and supply physiologically active substances such as polyamines or α-glucosidase inhibitors, so that separate fermentation costs and processing costs for the production and supply of useful substances are not required, thereby reducing production costs. In addition, it can be confirmed that there is an advantage in that an effective concentration can be maintained constantly by continuously producing in the body. In addition, since the spores of useful bacteria are used, problems such as fermentation odor do not occur, and since they can be used as additives to processed foods in the form of spores with negligible volume, they can be applied to various types of foods.

[0093] Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)

[0094] Accession number: KCTC 15644BP

[0095] Date of acceptance: 20231016

[0096]

[0097] Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)

[0098] Accession number: KCTC 15645BP

[0099] Date of acceptance: 20231016

Claims

1. A Bacillus licheniformis strain produced by mutation breeding from Bacillus licheniformis 203 (accession number KCTC 13021BP) strain, which produces at least one selected from the group consisting of polyamines and α-glucosidase inhibitors.

2. A strain according to claim 1, wherein the strain has a 16s rRNA sequence represented by sequence number 1.

3. In the first paragraph, the strain is a Bacillus licheniformis BO104 (accession number KCTC 15644BP) strain or a Bacillus licheniformis BO825 (accession number KCTC 15645BP) strain.

4. In the third paragraph, the Bacillus licheniformis BO104 strain is a strain that produces a high level of α-glucosidase inhibitor and polyamine in the intestine.

5. In the third paragraph, the Bacillus licheniformis BO825 strain is a strain that produces a high level of polyamine in the intestine.

6. A strain according to claim 4, wherein the α-glucosidase inhibitor produced in the intestine is excreted from the organism within 2 to 4 weeks after administration of the strain.

7. In the third paragraph, the Bacillus licheniformis BO104 (accession number KCTC 15644BP) strain is a strain produced by mutation breeding in which the Bacillus licheniformis 203 (accession number KCTC 13021BP) strain is treated with a chemical mutagen selected from the group consisting of nitrosoguanidine, ethylmethanesulfonic acid, and a combination thereof.

8. In the third paragraph, the Bacillus licheniformis BO825 (accession number KCTC 15645BP) strain is a strain produced by mutation breeding in which the Bacillus licheniformis BO104 (accession number KCTC 15644BP) strain is treated with a chemical mutagen selected from the group consisting of nitrosoguanidine, ethylmethanesulfonic acid, and a combination thereof.

9. A strain characterized in that the polyamine in paragraph 1 is spermidine.

10. A strain according to claim 1, characterized in that the strain is anaerobic.

11. A strain according to claim 1, characterized in that the strain is aerobic.

12. A culture or spore containing at least one selected from the group consisting of polyamines and α-glucosidase inhibitors, obtained by fermentation or culturing using the strain of paragraph 1.

13. A food composition comprising the strain of paragraph 1, its spores, its culture or its fermented product.

14. A health functional food composition comprising the strain of paragraph 1, its spores, its culture, or its fermented product.

15. A feed composition comprising the strain of paragraph 1, its spores, its culture or its fermented product.

16. A method for producing at least one selected from the group consisting of polyamines and α-glucosidase inhibitors, comprising a fermentation step using the strain of paragraph 1.

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