Polyamine-high-producing Bacillus coagulans strain

Abstract

The present invention relates to a Bacillus coagulans PA strain that mass-produces polyamines and a method for producing polyamines using the same. Since the Bacillus coagulans PA strain of the present invention grows in the anaerobic intestine when ingesting the spores themselves and can continuously produce a large amount of polyamines to supply the human body, the strain of the present invention, its spores, its culture or its ferment can be applied to foods, feeds, etc. that can produce polyamines, particularly spermidine, with high efficiency and can be usefully used as a functional raw material for promoting hair growth and improving skin.

Description

Technical Field

[0001] The present invention relates to a Bacillus strain that produces a large amount of polyamine under anaerobic conditions. More specifically, it relates to the Bacillus coagulans PA strain that grows in the intestine of animals under anaerobic conditions, produces polyamine, and directly supplies it to animals. Through this process, polyamine is continuously produced in the intestine and has the characteristic of being continuously absorbed into the body.

Background Art

[0002] Polyamine is a substance that exists in large amounts inside cells. Recently, various physiological meanings of polyamine have been elucidated. Physiological effects expected by the intake of polyamine include anti-aging, life extension, anti-inflammation, cell revival effects, etc. Along with this, the interest in polyamine or polyamine compositions has increased, and efforts have been made for their application to daily processed foods, health foods, cosmetics, etc.

[0003] Conventionally, technical content regarding a method of extracting polyamine from raw materials containing a large amount of polyamine, such as yeast cells and animal organs, and manufacturing it into an ingestible food has been disclosed.

[0004] As an example of the technology for manufacturing food polyamine, methods of extracting polyamine from fish testes (Japanese Patent Laid-Open Publication No. Hei 8-238094), yeast cells (Japanese Patent Laid-Open Publication Nos. 2001-008663, 2001-095483), soybeans or rice germ (Japanese Patent Laid-Open Publication Nos. Hei 10-101624, 2007-291027), etc. have been presented.

[0005] Apart from the method of supplying polyamines purified by direct extraction from natural substances, the use of fermented products rich in polyamines has been proposed. By fermenting soybean germ with a high polyamine content using a high polyamine-producing strain (Korean Registered Patent 10-2288725), a method for increasing the productivity of polyamines and the purity of spermidine has also been proposed.

[0006] However, the methods disclosed as extraction methods from animals, plants, and various materials require complex extraction and separate purification processes for the production of polyamines, resulting in high production costs. Especially when using plants as raw materials, putrescine, a polyamine with a strong unpleasant odor, is contained in a large amount. Therefore, processed products using this as a raw material have problems to be solved, such as a decrease in palatability and content.

[0007] Apart from the method of supplying polyamines purified by extraction from natural substances, research has also been conducted on methods of ingesting foods with increased polyamine content (Korean Registered Patent 10-1794772, Japanese Registered Patent 6054052). According to this method, natto with a polyamine content five times higher than ordinary natto can be ingested as a food. However, this also limits the general intake method due to the preference for specific fermented products.

[0008] The supply of polyamines in the body is either synthesized intracellularly from precursors such as ornithine, or polyamines contained in foods or synthesized by intestinal bacteria are absorbed into the body through the intestinal tract and become important sources. In growing young individuals, polyamine synthesis occurs actively, but as they age, the polyamine synthesis ability decreases. Therefore, ingesting foods rich in polyamines or polyamines synthesized by intestinal bacteria becomes important in maintaining the polyamine level in the body.

[0009] Therefore, as a result of researching strains capable of highly producing polyamines from facultative anaerobic strains that can grow in the intestine, the present inventors developed a Bacillus coagulans PA strain, which is a new strain with a dramatically improved polyamine production ability, and completed the present invention.

Summary of the Invention

Problems to be Solved by the Invention

[0010] An object of the present invention is to provide a novel strain having the ability to produce a large amount of polyamines in the human intestine and supply them to the human body.

Means for Solving the Problems

[0011] To achieve the above object, the present invention provides a Bacillus coagulans PA strain (Accession No. KCTC 14266BP) having the ability to produce polyamines.

[0012] In one embodiment, the strain is characterized by having a 16S rRNA sequence represented by SEQ ID NO: 1, and can produce polyamines, particularly spermidine, with high content and high efficiency in aerobic and anaerobic states.

[0013] The present invention also provides a culture or spore containing polyamines obtained by fermentation or culture using the above strain.

[0014] Furthermore, the present invention provides a food composition, a feed composition, and a cosmetic composition containing the above strain, its spore, its culture, or its fermented product.

[0015] Furthermore, the present invention provides a method for producing polyamines, which includes a step of fermenting using the above strain.

[0016] In addition, the present invention provides a hair growth promoting composition and a skin improving composition containing, as an active ingredient, any one or more selected from the group consisting of the above-mentioned strain, its culture, its spores, and its fermented product.

[0017] In addition, the present invention provides a beverage with an increased polyamine content fermented using the above-mentioned strain.

[0018] In addition, the present invention provides a hair growth promoting method including the step of administering to an individual an effective amount of any one or more selected from the group consisting of the above-mentioned strain, its culture, its spores, and its fermented product.

[0019] In addition, the present invention provides a skin improving method including the step of administering to an individual an effective amount of any one or more selected from the group consisting of the above-mentioned strain, its culture, its spores, and its fermented product.

Effects of the Invention

[0020] The Bacillus coagulans PA strain according to the present invention can continuously produce polyamines not only under aerobic conditions but also in the anaerobic intestine, so it can show the effect of being absorbed in the intestinal tract and increasing the blood polyamine concentration. It shows the characteristic of temporarily inhabiting in the intestine, and the administered strain can be excreted as needed and can be used safely.

[0021] In addition, since the Bacillus coagulans PA strain of the present invention can form spores in an inexpensive medium and be supplied in the form of spores, separate fermentation costs and processing costs are not required for the production of polyamines, the production cost can be reduced, problems such as fermentation odor do not occur, and it can be applied to various forms of food.

[0022] Furthermore, since the Bacillus coagulans PA strain of the present invention has the effect of promoting hair growth, it is also useful as a pharmaceutical, food, or cosmetic composition for promoting hair growth and a composition for improving the skin.

Brief Description of the Drawings

[0023]

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BEST MODE FOR CARRYING OUT THE INVENTION

[0024] Hereinafter, the present invention will be described in detail.

[0025] The inventors of the present invention conducted research on methods for efficiently supplying polyamines to the body and found that some edible facultative anaerobic bacteria have a high ability to synthesize polyamines not only under aerobic conditions but also under anaerobic conditions. The fact that they exhibit a high polyamine synthesis ability under anaerobic conditions means that a new and simple method can be switched to mass-produce and supply polyamines in the human intestine. Therefore, in order to mass-produce polyamines in the human intestine and supply them to the human body, an edible strain that mass-produces polyamines under anaerobic conditions was searched for, and the Bacillus coagulans PA strain with significantly improved polyamine productivity was completed through mutation breeding and deposited with the Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (Accession No. KCTC 14266BP).

[0026] Bacillus coagulans PA, which is a new strain of the present invention, grows well in the intestinal tract. As a result, it has the effect of increasing the concentration of polyamines in the blood, and the strain or its spores themselves can be directly administered, providing an economical and simple method for supplying polyamines to the body.

[0027] In one aspect, the present invention provides a Bacillus coagulans PA strain (Accession No. KCTC 14266BP) having the ability to produce polyamines.

[0028] The strain of the present invention can be obtained by separating facultative anaerobic bacteria that form heat-resistant spores from rice straw and hay, and through a mutagenesis process, selecting a strain that mass-produces high-content polyamines. The selected strain can produce polyamines, particularly spermidine, under aerobic or anaerobic conditions. Preferably, it can produce spermidine under both aerobic and anaerobic conditions.

[0029] According to an embodiment of the present invention, it was confirmed that using the strain of the present invention, spermidine with a high content of 120 μg / mL or more can be produced by aerobic culture and about 90 μg / mL or more by anaerobic culture in a 5% soybean medium.

[0030] The strain of the present invention is characterized by having a 16S rRNA sequence represented by SEQ ID NO: 1.

[0031] The strain of the present invention can be provided in various forms. For example, it can be provided in the form of a culture containing polyamines obtained by fermentation or culture using the strain, or in the form of spores.

[0032] In one aspect, the present invention provides a food composition, a feed composition, and a cosmetic composition containing the Bacillus coagulans PA strain, its spores, its culture, or its fermented product.

[0033] In the present invention, the term "spores" refers to resting cells of bacteria, and for the purpose of the present invention, spores mean resting cells of Bacillus coagulans PA.

[0034] The Bacillus coagulans PA strain provided by the present invention is useful in that the spores germinate in the animal intestine and grow into vegetative cells, directly produce polyamines, and continuously supply them to the host.

[0035] In the present invention, the term "culture" means a product obtained after culturing the strain, which may be a culture stock solution containing bacterial cells, or bacterial cells obtained by removing or concentrating the cultured strain and the culture supernatant. The composition of the culture may further contain not only the components necessary for normal Bacillus coagulans culture but also components that synergistically act on the growth of Bacillus coagulans, and the composition thereof can be easily selected by those skilled in the art.

[0036] The newly isolated Bacillus coagulans PA of the present invention can be cultured by the culturing method of ordinary Bacillus coagulans strains, but is not limited thereto. As the medium, a natural medium or a synthetic medium can be used. As the carbon source of the medium, for example, glucose, sucrose, dextrin, glycerol, starch, etc. can be used, and as the nitrogen source, 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, but are not limited to these components. As the inorganic salts contained in the medium, magnesium, manganese, calcium, iron, phosphorus, etc. can be used, but are not limited thereto. In addition to the components of the carbon source, nitrogen source and inorganic salts, amino acids, vitamins, nucleic acids and related compounds may be added to the medium.

[0037] In the present invention, the term "fermented product" means a medium obtained by inoculating and culturing (fermenting) the Bacillus coagulans PA strain in a medium containing the main raw material of food, or a culture containing the microorganism cultured together with the medium. Further, the fermented product may be a culture obtained by inoculating and culturing the Bacillus coagulans PA strain in a food raw material. The food raw material may be cereals, foods derived from cereals, etc., and the cereals may be beans, rice, barley, wheat, corn, etc., and preferably may be soy milk or green tea, but is not limited thereto.

[0038] The fermented product can be comprehensively interpreted to include all of the filtrate, diluent, concentrated solution, crude purification product, purified product, dried product, pulverized product, etc. of the medium or culture, but is not limited thereto.

[0039] The food composition, feed composition and cosmetic composition according to the present invention may exhibit a hair growth promoting effect and a skin improving effect.

[0040] In the present invention, the skin improvement may mean improvement of skin aging, improvement of wrinkles, skin regeneration, or scalp improvement, and preferably may mean improvement of wrinkles.

[0041] In the present invention, the "food" means a natural product or processed product containing one or more nutrients, preferably a product that has undergone a certain degree of processing and can be directly eaten, and in the ordinary sense, may include all of foods, food additives, functional foods, and beverages.

[0042] The "functional food" means a food group or food composition in which the functions of foods are given added value so as to act and be expressed for specific purposes, and is designed and processed so that the in-vivo regulatory functions related to biological defense rhythm regulation, disease prevention and recovery, etc. are fully expressed in the living body. The functional food may further contain food auxiliaries, carriers, excipients, and diluents that are acceptable in food science.

[0043] In the present invention, the "feed composition" may include both animal feeds and additives for animal feeds. The feed composition of the present invention may be in a dry or liquid preparation form, and in addition to the strain, its spores, its culture, or its fermented product, may further contain other non-pathogenic microorganisms or enzyme preparations. As raw materials for feeds, various grains, soybean proteins, peanuts, peas, sugar beets, pulp, grain by-products, animal offal powder, fish meal, etc. can be used, and these can be used without limitation in their unprocessed or processed forms.

[0044] The "cosmetic composition" can be used as an external preparation for the skin and can be directly applied to the necessary site. For example, it can be produced in various dosage forms such as ointments, creams, emulsions, shampoos, rinses, soaps, etc. The cosmetic composition of the present invention may further contain at least one additive selected from the group consisting of purified water, oil, surfactant, humectant, stabilizer, alcohol, thickener, chelating agent, pigment, preservative, and fragrance.

[0045] The food composition, feed composition, or cosmetic composition containing the strain, its spores, its culture, or its ferment of the present invention may contain the strain in the form of spores, and may contain 10 4 ~10 14 spores per gram.

[0046] On one aspect, the present invention provides a method for producing polyamine, which includes a step of fermenting using the strain.

[0047] In the method for producing polyamine, the fermentation may be the fermentation of soy milk or green tea, and the polyamine may be spermidine. At this time, the strain may be inoculated into the soy milk or green tea at 10 1 ~10 9 cell / mL for fermentation, and preferably may be inoculated at 10 4 ~10 8 cell / mL for fermentation.

[0048] In the method for producing polyamine, the fermentation may be carried out at 30 to 50 °C for 20 to 80 hours, and preferably may be cultured at 37 to 47 °C for 24 to 72 hours after inoculating the strain.

[0049] On one aspect, the present invention provides a composition for promoting hair growth, which contains any one or more selected from the group consisting of the strain, its culture, its spores, and its ferment as an active ingredient.

[0050] In the present invention, the composition for promoting hair growth may be a food composition for promoting hair growth, a cosmetic composition, a feed composition, or a pharmaceutical composition.

[0051] On one aspect, the present invention provides a composition for improving skin, which contains any one or more selected from the group consisting of the strain, its culture, its spores, and its ferment as an active ingredient.

[0052] In the present invention, the composition for improving skin may be a food composition for improving skin, a cosmetic composition, a feed composition or a pharmaceutical composition.

[0053] In the composition for promoting hair growth and the composition for improving skin of the present invention, the specific meanings of the strain, the food composition, the cosmetic composition and the feed composition are as described above.

[0054] The pharmaceutical composition for promoting hair growth and the pharmaceutical composition for improving skin according to the present invention may provide the viable cells of the strain of the present invention, the form of the dried strain, spores, the culture of the strain, the disrupted product of the strain, the fermented product of the strain itself as a composition, or may be provided as a composition that can be combined with a pharmaceutically acceptable carrier or medium.

[0055] The pharmaceutical composition for promoting hair growth according to the present invention may mean a pharmaceutical composition for treating or preventing hair loss.

[0056] The pharmaceutical composition for improving skin according to the present invention may mean a pharmaceutical composition for treating or preventing skin aging diseases or skin wrinkles.

[0057] The pharmaceutical composition for promoting hair growth and the pharmaceutical composition for improving skin of the present invention can be formulated by using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after being administered to mammals, respectively. The dosage form may be in the form of powder, granule, tablet, emulsion, syrup, aerosol, soft or hard gelatin capsule, sterile injectable solution, sterile powder, or may be provided as an enteric-coated enteric preparation as an oral unit dosage form. In addition, the pharmaceutical composition according to the present invention can be administered through several routes including oral, transdermal, subcutaneous, intravenous or intramuscular, and the dosage of the active ingredient can be appropriately selected based on various factors such as the administration route, the age, sex, weight of the patient and the severity of the patient, and the pharmaceutical composition according to the present invention can be administered in parallel with a known therapeutic agent having an effect of preventing, improving or treating the symptoms of hair loss diseases.

[0058] The pharmaceutical composition for promoting hair growth and the pharmaceutical composition for improving skin of the present invention can also be provided as an external preparation for promoting hair growth and an external preparation for improving skin.

[0059] In addition, the quasi-drug may include skin external preparations and personal hygiene products. Preferably, it may be, but is not limited to, a disinfectant cleaner, body soap, oral antiseptic solution, wet tissue, detergent, soap, hand soap, or ointment.

[0060] The individuals to whom the pharmaceutical composition or quasi-drug composition of the present invention can be administered include all animals including humans. For example, it may be, but is not limited to, mammals such as dogs, cats, and mice.

[0061] In one aspect, the present invention provides a beverage with an increased polyamine content fermented using the strain.

[0062] The beverage may mean, but is not limited to, fermented beverages such as fermented soy milk, fermented tea, fermented milk, soy fermented beverage, and soy milk-yogurt.

[0063] In addition, the present invention provides a method for promoting hair growth, comprising the step of administering to an individual an effective amount of any one or more selected from the group consisting of the strain, its culture, its spores, and its ferment.

[0064] In addition, the present invention provides a method for improving skin, comprising the step of administering to an individual an effective amount of any one or more selected from the group consisting of the strain, its culture, its spores, and its ferment.

[0065] The hair growth promoting method or skin improving method of the present invention includes administering to an individual an effective amount of the strain, its culture, its spores or its fermented product. The specific effective amount for a particular individual varies depending on the type and degree of the reaction to be achieved, whether different formulations are used in some cases, as well as the specific composition, the age, weight, general health status, gender and diet of the individual, the administration time, the administration route and the secretion rate of the composition, the administration period, drugs used with or simultaneously with the specific composition, and various factors including well-known similar factors in the field of health functional foods. The daily dosage is 0.0001 to 100 mg / kg based on the amount of the active ingredient of the present invention, preferably 0.01 to 100 mg / kg, and may be administered 1 to 6 times a day. However, it is obvious to those skilled in the art that the dosage or administration amount of each active ingredient must not be so high as to cause side effects by excessively containing the content of each active ingredient. Therefore, it is desirable to determine the effective amount of the composition suitable for the purpose of the present invention in consideration of the above-mentioned matters.

Mode for Carrying Out the Invention

[0066] Hereinafter, the present invention will be described more specifically by way of examples. However, the following examples are for illustrative purposes of the present invention, and the scope of the present invention is not limited thereby.

[0067] Example 1. Search and Selection of Strains Producing Polyamines

[0068] 1-1. Screening of Strains

[0069] Approximately 400 samples of hay and silage collected in South Korea were used as samples to isolate microorganisms. After adding a small amount of each sample to sterile physiological saline and suspending it, the spore solution obtained by heat treatment in a constant temperature water bath at 80°C for 20 minutes was spread on MRS plate medium and cultured at 55°C for 48 - 72 hours. Then, this was spread on MRS plate medium containing 0.7% calcium carbonate and cultured in an anaerobic incubator at 45°C for 3 - 5 days to isolate the bacteria that form a clear zone. The isolated bacteria were examined for polyamine production according to the following examples.

[0070] 1-2. Quantification of Polyamines

[0071] To examine the polyamine production of the bacteria isolated in Example 1 above, the strain was inoculated into a medium suspended with 5 ml of 5% soybean powder and cultured aerobically and anaerobically at 45°C for 48 hours. After adding 1 ml of 5% HClO4 to 1 ml of the 5% soybean medium after the culture was completed and shaking for 6 hours, the culture solution was centrifuged and the amount of polyamine was measured from the supernatant.

[0072] Specifically, 1 ml of the supernatant was taken, 2 ml of saturated Na2HCO3 and 2 ml of 1% dansyl chloride were added, and the reaction was carried out at 70°C for 1 hour. Then, 2 ml of 1% proline was added and the reaction was carried out at 70°C for 30 minutes. 0.5 ml of toluene was added to 0.5 ml of the reaction solution, vortex stirred, and then centrifuged to make the supernatant the TLC analysis sample. The analysis using TLC (stationary phase: silica gel) was carried out using cyclohexane:ethyl acetate = 3:2 (v / v) as the developing solvent. After loading 1 - 3 μl of the dansyl - treated sample and 1 - 3 μl of the spermidine standard substance and developing, it was photographed under ultraviolet light, the intensity of the spot was measured with a densitometer, and the amount of spermidine was quantified from the standard curve.

[0073] In addition, in order to analyze the dansyl derivatives of polyamines produced by the strain, analysis was performed by high performance liquid chromatography (HPLC). The analysis using HPLC (Shiseido SP-LC, Shiseido, Japan) was carried out with the dansyl derivative on an HPLC column (Imtakt Unison UK-C18 Column, 75×2 mm, particle size 3 μm) at 25 °C with a mobile phase of H2O:MeOH composition eluted at a flow rate of 0.2 ml / min with a concentration gradient from 50:50 to 0:100 (gradient elution), and HPLC was performed under the conditions shown in Table 1 below.

[0074]

Table 1

[0075] 1-3. Selection of Strains

[0076] After primary selection of about 30 kinds of bacteria as polyamine-high-producing bacteria by the above process, six kinds of bacteria classified as Bacillus coagulans were secondarily selected according to the taxonomic characteristic analysis method of Bacillus strains based on Bergey’s Manual of Systematic Bacteriology (2002). These six kinds of strains were each analyzed for 16s RNA and classified as Bacillus coagulans, and the Phy53 strain with the highest productivity of polyamine (spermidine, μg / mL) was selected (Table 2).

[0077]

Table 2

[0078] Example 2. Production of polyamine-high-producing mutant strains

[0079] The Phy53 strain cultured in MRS liquid medium at 37°C for 30 hours was centrifuged to collect the cells, which were then suspended in 0.8% saline at pH 7.0. Nitrosoguanidine (NTG) was added thereto at 100 μg / ml, and the treatment time was adjusted to induce mutations so that the killing rate reached 99.9%. After that, 10 5 cells per plate were smeared on Spizizen minimal medium containing 5 mM dicyclohexylamine and cultured at 37°C for 5 days. The surviving bacteria were cultured in 5% soybean medium for 36 to 72 hours, and then the amount of polyamine produced was measured. Among the 15 mutant strains with increased polyamine production, heat-resistant spores were formed, and the strain with the highest polyamine production was selected and named Bacillus coagulans Phy53`. Thereafter, mutations were induced in the Bacillus coagulans Phy53` strain in the same process as described above, and the strain with the highest polyamine production was finally selected and named Bacillus coagulans PA.

[0080] Example 3. Analysis of morphological and biochemical characteristics of the isolated mutant strains

[0081] 3-1. Morphological Characteristics of PA Strains

[0082] Regarding the Bacillus coagulans PA strain finally bred through the isolation process of Example 2 above, the morphological characteristics were first analyzed.

[0083] The colonies formed on the MRS agar plate medium of the Bacillus coagulans PA strain were small and flat, with smooth and round edges, and the surface showed a white appearance without luster. As a result of microscopic observation during the logarithmic growth phase of the cell growth using MRS liquid medium, the cells were in a short rod shape, similar to the comparative strain Bacillus coagulans. The strain was Gram-positive, the spores were located in the center of the cells, and the cells swelled with the formation of spores.

[0084] 3-2. Biochemical Characteristics of PA Strains

[0085] Using the API 50CHB kit (BioMerieux, France), the biochemical characteristics of the isolated strains were examined. As a result, it was confirmed that the strains had the biochemical characteristics (API test results) as shown in Tables 3 and 4 below. Regarding the effect of temperature on the growth of the bacteria, good growth was observed in the range of 25°C or higher and 55°C or lower, and no growth of the bacterial cells was confirmed at 60°C or higher. The PA strain of the present invention was positive for nitrate reduction ability, and negative for citrate utilization and the Voges-Proskauer test. In addition, it decomposed casein, gelatin, and starch, produced acid from glucose, did not produce acid from arabinose, xylose, and mannitol, was catalase positive, and grew under aerobic and anaerobic conditions.

[0086]

Table 3

[0087]

Table 4

[0088] 3-3. Taxonomic Characteristics of PA Strains

[0089] In addition, based on Bergey’s Manual of Systematic Bacteriology (2002), the taxonomic characteristics of Bacillus strains were analyzed. The analyzed strain was identified as Bacillus coagulans and named Bacillus coagulans PA. It was confirmed that the strain had the 16S rRNA sequence represented by SEQ ID NO: 1 (Figure 1), and the strain sequence was deposited with the Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC) on August 6, 2020 (Deposit No.: KCTC14266BP). Also, based on the above results, the taxonomic position of Bacillus coagulans PA strain is shown in Figure 2.

[0090] 3-4. Polyamine Production Characteristics of PA Strains

[0091] By the method of Example 1 above, the Bacillus coagulans PA strain of the present invention and the Bacillus coagulans ATCC7050 strain, which is a strain that produces a small amount of polyamine at a normal level, were cultured under aerobic or anaerobic conditions respectively, and polyamine production was confirmed by TLC. After developing the dansyl derivative of the strain culture by TLC, the spots that appeared under ultraviolet light were compared with the migration distance of the polyamine standard substance, and the results are shown in Figure 3. As a result of the analysis, polyamine spots such as spermine and putrescine were confirmed from the cultures (1, 2, 6, 7) of each strain. In particular, when the Bacillus coagulans PA strain of the present invention was cultured aerobically (2) and anaerobically (7), spots of spermidine that could not be confirmed in the ATCC7050 strain (1, 6) appeared at a very high concentration.

[0092] Furthermore, HPLC was performed in the same manner as in Example 1 above and under the conditions of Table 1. As a result of comparative analysis of the dansyl derivative of the polyamine contained in the culture solution of Bacillus coagulans PA with a standard substance, it was confirmed that a spermidine peak was detected at a retention time of 27 minutes, and it was confirmed that the main polyamine produced was spermidine (Figure 4).

[0093] Example 4. Production of spores of the isolated mutant strain

[0094] Since the processed product of the B. coagulans PA strain of Example 2 of the present invention is mainly used in a spore state, it is advantageous to form a high concentration of spores in the culture solution. Therefore, as an example, spores were obtained through the following process.

[0095] 4-1. Pre-culture

[0096] Put a medium composed of 2% soybean extract, 1% Glycerol, and 1% Yeast extract into a flask, and inoculate it with B. coagulans PA at a concentration of 10 3 ~10 5 cells / mL, and perform preculture at 37°C with a shaking speed of 200 rpm for 2 days.

[0097] 4-2. Main Culture

[0098] Inoculate 3% of the preculture solution into the main culture medium composed of 5% soybean extract, 0.5% Glucose, 0.5% Yeast extract, 0.1% Potassium phosphate dibasic, and 0.05% Sodium phosphate dibasic in a fermenter, and culture at 37°C with a dissolved oxygen amount (OD) of 5 mg / L or more and a rotation speed of 360 rpm. After culturing for 2 - 4 days, centrifuge to recover the bacteria. Suspend this in sterilized water, then perform heat treatment at 80°C for 30 minutes, and then centrifuge to recover the spores, and wash twice with distilled water to obtain a spore solution. The spore solution was used as a spore suspension, or after centrifuging to recover the spores, it was freeze-dried.

[0099] Example 5. Polyamine production amount by anaerobic or aerobic culture

[0100] 5-2. Comparison of Polyamine Production Amounts of Mutant Strains

[0101] First, for each of Bacillus coagulans Phy53, Bacillus coagulans Phy53`, and Bacillus coagulans PA in Example 2 above, in order to compare the polyamine production amounts under anaerobic conditions such as in the animal intestine and other aerobic conditions, 10 3 spores per mL of 5% soybean medium were inoculated respectively, and then cultured with shaking at 37°C for 48 hours or statically cultured in an anaerobic chamber, and the polyamine (spermidine) production amount was measured and shown in Table 5 below.

[0102]

Table 5

[0103] As a result, it was confirmed that the Bacillus coagulans PA strain, which is a new strain of the present invention, significantly increased the production amount of spermidine under both aerobic and anaerobic conditions.

[0104] 5-2. Comparison of Polyamine Production Amounts of Conventional Strains and Mutant Strains

[0105] In order to compare the polyamine productivity of the Bacillus coagulans PA strain of the present invention with that of other aerobic strains under anaerobic conditions such as in the animal intestine, 10 3 spores per 1 mL of 5% soybean medium were inoculated respectively, and after shaking culture at 37 °C for 48 hours or static culture in an anaerobic chamber, the produced polyamine (spermidine) was measured and shown in Table 6 below.

[0106]

Table 6

[0107] The conventional Bacillus subtilis EE5, which is known as a natto bacterium that produces a large amount of polyamine, produces a large amount of polyamine under aerobic conditions, and Bacillus subtilis var. natto Miura, a typical natto strain, only produces polyamine to the same extent as general natto under aerobic conditions. On the other hand, since both of the above strains hardly produced polyamine under anaerobic conditions, it was confirmed that Bacillus subtilis is unsuitable for polyamine production under anaerobic conditions.

[0108] Bacillus coagulans ATCC7050 is a general facultative anaerobic Bacillus coagulans bacterium that produces a small amount of polyamine under aerobic and anaerobic conditions.

[0109] In contrast, Bacillus coagulans PA produces 141 μg / mL of spermidine under aerobic conditions and 105 μg / mL under anaerobic conditions, showing significantly better productivity than conventional general strains under both aerobic and anaerobic culture conditions.

[0110] Example 6. Changes in blood polyamine concentration by administration of PA strain

[0111] To measure the changes in blood polyamine concentration by administration of the spores of the Bacillus coagulans PA strain (Example 4) and the spores of the conventional strain in Example 2 above, 10-week-old male mice were used as subjects and 10 mice were administered each spore sample of each strain. At this time, the microbial administration group was administered 10 7 cells three times a week, and the polyamine administration group was administered 30 μg and 60 μg of spermidine three times a week as the respective doses. The microbial administration group and the polyamine administration group diluted the respective doses in 200 μL of sterilized water and administered them orally three times a week. As a control group, 200 μL of physiological saline was administered orally three times a week instead of the strain spore sample. After 4 weeks of administration, blood was collected and the concentration of blood polyamine (spermidine) was measured and shown in Table 7 below.

[0112]

Table 7

[0113] As shown in Table 7 above, in the change of blood polyamine concentration, when spores of Bacillus coagulans ATCC7050 strain were administered, no significant difference was found compared with the control group. In contrast, when spores of Bacillus coagulans PA strain of the present invention were administered, it showed an effect similar to that when polyamine (spermidine) was directly administered, and it was confirmed that it was effective in growing in the intestine of animals and producing polyamine to continuously supply the host.

[0114] Example 7. Hair growth promoting effect by administration of PA strain

[0115] The Bacillus coagulans PA (Bacillus coagulans PA) strain obtained in Example 2 above was orally administered once a day in powder form to a total of 5 men aged 40 to 60 with hair loss symptoms, 10 9 spores (Example 4) each day for 4 months.

[0116] In the above-mentioned men, after administration of the strain of the present invention, changes 10 days, 30 days and 100 days later were photographed. Some of the results are shown in Fig. 5.

[0117] As a result, it was confirmed that there was an effect of improving the amount and quality of hair, such as new hair growing on the forehead or existing hair becoming thicker after administration.

[0118] Example 8. Skin improvement effect by administration of PA strain

[0119] The spores of Bacillus coagulans PA (Bacillus coagulans PA) strain obtained in Example 4 above were taken orally 10 9 cells each day for 4 weeks, and then the change of the wrinkles under the eyes was photographed. Some of the results are shown in Fig. 6.

[0120] As a result, it was confirmed that there was an effect of improving the wrinkles under the eyes after administration of the PA strain spores of the present invention.

[0121] Production Example 1. Production of Beverage Containing PA Strain

[0122] 1-1. Probiotics Powdered Green Juice

[0123] 1 g of the dried product of one or more mixture materials that can be processed into powder green juice such as young barley leaves, kale, and ashitaba contains 10 4 to 10 14 Bacillus coagulans PA spores (Example 4) and provided as a probiotic product. Since Bacillus coagulans PA spores are resistant to drying, heat, etc., they have the advantage of maintaining the number of bacteria at room temperature for more than one year.

[0124] 1-2. Probiotics Green Tea

[0125] 1 to 2 g of green tea contains 10 4 to 10 14 Bacillus coagulans PA spores (Example 4) are mixed, put into a tea bag, and provided so that Bacillus coagulans bacteria can be easily ingested as a probiotic.

[0126] 1-3. Soybean Yogurt

[0127] Sterilized soy milk is inoculated with Bacillus coagulans PA bacteria (Example 2) to reach 10 8 cells per mL of soy milk, and then cultured at 37 - 45 °C for 24 - 72 hours. At this time, for the purpose of improving the flavor, it may be fermented together with lactic acid bacteria such as the genus Bifidobacterium and the genus Lactobacillus. Here, sugar, concentrated fruit juice, thickener, acidulant, etc. are added to provide polyamine-rich soy milk yogurt in which Bacillus coagulans PA bacteria are alive.

[0128] JPEG2025522106000009.jpg220170

Claims

1. A Bacillus coagulans PA (Bacillus coagulans PA) strain (Accession No. KCTC 14266BP) having the ability to produce polyamine.

2. The strain according to Claim 1, wherein the strain has a 16S rRNA sequence represented by SEQ ID NO:

1.

3. The strain according to Claim 1, wherein the strain produces polyamine in an aerobic or anaerobic state.

4. The strain according to Claim 1, wherein the polyamine is spermidine.

5. A culture or spore containing polyamine obtained by fermentation or culture using the strain according to Claim 1.

6. A food composition containing the strain according to Claim 1, its spore, its culture or its fermented product.

7. A feed composition containing the strain according to Claim 1, its spore, its culture or its fermented product.

8. A cosmetic composition containing the strain according to Claim 1, its spore, its culture or its fermented product.

9. A method for producing polyamine comprising a step of fermenting using the strain according to Claim 1.

10. The production method according to Claim 9, wherein the polyamine is spermidine.

11. A composition for promoting hair growth, comprising any one or more selected from the group consisting of the strain according to Claim 1, its culture, its spore and its fermented product as an active ingredient.

12. A composition for improving skin, comprising any one or more selected from the group consisting of the strain according to Claim 1, its culture, its spore and its fermented product as an active ingredient.

13. A beverage with an increased polyamine content fermented using the strain according to Claim 1.

14. A method for promoting hair growth, comprising the step of administering an effective amount of any one or more selected from the group consisting of the strain according to Claim 1, its culture, its spore and its fermented product to an individual.

15. A method for improving skin, comprising the step of administering an effective amount of any one or more selected from the group consisting of the strain according to Claim 1, its culture, its spore and its fermented product to an individual.

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