Bacillus coagulans strain with high polyamine yield

Abstract

The present invention relates to a Bacillus coagulans PA strain for mass production of polyamines and a method for preparing polyamines using the same. The Bacillus coagulans PA strain according to the present invention can be supplied to the human body by proliferating in the intestine, which is anaerobic, when spores are ingested, and continuously producing a large amount of a polyamine, and can be used as a food, feed, or the like, in which the polyamine can be efficiently produced. Particularly, the composition can be used as food, feed and the like capable of efficiently producing spermidine, and can also be effectively 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 polyamines under anaerobic conditions. More specifically, it relates to a Bacillus coagulans PA strain that grows in the intestinal tract of an animal under anaerobic conditions and produces polyamines to directly provide the animal with the polyamines. Through this process, the strain has the characteristics of continuously producing polyamines in the intestinal tract and continuously absorbing them into the body. Background Art

[0002] Polyamines are substances that exist in large quantities in cells, and the various physiological significances of polyamines have been recently identified. By taking polyamines, the following physiological effects can be expected, namely, inhibition of aging, extension of life, anti-inflammation, cell resuscitation, etc. Therefore, as the attention to polyamines or polyamine compositions increases, efforts are being made to use them as ordinary processed foods, health foods, cosmetics, etc.

[0003] Conventionally, there have been disclosed technical contents related to methods for extracting polyamines from raw materials containing a large amount of polyamines, such as yeast cells or animal organs, and preparing the extracted polyamines into foods that can be ingested.

[0004] As an example of a technology for preparing polyamines for food, a method for extracting polyamines from fish testicles (Japanese Patent Gazette No. 8-238094), yeast cells (Japanese Patent Gazette No. 2001-008663, Japanese Patent Gazette No. 2001-095483), soybean or rice germ (Japanese Patent Gazette No. 10-101624, Japanese Patent Gazette No. 2007-291027), etc. has been proposed.

[0005] Different from the method of directly extracting and refining polyamines from natural products, a method of using fermented products containing a large amount of polyamines has been proposed. A method of increasing the productivity of polyamines and the purity of spermidine by fermenting soybean germs with a high polyamine content using a strain that produces a large amount of polyamines (Korean Patent No. 10-2288725) has also been proposed.

[0006] However, the disclosed method for extracting polyamines from plants, animals and various materials requires a complex extraction process and a separate refining process for the production of polyamines, and thus has a problem of high production costs. In particular, when plants are used as raw materials, since they contain a large amount of putrescine, a polyamine with a high irritability index, the likeability or content of processed products made from the raw materials may decrease.

[0007] Different from the method of providing polyamines extracted from natural products and then refined, a method of ingesting foods with increased polyamine content has also been studied (Korean Patent No. 10-1794772, Japanese Patent No. 6054052). According to this method, high-polyamine natto with a content five times higher than that of ordinary natto can be ingested as a food. However, this also has the problem that the degree of preference for specific fermented products limits the ordinary intake method.

[0008] The supply of polyamines in the body is an important source of self-synthesis in cells from precursors such as ornithine, or polyamines contained in food or polyamines synthesized by intestinal bacteria are absorbed into the body through the intestines. Although polyamines are actively synthesized in growing young individuals, the ability to synthesize polyamines decreases with age. Therefore, ingesting foods containing a large amount of polyamines or polyamines synthesized by intestinal bacteria is important in maintaining the level of polyamines in the body.

[0009] Therefore, the present inventors have studied strains capable of producing large amounts of polyamines among facultative anaerobic strains that can proliferate in the intestine, and ultimately developed Bacillus coagulans PA strain as a novel strain having significantly improved polyamine production capacity, thereby completing the present invention. Summary of the invention

[0010] Technical issues

[0011] An object of the present invention is to provide a novel bacterial strain having the ability to produce a large amount of polyamines in the human intestine and supply the polyamines to the human body.

[0012] Technical Solution

[0013] In order to achieve the above object, the present invention provides a Bacilluscoagulans PA strain (Deposit No. KCTC 14266BP) having the ability to produce polyamines.

[0014] In a specific embodiment, the present invention is characterized in that the strain has a 16s rRNA sequence represented by SEQ ID NO: 1, and can produce polyamines, especially spermidine, at a high content and with high efficiency under aerobic and anaerobic conditions.

[0015] Furthermore, the present invention provides a culture or spores containing a polyamine obtained by fermentation or cultivation using the strain.

[0016] Furthermore, the present invention provides a food composition, a feed composition and a cosmetic composition comprising the strain, its spores, its culture or its fermentation product.

[0017] Furthermore, the present invention provides a method for preparing polyamines, which includes the step of fermenting using the strain.

[0018] Furthermore, the present invention provides a hair growth promoting composition and a skin improving composition comprising, as an active ingredient, at least one selected from the group consisting of the strain, a culture thereof, a spore thereof, and a fermentation product thereof.

[0019] Furthermore, the present invention provides a beverage having an increased polyamine content obtained by fermentation using the strain.

[0020] Furthermore, the present invention provides a method for promoting hair growth, comprising the step of administering to an individual an effective amount of at least one selected from the group consisting of the strain, its culture, its spores and its fermentation product.

[0021] Furthermore, the present invention provides a method for improving skin comprising the step of administering to an individual an effective amount of at least one selected from the group consisting of the strain, its culture, its spores and its fermentation product.

[0022] Effects of the Invention

[0023] The Bacillus coagulans PA strain of the present invention continuously produces polyamines in the aerobic and anaerobic intestines, and thus can exhibit the effect of increasing the concentration of polyamines in the blood by being absorbed by the intestinal tract. It exhibits the characteristic of temporarily inhabiting the intestines, thereby being able to excrete the strains administered as needed, and can therefore be used safely.

[0024] Furthermore, the Bacillus coagulans PA strain of the present invention can be provided in a spore form by forming spores in a low-cost culture medium, and thus can reduce production costs because separate fermentation costs and processing costs for producing polyamines are not required. Moreover, since problems such as fermentation odor are not generated, the strain can be applied to a variety of food forms.

[0025] Furthermore, the Bacillus coagulans PA strain of the present invention has a hair growth promoting effect and is therefore useful as a hair growth promoting pharmaceutical, food or cosmetic composition and a skin improving composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The 16s rRNA base sequence represented by SEQ ID NO: 1 of Bacillus coagulans PA strain is shown.

[0027] Figure 2 A simplified diagram showing the taxonomic position of Bacillus coagulans PA strains.

[0028] Figure 3The results of TLC analysis of polyamines produced by Bacillus coagulans PA strains cultured under aerobic or anaerobic conditions are shown below: 1 is cultured aerobically by Bacillus coagulans ATCC7050 strain, 2 is cultured aerobically by B. coagulans PA strain, 3 to 5 are polyamine standard substances with different concentrations, 6 is cultured anaerobically by Bacillus coagulans ATCC7050 strain, and 7 is cultured anaerobically by Bacillus coagulans PA strain. Bacillus coagulans ATCC7050 strains 1 and 6 are strains that produce polyamines in small amounts at a normal level.

[0029] Figure 4 This is a chromatogram of dansyl chloride derivatives of polyamines produced by the strain culture of Bacillus coagulans PA by HPLC (A: spermidine standard substance, B: Bacillus coagulans PA culture solution) in order to confirm the polyamines produced by the strain. In this case, the peak at a retention time of about 27 minutes is spermidine.

[0030] Figure 5 The comparison time of hair at 10 days, 30 days and 100 days after taking the Bacillus coagulans PA strain of the present invention.

[0031] Figure 6 For pressing 10 9 The comparison time of fundus wrinkle improvement after taking the Bacillus coagulans PA strain spores of the present invention every day for 4 weeks. DETAILED DESCRIPTION

[0032] Best Mode for Carrying Out the Invention

[0033] Hereinafter, the present invention will be described in detail.

[0034] The inventors of the present invention have discovered that some edible facultative anaerobic bacteria have high polyamine production and synthesis performance under aerobic and anaerobic conditions during the process of studying the scheme of effectively supplying polyamines to the body. The high polyamine production and synthesis performance under anaerobic conditions means that it can be converted into a new and simple method for mass production and supply of polyamines in the human intestine. Therefore, in order to mass produce polyamines in the human intestine and supply them to the human body, edible strains that mass produce polyamines under anaerobic conditions were explored, and a Bacillus coagulans PA strain with greatly improved polyamine productivity was completed through mutation breeding, and it was entrusted to the Bioresource Center of the Korea Institute of Life Engineering (deposit number KCTC 14266BP).

[0035] Bacillus coagulans PA, a novel strain of the present invention, proliferates well in the intestine and ultimately has the effect of increasing the polyamine concentration in the blood. Therefore, the strain or its spores can be directly administered, providing a method for economically and simply supplying polyamines to the body.

[0036] According to one embodiment, the present invention provides a Bacilluscoagulans PA strain (Deposit No. KCTC 14266BP) having the ability to produce polyamines.

[0037] The strain of the present invention can be obtained by separating a facultative anaerobic strain that forms heat-resistant spores from rice straw and hay, and screening a strain that produces a large amount of high-content polyamines through a mutation induction process. The screened strain can produce polyamines, especially spermidine, under aerobic or anaerobic conditions. Preferably, spermidine can be produced under both aerobic and anaerobic conditions.

[0038] According to one embodiment of the present invention, it was confirmed that the strain of the present invention can produce 120 μg / mL or more of spermidine in aerobic culture in 5% soybean medium and 90 μg / mL or more of spermidine in anaerobic culture.

[0039] The strain of the present invention is characterized by having a 16s rRNA sequence represented by SEQ ID NO:1.

[0040] 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 polyamine obtained by fermentation or cultivation using the strain.

[0041] In one embodiment, the present invention provides a food composition, a feed composition and a cosmetic composition comprising the Bacillus coagulans PA strain, its spores, its culture or its fermentation product.

[0042] The term "spore" in the present invention refers to resting cells of bacteria, and for the purpose of the present invention, the spore means resting cells of Bacillus coagulans PA.

[0043] The Bacillus coagulans PA strain provided by the present invention is useful in this regard because it proliferates into vegetative cells by germinating spores in the intestine of an animal and continuously supplies polyamines to the host by directly producing polyamines.

[0044] The term "culture" in the present invention refers to the product obtained after culturing the strain, which may be a culture stock solution containing bacterial cells, or bacterial cells after removing the culture strain, removing or concentrating the culture supernatant. The composition of the culture not only includes the components required for conventional Bacillus coagulans culture, but also includes components that play a synergistic role in the growth of Bacillus coagulans, and the composition based on this can be easily selected by a person skilled in the art.

[0045] The novel isolated Bacillus coagulans PA of the present invention can be cultured by a common culture method for Bacillus coagulans strains, but is not limited thereto. The culture medium can use a natural culture medium or a synthetic culture medium. Examples of the carbon source of the culture medium include glucose, sucrose, dextrin, glycerol, starch, etc., and the nitrogen source can use soybean hot water extract, peptone, meat extract, yeast extract, defatted soybean cake, ammonium salt, nitrate and other organic nitrogen compounds or inorganic nitrogen compounds, but are not limited to such components. The inorganic salt contained in the culture medium can use magnesium, manganese, calcium, iron, phosphorus, etc., but is not limited thereto. In addition to the carbon source, nitrogen source and inorganic salt components, amino acids, vitamins, nucleic acids and multiple compounds related thereto can also be added to the culture medium.

[0046] The term "fermented product" in the present invention means a culture medium obtained by inoculating and culturing (fermenting) Bacillus coagulans PA strain into a culture medium containing a main raw material of a food, or a culture of microorganisms cultured together with the culture medium. Furthermore, the fermented product may be a culture obtained by inoculating and culturing Bacillus coagulans PA strain into a food raw material. The food raw material may be cereals, foods derived from cereals, etc. The cereals may be soybeans, rice, barley, wheat, corn, etc., preferably soy milk or green tea, but not limited thereto.

[0047] The fermented product can be generally interpreted as including all of the filtrate, dilution, concentrate, adjusted product, refined product, dried product, pulverized product, etc. of the culture medium or culture, but is not limited thereto.

[0048] The food composition, feed composition and cosmetic composition of the present invention can exhibit hair growth promoting effects and skin improving effects.

[0049] In the present invention, the skin improvement may mean improvement of skin aging, improvement of wrinkles, skin regeneration or improvement of scalp, and may preferably mean improvement of wrinkles.

[0050] In the present invention, the "food" means a natural or processed product containing one or more nutrients, preferably means a state that can be eaten directly after a certain degree of processing. As a general meaning, it can include all foods, food additives, health functional foods and beverages.

[0051] The "healthy functional food" means a food group that gives added value in a way that allows the function of the food to work and express for a specific purpose, or a food that is designed and processed in a way that allows the in vivo regulatory functions related to the biological defense rhythm regulation, disease prevention and recovery of the food composition to be fully expressed in the organism. The functional food may also contain food auxiliary additives, appropriate carriers, excipients and diluents that are permitted in food science.

[0052] In the present invention, the "feed composition" includes all animal feeds and animal feed additives. The feed composition of the present invention can be formed into a dry or liquid preparation form, and in addition to the strain, its spores, its culture or its fermentation product, it can also include other non-pathogenic microorganisms or enzyme preparations. Feed raw materials can use various grains and soybean protein such as peanuts, peas, beets, meat parts, grain by-products, animal viscera powder and fish meal, etc., which can be used unprocessed or processed without limitation.

[0053] The "cosmetic composition" can be used as an external skin preparation and can be directly applied to the necessary part. For example, it can be made into various dosage forms such as ointment, cream, lotion, shampoo, conditioner, soap, etc. The cosmetic composition of the present invention may also contain one or more additives selected from the group consisting of purified water, oil, surfactant, moisturizer, stabilizer, ethanol, thickener, chelating agent, pigment, preservative and fragrance.

[0054] The food composition, feed composition or cosmetic composition comprising the strain, its spores, its culture or its fermented product of the present invention comprises the strain in the form of spores, and each 1g comprises 10 4 to 10 14 Spores.

[0055] According to one embodiment, the present invention provides a method for preparing polyamines, comprising the step of fermenting using the strain.

[0056] In the method for preparing the polyamine, the fermentation may be fermentation of soy milk or green tea, and the polyamine may be spermidine. In this case, the soy milk or green tea may be inoculated with 10 1 ~10 9 cell / mL of the strain for fermentation, preferably, by inoculating 10 4 ~10 8 cell / mL for fermentation.

[0057] In the method for preparing the polyamine, the fermentation can be carried out at a temperature of 30° C. to 50° C. for 20 to 80 hours, preferably, it can be achieved by culturing at a temperature of 37° C. to 47° C. for 24 to 72 hours after strain inoculation.

[0058] According to one embodiment, the present invention provides a composition for promoting hair growth comprising, as an active ingredient, at least one selected from the group consisting of the strain, a culture thereof, a spore thereof, and a fermentation product thereof.

[0059] In the present invention, the hair growth promoting composition may be a hair growth promoting food composition, a cosmetic composition, a feed composition or a pharmaceutical composition.

[0060] According to one embodiment, the present invention provides a skin-improving composition comprising, as an active ingredient, at least one selected from the group consisting of the above-mentioned strain, a culture thereof, a spore thereof, and a fermentation product thereof.

[0061] In the present invention, the skin-improving composition may be a skin-improving food composition, a cosmetic composition, a feed composition or a pharmaceutical composition.

[0062] In the hair growth promoting composition and skin improving composition of the present invention, the specific meanings of the strain, food composition, cosmetic composition and feed composition are the same as those described above.

[0063] The pharmaceutical composition for promoting hair growth and the pharmaceutical composition for improving skin of the present invention can be a composition using the living bacteria of the strain of the present invention, the form of the dried strain, the spores, the culture of the strain, the crushed product of the strain, and the fermentation product of the strain itself, or can be a composition combined with a pharmaceutically acceptable carrier or medium.

[0064] The pharmaceutical composition for promoting hair growth of the present invention may mean a pharmaceutical composition for treating or preventing hair loss.

[0065] The skin-improving pharmaceutical composition of the present invention may refer to a pharmaceutical composition for treating or preventing skin aging diseases or skin wrinkles.

[0066] The pharmaceutical composition for promoting hair growth and the pharmaceutical composition for improving skin of the present invention can be formulated by methods known in the art so as to provide rapid, continuous or delayed release of active ingredients after administration to mammals. The dosage form can be in the form of powder, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injection solutions, sterile powders. As a unit dosage form for oral use, an enteric preparation wrapped in an enteric coating can be provided. In addition, the pharmaceutical composition of the present invention can be administered by a variety of routes including oral, transdermal, subcutaneous, intravenous or intramuscular, and the dosage of the active ingredient can be appropriately selected according to a variety of factors such as the route of administration, the patient's age, sex, weight and the severity of the patient. The pharmaceutical composition of the present invention can be administered together with a known therapeutic agent that has the effect of preventing, improving or treating the symptoms of alopecia.

[0067] The hair growth promoting pharmaceutical composition and the skin improving pharmaceutical composition of the present invention can also be provided as a hair growth promoting external pharmaceutical composition and a skin improving external pharmaceutical composition.

[0068] Furthermore, the external medicine may include skin external preparations and personal hygiene products, preferably, disinfectant cleaners, shower foam, mouthwash, wet wipes, detergents, soaps, hand sanitizers or ointments, but are not limited thereto.

[0069] Individuals to whom the pharmaceutical composition or external medicine composition of the present invention can be administered include all animals including humans, for example, mammals such as dogs, cats, and mice, but are not limited thereto.

[0070] According to one embodiment, a beverage having an increased polyamine content obtained by fermentation using the strain is provided.

[0071] The beverage may refer to fermented beverages such as fermented soybean milk, fermented tea, fermented milk, fermented soybean beverage, and soybean milk-yogurt, but is not limited thereto.

[0072] Furthermore, the present invention provides a method for promoting hair growth, comprising the step of administering to an individual an effective amount of at least one selected from the group consisting of the strain, its culture, its spores and its fermentation product.

[0073] Furthermore, the present invention provides a method for improving skin comprising the step of administering to an individual an effective amount of at least one selected from the group consisting of the strain, its culture, its spores and its fermentation product.

[0074] The method for promoting hair growth or improving skin of the present invention comprises administering the strain, its culture, its spores or its fermentation product to an individual in an effective amount. Preferably, the specific effective amount for a specific individual should be adopted differently according to the type and degree of the reaction to be achieved, whether to use specific compositions such as other preparations according to the situation, the individual's age, weight, general health status, gender and diet, administration time, administration route and release rate of the composition, administration time, drugs used together with or simultaneously with the specific composition and similar factors known in the field of health functional foods. The amount of the active ingredient of the present invention is used as a benchmark, and the daily dosage is 0.0001 mg / kg to 100 mg / kg, preferably 0.01 mg / kg to 100 mg / kg, and can be administered 1 to 6 times a day. However, the dosage or dosage of each active ingredient should not reach the extent of causing side effects due to excessive content of each active ingredient, which is a matter of course for those skilled in the art. Therefore, preferably, the effective amount of the composition that meets the purpose of the present invention should be determined in consideration of the above matters. DETAILED DESCRIPTION

[0076] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0077] Example 1. Exploration and screening of polyamine-producing strains

[0078] 1-1. Exploration of strains

[0079] Microorganisms were isolated from approximately 400 hay and silage collected in Korea as feed. Various samples were added in small amounts to sterile saline and suspended, and then heat-treated in a constant temperature water tank at 80°C for 20 minutes to obtain spore liquid. The spore liquid was applied to an MRS plate medium and cultured at 55°C for 48 to 72 hours. The spore liquid was then applied to an MRS plate medium containing 0.7% calcium carbonate, and cultured in an anaerobic medium at 45°C for 3 to 5 days to isolate bacteria that form a sterile zone (clear zone). The isolated bacteria were used to investigate the production of polyamines in the following examples.

[0080] 1-2. Quantification of polyamines

[0081] In order to investigate the production amount of polyamines produced by the bacteria isolated in Example 1, the strain was inoculated into a medium in which 5 ml of 5% soybean powder was suspended, and cultured aerobically and anaerobically at 45° C. for 48 hours. 1 ml of 5% HClO4 was added to 1 ml of the 5% soybean medium after the culture was completed, and the culture was shaken for 6 hours, after which the culture solution was centrifuged to measure the amount of polyamines from the supernatant.

[0082] Specifically, 1 ml of the upper layer was taken and 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. 2 ml of 1% proline was added to this 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, and the mixture was vortex-stirred and centrifuged, and the upper layer was used as a sample for thin layer chromatography (TLC) analysis. The analysis was performed by thin layer chromatography (TLC, stationary phase: silica gel) using cyclohexane:ethylacetate = 3:2 (v / v) as a developing solvent. After loading 1 to 3 μl of a sample treated with dansyl chloride and 1 to 3 μl of a spermidine standard and developing, the sample was photographed under ultraviolet light and the intensity of the spot was measured by a densitometer, thereby quantifying the amount of spermidine from the standard curve.

[0083] In addition, in order to analyze the dansyl chloride derivatives of the polyamines produced by the strain, analysis was performed by high performance liquid chromatography (HPLC). The analysis performed by high performance liquid chromatography (HPLC, Shiseido SP-LC, Shiseido, Japan) was as follows: for the dansyl chloride derivatives, a high performance liquid chromatography column (HPLC Column, Imtakt Unison UK-C18 Column, 75X 2mm, particle size 3μm) was used at a temperature of 25°C and a flow rate of 0.2ml / min to perform a 50:50→0:100 concentration gradient elution with a mobile phase composed of H2O:MeOH, and high performance liquid chromatography was performed under the conditions shown in Table 1 below.

[0084] [Table 1]

[0085]

[0086] 1-3. Screening of strains

[0087] After about 30 bacteria were screened as polyamine high-producing bacteria in the above steps, six bacteria classified as Bacillus coagulans were screened again according to the taxonomic characteristics analysis method of Bacillus strains based on Bergey's Manual of Systematic Bacteriology (Bergey's Manual of Systematic Bacteriology, 2002). These six strains were classified as Bacillus coagulans by 16sRNA analysis, thereby screening the Phy53 strain with the highest polyamine productivity (spermidine, μg / mL) (Table 2).

[0088] [Table 2]

[0089] Strain name JS18 JC202 Phy02 Phy14 Phy53 KC147 Spermidine (μg / mL) 50 54 49 55 67 52

[0090] Example 2. Preparation of mutant strains with high polyamine production

[0091] Phy53 strains were cultured in MRS liquid medium at 37°C for 30 hours, centrifuged and collected, and then suspended in 0.8% saline at pH 7.0. 100 μg / ml of nitrosoguanidine (NTG) was added to adjust the treatment time so that the killing rate reached 99.9% to induce mutations, and then 100 μg / ml of nitrosoguanidine (NTG) was added to Spizizen minimal medium containing 5 mM dicyclohexylamine.5 After only applying, the culture was cultured in a 5% soybean medium at 37°C for 5 days. After culturing the surviving bacteria for 36 to 72 hours, the amount of polyamine produced was measured. Among the 15 mutant strains with improved polyamine production, heat-resistant spores were formed, and the strain with the highest polyamine production was selected and named Bacillus coagulans Phy53′. Subsequently, the Bacillus coagulans Phy53′ strain was used as a target to induce mutations in the same procedure as above, and finally the strain with the highest polyamine production was selected and named Bacillus coagulans PA.

[0092] Example 3. Analysis of morphological and biochemical characteristics of isolated mutant strains

[0093] 3-1. Morphological characteristics of PA strains

[0094] The morphological characteristics of the Bacilluscoagulans PA strain finally bred through the separation steps of Example 2 were first analyzed.

[0095] The bacterial colonies formed on the MRS plate count agar medium of the Bacillus coagulans PA strain were very small and flat, with smooth round edges and a matte white surface. The results of microscopic observation of the bacterial cells in the logarithmic phase of growth using the MRS liquid medium showed that the appearance of the cells was very short rods, similar to the comparative strain Bacillus coagulans. The strain was a Gram-positive bacterium, with spores located in the center of the cell, and the cell swelled as the spores were generated.

[0096] 3-2. Biochemical characteristics of PA strains

[0097] The results of investigating the biochemical characteristics of the isolated strain using the API50CHB kit (BioMerieux, France) showed that the biochemical characteristics (API test results) shown in Tables 3 and 4 were confirmed. In terms of the temperature effect on bacterial growth, good growth was observed in the range of 25°C to 55°C, and no bacterial growth was confirmed at temperatures above 60°C. The PA strain of the present invention has a good nitrate reducing power, and is negative in citric acid and Voges-Proskauer tests. In addition, it decomposes casein, gelatin, and starch, produces acid from glucose, does not produce acid from arabinose, xylose, and mannitol, is catalase positive, and grows under aerobic and anaerobic conditions.

[0098] [Table 3]

[0099]

[0100] [Table 4]

[0101]

[0102]

[0103] 3-3. Taxonomic characteristics of PA strains

[0104] Furthermore, the taxonomic characteristics of the Bacillus strains based on Bergey's Manual of Systematic Bacteriology (2002) were analyzed. The analyzed strain was identified as Bacillus coagulans and named Bacillus coagulans PA, and it was confirmed that the strain had a 16s rRNA sequence represented by SEQ ID NO: 1 ( Figure 1 ), and entrusted the strain sequence to the Bioresource Center of the Korea Institute of Biotechnology (KCTC, entrustment number: KCTC14266BP) on August 6, 2020. And, based on the results, Figure 2 The taxonomic position of Bacillus coagulans PA strains is shown.

[0105] 3-4. Polyamine production characteristics of PA strain

[0106] The Bacillus coagulans PA strain of the present invention and the Bacillus coagulans ATCC7050 strain, which is a strain producing polyamines at a conventional level in a small amount, were cultured under aerobic or anaerobic conditions by the method of Example 1, and polyamine production was confirmed by TLC. The spots shown by irradiation with ultraviolet rays after developing the strain culture by TLC were compared with the migration distances of polyamine standard substances. Figure 3 The results are shown in the accompanying drawings. The analysis results showed that polyamine sites such as spermine and putrescine were confirmed in the cultures of the respective strains (1, 2, 6, 7), and in particular, when the Bacillus coagulans PA strain of the present invention was cultured under aerobic (2) and anaerobic (7) conditions, spermidine sites that did not appear in the ATCC7050 strains (1, 6) showed very high concentrations.

[0107] Next, high performance liquid chromatography (HPLC) was performed in the same manner as in Example 1 and under the conditions shown in Table 1 to compare the dansyl chloride derivatives of polyamines contained in the culture solution of Bacillus coagulans PA with standard substances. As a result, it was confirmed that a spermidine peak was detected at a retention time of 27 minutes, thereby confirming that the main polyamine produced was spermidine ( Figure 4 ).

[0108] Example 4. Production of spores of isolated mutant strains

[0109] Since the processed product of the Bacillus coagulans PA strain of Example 2 of the present invention is mainly used in the form of spores, it is advantageous to form a high concentration of spores in the culture solution. As an example, the spores were obtained by the following steps.

[0110] 4-1. Pre-culture

[0111] A medium consisting of 2% soybean extract, 1% glycerol, and 1% yeast extract was placed in the flask and stirred at a temperature that could reach 10 3 ~10 5 Bacillus coagulans PA strain was inoculated at a concentration of 1 cell / mL and pre-cultured at 37°C and shaking speed of 200 rpm for 2 days.

[0112] 4-2. Formal training

[0113] In a fermentation tank, 3% of the pre-culture solution was inoculated into a formal culture medium consisting of 5% soybean extract, 0.5% glucose, 0.5% yeast extract, 0.1% potassium phosphate dibasic, and 0.05% sodium phosphate dibasic, and cultured at a temperature of 37°C, a dissolved oxygen content (OD) of 5 mg / L or more, and a rotation speed of 360 rpm. After culturing for 2 to 4 days, the bacteria were collected by centrifugation. After suspending it in sterile water, it was heat-treated at 80°C for 30 minutes, and then the spores were recovered by centrifugation and washed twice with distilled water to obtain a spore liquid. The spore suspension was used directly as the spore liquid, or the spores were recovered by centrifugation and freeze-dried for use.

[0114] Example 5. Production of polyamines based on anaerobic or aerobic culture

[0115] 5-1. Comparison of polyamine production by mutant strains

[0116] First, in order to compare the polyamine production of Bacillus coagulans Phy53, Bacillus coagulans Phy53′, and Bacillus coagulans PA of Example 2 under anaerobic conditions similar to those in the intestinal tract of animals and other aerobic conditions, 10 μg / ml of each of the above-mentioned strains were inoculated into a 5% soybean culture medium. 3 After the cells were incubated with spores of 100 μg / mL, shaking culture was performed at 37°C for 48 hours or static culture was performed in an anaerobic chamber. The polyamine (spermidine) was measured and the content is shown in Table 5.

[0117] [Table 5]

[0118] Strain name Phy53 Phy53′ PA Spermidine (μg / mL) 66 120 141

[0119] Finally, it was confirmed that the Bacillus coagulans PA strain, which is a novel strain of the present invention, showed a significant increase in the production of spermidine under both aerobic and anaerobic conditions.

[0120] 5-2. Comparison of polyamine production by existing strains and mutant strains

[0121] In order to compare the productivity of polyamines between the Bacillus coagulans PA strain of the present invention and other aerobic strains under anaerobic conditions similar to those in the intestinal tract of animals, 103 spores were inoculated into 1 ml of a 5% soybean medium, followed by shaking culture at 37°C for 48 hours or static culture in an anaerobic chamber, and then the produced polyamines (spermidine) were measured and shown in Table 6.

[0122] [Table 6]

[0123]

[0124] The existing Bacillus subtilis EE5, which is known as a natto strain that produces a large amount of polyamines, produces a large amount of polyamines under aerobic conditions, while the representative natto strain Bacillus subtilis var. natto Miura only produces polyamines at the level of ordinary natto under aerobic conditions. In contrast, both strains hardly produce polyamines under anaerobic conditions, thus confirming that Bacillus subtilis is not compatible with the production of polyamines under anaerobic conditions.

[0125] Bacillus coagulans ATCC 7050, a facultative anaerobic common Bacillus coagulans, produces small amounts of polyamines under aerobic and anaerobic conditions.

[0126] In comparison, Bacillus coagulans PA produced 141 μg / mL of spermidine under aerobic conditions and 105 μg / mL under anaerobic conditions, showing higher productivity than existing conventional strains under both aerobic and anaerobic culture conditions.

[0127] Example 6. Changes in blood polyamine concentrations based on administration of PA strains

[0128] In order to measure the blood polyamine concentration after administration of spores of the Bacillus coagulans PA strain (Example 4) and spores of existing strains based on Example 2, 10 male mice aged 10 weeks were used as subjects and spore samples of each strain were administered to 10 mice. In this case, the dosage of the microorganism administration group was as follows: 10 7 The spores of Bacillus coagulans PA or Bacillus coagulans ATCC 7050 were administered three times a week, and the dosage of the polyamine administration group was 30 μg and 60 μg of spermidine three times a week. The microorganism administration group and the polyamine administration group diluted the dosage to 200 μL of sterile water and administered orally three times a week, and the control group was administered orally three times a week with 200 μL of saline instead of the strain spore sample. After 4 weeks of administration, the concentration of polyamine (spermidine) in the blood was measured by blood sampling and is shown in Table 7.

[0129] [Table 7]

[0130]

[0131]

[0132] As shown in Table 7, when the spores of Bacillus coagulans ATCC 7050 were administered, there was no significant difference in the change of the polyamine concentration in the blood compared with the control group. However, when the spores of the Bacillus coagulans PA strain of the present invention were administered, an effect similar to that of the case of direct administration of polyamine (spermidine) was shown, and it was confirmed that polyamines were proliferated and produced in the intestines of animals, and were effectively and continuously supplied to the host.

[0133] Example 7. Hair growth promoting effect based on administration of PA strains

[0134] A total of 10% of the Bacillus coagulans PA strain obtained in Example 2 were administered in powder form to 5 males aged 40 to 60 with hair loss symptoms. 9 The spores (Example 4) were supplied for 4 months and administered orally once a day.

[0135] For the male, the changes after 10 days, 30 days and 100 days of administration of the strain of the present invention were photographed. Figure 5 Some results are shown.

[0136] Finally, it was confirmed that after taking the product, new hair grew on the forehead and the thickness of existing hair became thicker, and the hair volume and quality improved.

[0137] Example 8. Skin improvement effect based on PA strain administration

[0138] 10 mg of the Bacilluscoagulans PA strain obtained in Example 4 was taken daily in the form of powder. 9 After the cell spores reached 4 weeks, the changes in eye wrinkles were photographed. Figure 6 Some results are shown.

[0139] Finally, it was confirmed that taking the PA strain spores of the present invention has an improving effect on fundus wrinkles.

[0140] Preparation Example 1. Preparation of PA-containing beverage

[0141] 1-1. Probiotic powder vegetable juice

[0142] 1 g of a dry substance of one or more of a mixture of barley malt, kale, ashitaba, etc., which can be processed into a powdered vegetable juice, contains 104 Up to 10 14 The probiotic product is provided by mixing the spores of Bacillus coagulans PA (Example 4). Since the spores of Bacillus coagulans PA are resistant to drying and heat, they have the advantage of being able to maintain the bacterial count for more than one year at room temperature.

[0143] 1-2. Probiotic Green Tea

[0144] Mix 10 for every 1-2g of green tea. 4 Up to 10 14 The invention provides Bacillus coagulans PA spores (Example 4) in a tea bag to facilitate ingestion of Bacillus coagulans as a probiotic.

[0145] 1-3. Soy milk yogurt

[0146] In 1mL of soy milk containing 10 8 After inoculating the sterilized soy milk with Bacillus coagulans PA bacteria (Example 2) in a cell method, the soy milk was cultured at a temperature of 37 to 45°C for 24 to 72 hours. In this case, it can also be fermented with lactic acid bacteria such as Bifidobacterium and Lactobacillus to enhance the flavor. Sugar, concentrated fruit juice, thickener, acidulant, etc. are added to provide soy milk yogurt containing a large amount of polyamines in which Bacillus coagulans PA bacteria survive.

[0147]

Claims

1. A Bacillus coagulans PA strain, whose deposit number is KCTC 14266BP, characterized in that: Possesses the ability to produce polyamines.

2. The strain according to claim 1, characterized in that The strain has a 16srRNA sequence represented by SEQ ID NO:

1.

3. The strain according to claim 1, characterized in that The strain produces polyamines under aerobic or anaerobic conditions.

4. The strain according to claim 1, characterized in that The polyamine is spermidine.

5. A culture or spore, characterized in that The invention comprises a polyamine obtained by fermentation or cultivation using the strain according to claim 1.

6. A food composition, characterized in that The method comprises the strain according to claim 1, its spores, its culture or its fermentation product.

7. A feed composition, characterized in that The method comprises the strain according to claim 1, its spores, its culture or its fermentation product.

8. A cosmetic composition, characterized in that The method comprises the strain according to claim 1, its spores, its culture or its fermentation product.

9. A method for preparing polyamines, characterized in that: The method comprises the step of fermenting using the strain according to claim 1.

10. The preparation method according to claim 9, characterized in that: The polyamine is spermidine.

11. A composition for promoting hair growth, characterized in that: The method comprises at least one selected from the group consisting of the strain according to claim 1, a culture thereof, a spore thereof and a fermentation product thereof as an active ingredient.

12. A skin improving composition, characterized in that: The composition comprises at least one selected from the group consisting of the strain according to claim 1, a culture thereof, a spore thereof and a fermentation product thereof as an active ingredient.

13. A beverage, characterized in that The content of polyamines in the fermentation using the strain according to claim 1 is increased.

14. A method for promoting hair growth, characterized in that: comprising administering to a subject an effective amount of a One or more steps selected from the group consisting of the strain according to claim 1, its culture, its spores and its fermentation product.

15. A method for improving skin, characterized in that: comprising administering to a subject an effective amount of a One or more steps selected from the group consisting of the strain according to claim 1, its culture, its spores and its fermentation product.

Images