JP4935173B2 - Process for preparing polyamine compositions from plants - Google Patents

Description

The present invention relates to a method for efficiently and in large quantities producing a polyamine composition from a plant.

Polyamine is a generic term for aliphatic hydrocarbons having two or more primary amino groups, and is a natural product that exists universally in the living body, and more than 20 types of polyamines have been found. Typical polyamines include putrescine, spermidine and spermine. The main physiological functions of polyamines are as follows: (1) Stabilization and structural changes of nucleic acids by interaction with nucleic acids (2) Promotion of various nucleic acid synthesis systems (3) Activation of protein synthesis systems (4) Cell membrane Stabilization and enhancement of membrane permeability of substances (5) elimination of active oxygen (6) promotion of cell growth (7) anti-allergic action is known.

Polyamines are natural products that are ubiquitous in living organisms such as animals, plants, and microorganisms. Meat, cheese, and fermented foods have a relatively high polyamine content, but overall the content of polyamines in food is low. Is a level. In particular, the concentration of polyamine contained in plants is on the order of several tens to several hundreds nmol per gram in the living body (Non-patent Document 1). Under these circumstances, the preparation of polyamines not from plants but from animal-related materials having a high polyamine concentration has been studied.

The methods for preparing industrially available polyamines or polyamine compositions include fish larvae (Patent Literature 1), milk materials (Patent Literature 2 and Patent Literature 3), yeast (Patent Literature 4 and Patent Literature 5) and chemical synthesis ( Patent Document 6), chemical synthesis by enzyme reaction and microbial metabolism (Non-Patent Document 2) has been reported. However, when preparing a polyamine composition from other than plant origin, there are many problems and problems in safety and yield when preparing a polyamine composition by chemical synthesis or enzymatic reaction. For example, as a safety issue, a polyamine composition prepared by chemical synthesis is extremely difficult to apply to food applications because of the presence or absence of substances used in the synthesis reaction and catalyst.

There are no reports on methods for preparing polyamines or polyamine compositions from plants. Polyamines are very unstable and easily decomposed by polyamine oxidase, and it has been difficult to prepare polyamines or polyamine compositions in large quantities. Since plants have a strong cell wall, in order to recover the polyamine or polyamine composition present in the cells, it was necessary to damage the cell wall under stable conditions in which the polyamine or polyamine composition is not degraded. Furthermore, a large amount of impurities such as polysaccharides, polyphenols, and secondary metabolites are contained in plants and / or plant extracts, and these impurities are a major factor that hinders recovery of polyamines or polyamine compositions. It was.
Japanese Patent No. 3518778 Japanese Patent Laid-Open No. 2001-8663 Japanese Patent Laid-Open No. 2001-95483 Japanese Patent Laid-Open No. 10-52291 JP 2000-245493 A Japanese Patent Laid-Open No. 7-277917 Chemical regulation of plants, 35, 56-66, 2000 Metabolism, Vol.9, No.11, 1010-1017, 1972

  In recent years, the importance of polyamines has increased due to elucidation of various physiological actions. Polyamines or polyamine compositions are being used for health, cosmetics, food and pharmaceutical applications. However, a sufficient method for preparing polyamines or polyamine compositions has not been established. In particular, the preparation method of the polyamine or polyamine composition from a plant has not been established until now.

  An object of the present invention is to provide a method for preparing a polyamine or a polyamine composition in a high yield from a plant material with high safety.

As a result of diligent efforts to achieve the above object, the present inventors have found a method for efficiently and in large quantities preparing a polyamine or a polyamine composition that can be used from plants for health, cosmetics, food, and pharmaceutical applications. The invention has been completed. That is, the present invention has the following configuration.

1. (1) A method for preparing a polyamine composition, comprising: treating a plant and / or plant extract under acidic conditions; and (2) separating a liquid fraction.
2. 1. A method for preparing a polyamine composition according to 1, wherein an acid solution containing a mineral acid and / or an organic acid is added and treated under acidic conditions.
3. Mineral acid and / or organic acid is hydrochloric acid, sulfuric acid, nitric acid, acetic acid, phosphoric acid, trichloroacetic acid, perchloric acid, citric acid, lactic acid, propionic acid, butyric acid, succinic acid, malonic acid, succinic acid, fumaric acid, maleic acid 2. A method for preparing a polyamine composition according to 2, wherein the polyamine composition is at least one acid selected from the group consisting of malic acid, tartaric acid, benzoic acid, sulfosalicylic acid and formic acid.
4). The method for preparing a polyamine composition according to any one of 1 to 3, wherein the acid solution added so as to be in an acidic condition is hydrochloric acid and / or perchloric acid.
5. The method for preparing a polyamine composition according to any one of 1 to 4, wherein a polyphenol adsorbent is added and a liquid fraction is separated simultaneously with or after the plant and / or plant extract is treated under acidic conditions.
6). The plant and / or plant extract is at least one selected from the group consisting of soybean seed, soybean germ, soybean embryo, wheat seed, wheat germ, wheat embryo, soy milk and okara 1 to 6. The method for preparing a polyamine composition according to any one of 5 above.
7). The polyamine composition is 1,3-diaminopropane, putrescine, cadaverine, cardine, spermidine, homospermidine, aminopropyl cadaverine, theremin, spermine, thermospermine, canabalmine, aminopentylnorspermidine, N, N-bis (aminopropyl) 1. The polyamine composition according to any one of 1 to 6, comprising at least one compound selected from the group consisting of cadaverine, homospermine, cardopentamine, homocardopentamine, cardohexamine and homocardohexamine. Preparation method.
8). The polyamine composition comprises at least one compound selected from the group consisting of putrescine, cadaverine, spermidine, and spermine.
9. The method for preparing a polyamine composition according to any one of 1 to 8, wherein the step of separating the liquid fraction is centrifugation and / or filtration separation.
At least one treatment selected from the group consisting of an ion exchange method, a gel filtration method, a membrane fractionation method, and an electrodialysis method is further applied to the polyamine composition recovered by any one of the preparation methods of 10.1 to 9. A method for preparing a polyamine composition, characterized by being purified by the method.

  According to the present invention, it has become possible to provide a method for preparing a large amount of a polyamine or a polyamine composition in high yield from a plant material.

In the present invention, “plants and / or plant extracts” are various plants and plant extracts, and are not particularly limited. For example, dicotyledonous plants, monocotyledonous plants, herbaceous plants, and woody plants. Cucurbitaceae plant, solanaceous plant, gramineous plant, cruciferous plant, legume plant, mallow plant, asteraceae plant, red plant plant, legume plant, plant extract, plant extract, etc. . For example, sweet potato, tomato, cucumber, pumpkin, melon, watermelon, tobacco, Arabidopsis, pepper, eggplant, bean, taro, spinach, carrot, strawberry, potato, rice, corn, alfalfa, wheat, barley, soybean, rapeseed, sorghum, Eucalyptus, poplar, kenaf, Tochu, sugar cane, sugar beet, cassava, sago palm, red pepper, lily, orchid, carnation, rose, chrysanthemum, petunia, torenia, snapdragon, cyclamen, gypsophila, geranium, sunflower, shiba, cotton, enoki mushroom , Matsutake, shiitake mushrooms, ginseng, agaricus, turmeric, ginseng, citrus, green tea, tea, oolong tea, banana, kiwi, natto, soy milk, soybean extract, wheat extract, Bud extract, embryo extract, fruit juice, Ocala, rice germ, wheat germ, barley germ, soybean germ, corn germ, milo germ, such as sunflower germ, and the like.
Preferred are monocotyledonous plants and dicotyledonous plants, more preferred are grasses and legumes, and particularly preferred are corn, mushrooms, soybean, wheat, natto, soy milk, okara, wheat germ, soybean germ. Corn germ, soybean extract, wheat extract, germ extract, embryo extract are preferred. In particular, polyamines or polyamine compositions may be recovered from plants with a large amount of net food per person per year, for example, 6.7 kg of soybean per year in 2003, 32.6 kg of wheat per year in 2003.

  The plant tissue for recovering the polyamine composition is not particularly limited. Preferably, the seed is in the form of seed or growth process. Plants in the process of growing can be recovered from whole or partial tissues. Although it does not specifically limit as a site | part which can be collect | recovered, a whole tree, a flower, a cocoon, an ovary, a fruit, a leaf, a cotyledon, a stem, a bud, a root, a seed, a dry seed, an embryo, an embryo, a root, etc. Preferred are fruits, leaves, stems, buds, seeds, dried seeds, embryos, embryos, and particularly preferred are seeds, dried seeds, germs, embryos, and the like.

  One of the important disclosures in the present invention is to add an acid solution to the plant and / or plant extract so as to be in an acidic condition in the purification step. The inventors have found that polyamines can be highly stably purified by this process.

  In the present invention, “under acidic conditions” refers to conditions under which the pH is 6 or less. By effecting the pH under acidic conditions during purification, an efficient and stable polyamine composition recovery effect can be obtained from plant tissue. This effect is uniformly obtained as long as the pH is 6 or less, but is preferably 4 or less, particularly preferably 2 or less. The lower limit is not particularly limited because it may be the pH of the stock solution of the acid solution to be used, but is preferably pH 0-2.

The “plant extract” of the present invention refers to a product obtained from a plant or a processed product thereof. The preparation method is impregnation method under low temperature, room temperature and warm condition using water, organic solvent, mixture of water and organic solvent, distillation method, pressing method, ultrasonic method, supercritical fluid The extract is recovered by the method or subcritical fluid method. Furthermore, the processed material etc. which fermented the extract collect | recovered from the plant and the plant are included. For example, plant extracts, soy milk, okara, wheat flour, fermented extract, natto and the like can be mentioned.

  Acid solutions to be added under acidic conditions include mineral acids such as hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, propionic acid, butyric acid, succinic acid, malonic acid, succinic acid, fumaric acid, maleic acid, malic acid, lactic acid Organic acids such as tartaric acid, citric acid, benzoic acid and acidic water, but 0.01N to 6N hydrochloric acid, sulfuric acid, nitric acid, acetic acid, phosphoric acid, trichloroacetic acid, sulfosalicylic acid, formic acid, citric acid, lactic acid, Inorganic acids such as 0.1 to 10% perchloric acid and organic acids. Preferred are 0.0625 to 1N hydrochloric acid, 0.25 to 5% perchloric acid, and the like.

Another important disclosure in the present invention is to add a “polyphenol adsorbent” in the purification step. It has been found that the addition of a polyphenol adsorbent facilitates the recovery of the polyamine or polyamine composition.

In the present invention, the “polyphenol adsorbent” is not particularly limited as long as it is a substance capable of adsorbing polyphenols, but PVPP (polyvinyl polypyrrolidone), PVP (polyvinyl pyrrolidone), PEG (polyethylene glycol) and the like are preferably used. The Particularly preferred is PVPP (polyvinyl polypyrrolidone). These polyphenol adsorbents may use commercially available materials. For example, Polyclar (registered trademark), POLYCLAR (registered trademark) (manufactured by IS Corporation), Dowex (registered trademark) -1, PVP-40, or the like may be used. By adding a polyphenol adsorbent after adding an acid solution to a plant and / or plant extract, it can be expected that the recovered amount, yield, and purity of the polyamine composition are increased.
The amount of polyphenol adsorbent added is preferably 0.1 to 30% (w / v), more preferably 0.5 to 20% (w / v), still more preferably 1 to 10% (w / v). It is.

  By crushing, crushing, and mixing the plant and / or plant extract after adding the acid solution to the acidic condition or adding the acid solution to the acidic condition and then adding the polyphenol adsorbent The recovery amount of the polyamine composition can be increased. Particularly in the case of plant tissue, it is desirable to damage the cell wall because it has a cell wall. In the case of a plant extract or plant extract, it does not need to be crushed or crushed so as to damage the cell wall because it does not contain the cell wall. As a method for performing crushing or crushing, for example, a mixer, a blender, a homogenizer, a mortar, an ultrasonic crusher, or the like can be used.

  After sufficiently extracting the polyamine contained in the plant and / or plant extract into the acid solution (liquid fraction), the liquid fraction is separated from the residue and precipitate by centrifugation or filtration. The recovered liquid fraction contained a large amount of polyamine and was obtained as a “polyamine composition”.

  In the present invention, “polyamine” is a general term for aliphatic hydrocarbons having two or more primary amino groups, and is a natural product that exists universally in the living body, and more than 20 types of polyamines have been found. For example, 1,3-diaminopropane, putrescine, cadaverine, cardine, spermidine, homospermidine, aminopropyl cadaverine, theremin, spermine, thermospermine, canabalmin, aminopentylnorspermidine, N, N-bis (aminopropyl) cadaverine, homo Examples include spermine, cardopentamine, homocardopentamine, cardohexamine, and homocardohexamine. Typical polyamines include putrescine, spermidine and spermine.

  In the present invention, “putrescine” is one of typical polyamines and is a general natural product that exists universally in living organisms, and is an aliphatic hydrocarbon compound having two primary amino groups. . “Cadaverine” is one of the typical polyamines and is a general natural product that exists universally in living organisms, and is an aliphatic hydrocarbon compound having two primary amino groups. “Spermidine” is one of the typical polyamines and is a general natural product ubiquitously present in living organisms, and is an aliphatic hydrocarbon compound having three primary amino groups. “Spermine” is one of the typical polyamines and is a general natural product universally present in living organisms, and is an aliphatic hydrocarbon compound having four primary amino groups.

If necessary, the polyamine composition may be subjected to desalting treatment or purification treatment by ion exchange method, membrane fractionation method, gel filtration method, electrodialysis method, and by carrying out at least one of these methods. A higher purity polyamine composition can be obtained. For example, as an ion exchange method, a polyamine solution is passed through a column packed with an ion exchange resin to separate the polyamine from impurities such as amino acids, peptides, proteins, and sugars. As the ion exchange resin to be used, the ion exchange group may be a sulfonic acid group, sulfopropyl group, phosphoric acid group, carboxylmethyl group, aminoethyl group, diethylamino group, quaternary aminoethyl group, quaternary ammonium group, etc. Either a cation exchange resin or an anion exchange resin can be used. When a cation exchange resin is used, the polyamine is adsorbed on the cation exchange resin. Therefore, after sufficiently separating non-adsorbed substances, the polyamine is removed with an acidic solution such as sulfuric acid or hydrochloric acid, or a salt solution such as sodium chloride. Elute. When an anion exchange resin is used, the polyamine is not adsorbed on the anion exchange resin, and thus the non-adsorbed fraction containing the polyamine is collected. For example, as a membrane fractionation method, cellulose, cellulose acetate, polysulfone, polyamide, polyacrylonitrile, polytetrafluoroethylene, polyester, polypropylene and the like have a fractional molecular weight in the range of 1000 to 100,000. The polyamine composition is subjected to UF using an ultrafiltration (UF) membrane, and the permeate containing the polyamine is recovered. Further, NF of the polyamine solution is performed using a nanofiltration (NF) membrane having a salt rejection rate of 30 to 80% for desalting. For example, in the gel filtration method, the polyamine composition is neutralized and passed through a column packed with a gel filtration carrier to recover the polyamine by molecular weight fractionation. The gel filtration carrier used is a dextran-based, acrylamide-based, agarose-based, cellulose-based, polyvinyl-based, glass-based, polystyrene-based, or the like with a molecular weight cut-off in the range of 100 to 100,000. For example, in electrodialysis, electrodialysis is performed by alternately supplying a polyamine composition and saline between each membrane partitioned by a cation exchange membrane and an anion exchange membrane. The electrodialysis conditions include an initial current density of 0.5 to 15 A / dm ² and a voltage of 0.1 to 1.5 V / tank.

  The polyamine composition recovered in the present invention can be used in combination with quasi drugs, cosmetics for skin and hair, foods and drinks, pharmaceuticals, animal feeds and the like.

Specific examples of cosmetics and quasi-drugs include basic cosmetics such as internal and external pharmaceutical preparations, lotions, emulsions, creams, ointments, lotions, oils, packs, facial cleansers, skin cleansers, shampoos, etc. Hair conditioner, rinse, hair treatment, hair cream, pomade, hair spray, hair conditioner, permanent agent, hair nick, hair dye, hair restorer, hair restorer, hair restorer, foundation, white powder, funny, lipstick, blusher, eye Makeup cosmetics such as shadows, eyeliner, mascara, eyebrows and eyelashes, cosmetics for finishing such as nail care, perfumes, bath preparations, toothpastes, mouth fresheners, gargles, liquid odors and deodorants Agents, sanitary products, sanitary cotton, and wet tissue.

Examples of foods and beverages include soft drinks, carbonated drinks, nutrition drinks, fruit drinks, lactic acid drinks, ice cream, ice sherbet, shaved ice and other frozen desserts, buckwheat, udon, harsame, gyoza skin, shumai skin, chinese food Noodles such as noodles, instant noodles, candy, candy, gum, chocolate, tablet confectionery, snack confectionery, biscuits, jelly, jam, cream, baked confectionery, bakery confectionery, crab, salmon, clams, tuna, sardines, shrimp, Skipjack, mackerel, whale, oyster, saury, squid, red scallop, scallop, abalone, sea urchin, salmon roe, tofubushi and other marine products, fishery products such as kamaboko, ham, sausage, processed milk, infant formula, infant formula Milk products such as milk powder, baby food, fermented milk, salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream , Oils and fats such as dressings, processed foods such as sauces, sauces, sauces, curry, stew, oyakodon, rice bowls, miscellaneous cooking, Chinese rice bowls, and rice bowls, tempura, eel rice, hayashi rice, oden, mabodorf, beef bowl, meat sauce , Egg soup, omelet rice, dumplings, shumai, hamburger, meatballs and other retort pouch foods, various forms of health and nutritional supplements, health functional foods, tablets, capsules, drinks, troches and the like.

The polyamine composition recovered in the present invention is suitably applied to humans, but can also be applied to animals other than humans as long as each effect can be expected.

EXAMPLES The present invention will be described more specifically with reference to the following examples. However, these are merely examples and do not limit the scope of the present invention.

(Polyamine analysis method)
The content of polyamines contained in leaves, stems, roots, seeds, fruits, embryos, embryos, soy milk, natto, okara, etc. can be examined as plants or / and plant extracts. Polyamines include free polyamines, compound polyamines, and conjugated polyamines, which can be analyzed with different extraction methods (Plant Cell Physiol., 43 (2), 196-206, 2002). As a specific example, a method for analyzing seed free polyamine will be described in detail. About 0.1 to 1.0 g of soybean seeds were diluted with an internal standard solution (1,6-hexaneamine or 1,7-diaminoheptane, internal standard amount = 7.5 to 48 nmol) and a 5% aqueous perchloric acid solution (sample raw 5-20 ml per 1.0 g body weight) and thoroughly triturate and extract at room temperature using a Polytron mixer. The grinding solution is centrifuged at 4 ° C., 35,000 × g for 20 minutes to collect the supernatant, and this solution is used as a free polyamine solution. Add 100-400 μl of free polyamine solution (polyamine composition, purified polyamine composition), 200 μl of saturated sodium carbonate aqueous solution, 200 μl of dansyl chloride / acetone solution (10 mg / ml) to a microtube with a screw cap and mix gently. To do. After tightly closing the stopper of the tube, it is covered with aluminum foil and heated in a water bath at 60 ° C. for 1 hour for dansylation. After allowing the tube to cool, add 200 μl of an aqueous proline solution (100 mg / ml) and mix. Cover with aluminum foil and reheat in water bath for 30 minutes. After cooling, nitrogen gas is blown to remove acetone, and then 600 μl of toluene is added and mixed vigorously. After the tube is allowed to stand to separate into two phases, the upper toluene layer is dispensed into 300 μl microtubes. Nitrogen gas is blown onto the collected toluene to completely remove the toluene. 200 μl of methanol is added to the tube to dissolve the dansylated free polyamine. The amount of free polyamines of putrescine, spermidine, and spermine is analyzed by an internal standard method using high-performance liquid chromatography connected to a fluorescence detector (excitation wavelength: 365 nm, emission wavelength: 510 nm). As the HPLC column, μ Bondapak C18 (manufactured by Waters: 0273324, 3.9 × 300 mm, particle diameter 10 μm) is used. The polyamine content in the sample is calculated by obtaining the peak areas of each polyamine and the internal standard from the standard solution and the HPLC chart of the sample, respectively.

(Example 1) Preparation of polyamine composition from soybean seeds 100 g soybean seed (variety 'Fukuyutaka') diluted internal standard solution (1,7-diaminoheptane, internal standard amount = 1200 nmol) and 480 ml 5% perchloric acid An aqueous solution was added and left overnight at room temperature. Thereafter, 16 g of polyclar VT (ISP), which is a polyphenol adsorbent, was added, and the soybean seeds were sufficiently crushed with a blender mixer, and then left at room temperature for 30 minutes to extract a polyamine composition under acidic conditions. The crushed material was centrifuged at 2 ° C. and 22,000 × g for 20 minutes to collect a liquid fraction, which was neutralized with a 30% sodium hydroxide solution to obtain a polyamine composition. The polyamine composition contained 8.2 mg of putrescine, 12.4 mg of spermidine, and 6.1 mg of spermine as the polyamine, for a total of 26.7 mg. Further, the recovered polyamine composition was passed through a column packed with a cation exchange resin (AG 50W-X4, 200-400 mesh, H + type, manufactured by Bio-Rad), and the polyamine was adsorbed on the resin. The column was washed by flowing 0.7N NaCl / 0.1M sodium phosphate solution (pH 8.0), water and 1N hydrochloric acid sequentially. After removing the impurities, the polyamine was eluted with 6N hydrochloric acid and neutralized with 30% sodium hydroxide to obtain a purified polyamine crude product. The purified polyamine composition contained 6.4 mg of putrescine, 10.4 mg of spermidine and 5.1 mg of spermine as the polyamine, for a total of 21.9 mg. A chart of the HPLC analysis of the polyamine composition and the purified polyamine composition is shown in FIG. Sharpness of putrescine (detection time: 10.173 minutes, 10.175 minutes), cadaverine (detection time: 10.903 minutes, 10.890 minutes), spermidine (detection times: 16.050 minutes, 16.137 minutes), spermine (detection times: 20.100 minutes, 20.225 minutes) A single peak was detected in any composition. Impurity peaks other than polyamine were hardly detected, and it was clearly confirmed that the polyamine composition was highly pure and polyamine was the main component. When the polyamine purity was calculated from the peak area of HPLC analysis, the polyamine composition was 80 to 86%, and the purified polyamine composition was 88 to 93%. The reason for the high purity of the polyamine composition is greatly influenced by denaturation and adsorption removal of impurities such as seed protein by adding a polyphenol adsorbent and extracting it under acid conditions. The purified polyamine composition was desalted with an electrodialyzer (Acylizer S3, manufactured by Astom Corp.) and concentrated.

Diluted internal standard solution (1,7-diaminoheptane, internal standard amount = 1200 nmol) and 500 ml of 1N hydrochloric acid solution were added to 100 g of soybean seeds (variety 'Fukuyutaka') and allowed to stand overnight at low temperature (4 ° C.). Thereafter, 16 g of polyphenol VT (made by ISP), a polyphenol adsorbent, was added, and the soybean seeds were sufficiently crushed with a blender mixer, and then allowed to stand at low temperature (4 ° C.) for 30 minutes to form a polyamine composition under acidic conditions. Extracted. The crushed material was centrifuged at 2 ° C. and 22,000 × g for 20 minutes to collect a liquid fraction, which was neutralized with a 30% sodium hydroxide solution to obtain a polyamine composition. The polyamine composition contained 4.1 mg of putrescine, 9.1 mg of spermidine, and 2.9 mg of spermine as the polyamine, for a total of 16.1 mg.

From the above results, it was possible to recover a polyamine or a polyamine composition that can be used industrially or practically from soybean seeds. In particular, since it was confirmed that the polyamine or the polyamine composition can be recovered from the plant with 1N hydrochloric acid, the polyamine or the polyamine composition excellent in safety can be provided. If a polyamine or a polyamine composition is recovered with hydrochloric acid and neutralized with 30% sodium hydroxide, it becomes a neutral reaction by a very general ion, and is considered to be safer than trichloroacetic acid.

(Example 2) Preparation of polyamine composition from soybean buds Soybean seeds (variety 'Fukuyutaka') were allowed to absorb water and cultivated by culturing at 26 ° C for 2 days under light or dark conditions. About 50 g of bud parts of germinated soybeans cultured under light conditions or dark conditions were sampled. 8 g of diluted internal standard solution (1,7-diaminoheptane, internal standard amount = 600 nmol), 250 ml of 5% perchloric acid aqueous solution, polyphenol adsorbent polyclar VT (manufactured by ISP) was added to 50 g of soybean bud, and homogenizer Soy soybean buds were sufficiently crushed. Next, it was left to stand at room temperature for 30 minutes, and the polyamine composition was extracted under acidic conditions. The crushed material was centrifuged at 2 ° C. and 22,000 × g for 20 minutes to collect a liquid fraction, which was neutralized with a 30% sodium hydroxide solution to obtain a polyamine composition. Soybean buds germinated under light conditions contained 26.2 mg of putrescine and cadaverine as polyamines, 1.8 mg of spermidine, and 0.2 mg of spermine as a polyamine, for a total of 28.2 mg. . Soybean buds germinated under dark conditions contained 17.1 mg of putrescine and cadaverine as polyamines, 1.6 mg of spermidine and 0.2 mg of spermine as a polyamine, for a total of 18.9 mg. .

From the above results, it was revealed that soybean buds were particularly rich in putrescine and cadaverine. Among polyamines, when putrescine or cadaverine is used as an active ingredient, it is desirable to use soybean buds as an extraction material. Polyamines or polyamine compositions that can be used industrially or practically could be recovered from soybean buds.

(Example 3) Preparation of polyamine composition from soybean germ (soybean embryo) 100 g soybean germ (manufactured by Foryou) diluted internal standard solution (1,7-diaminoheptane, internal standard amount = 1200 nmol), 500 ml of 5% A perchloric acid aqueous solution was added and the mixture was allowed to stand at room temperature for 1 hour. Thereafter, 16 g of polyclar VT (ISP), which is a polyphenol adsorbent, was added, and the soybean germ was sufficiently crushed with a blender mixer, and then left at room temperature for 30 minutes to extract a polyamine composition under acidic conditions. The crushed material was centrifuged at 2 ° C. and 22,000 × g for 20 minutes to collect a liquid fraction, which was neutralized with a 30% sodium hydroxide solution to obtain a polyamine composition. The polyamine composition contained 23.5 mg of putrescine, 24.0 mg of spermidine and 9.6 mg of spermine as the polyamine, for a total of 57.1 mg. Further, the recovered polyamine composition was passed through a column packed with a cation exchange resin (AG 50W-X4, 200-400 mesh, H + type, manufactured by Bio-Rad), and the polyamine was adsorbed on the resin. The column was washed by flowing 0.7N NaCl / 0.1M sodium phosphate solution (pH 8.0), water and 1N hydrochloric acid sequentially. After removing the impurities, the polyamine was eluted with 6N hydrochloric acid and neutralized with 30% sodium hydroxide to obtain a purified polyamine crude product. The purified polyamine composition contained 20.4 mg of putrescine, 22.3 mg of spermidine and 7.7 mg of spermine as polyamines, for a total of 50.4 mg. The purified polyamine composition was desalted with an electrodialyzer (Acylizer S3, manufactured by Astom Corp.) and concentrated.

To 1 kg of soybean germ (manufactured by Foyu), a diluted internal standard solution (1,7-diaminoheptane, internal standard amount = 10 μmol) and 5 L of 1N hydrochloric acid solution were added and left at room temperature for 1 hour. Thereafter, 80 g of polyclar VT (ISP), which is a polyphenol adsorbent, was added, the soybean germ was sufficiently crushed with a homogenizer, and left at room temperature for 1 hour to extract a polyamine composition under acidic conditions. The crushed material was centrifuged at 2 ° C. and 22,000 × g for 30 minutes to collect a liquid fraction, which was neutralized with a 30% sodium hydroxide solution to obtain a polyamine composition. The polyamine composition contained 235.1 mg of putrescine as polyamine, 241.8 mg of spermidine, and 80.1 mg of spermine, for a total of 557.0 mg. A chart of the HPLC analysis of the polyamine composition is shown in FIG. Sharp single peaks of putrescine (detection time: 10.925 minutes), spermidine (detection time: 15.898 minutes), and spermine (detection time: 20.025 minutes) were detected. The single peak at 13.002 minutes is the diluted internal standard solution (1,7-diaminoheptane). Impurity peaks other than polyamine and diluted internal standard were hardly detected, and it was clearly confirmed that the polyamine composition was highly pure and polyamine was the main component. The reason for the high purity of the polyamine composition is that the use of only the embryo (embryo) part results in low contamination of seed proteins and the like, and addition of a polyphenol adsorbent and extraction under acidic conditions such as seed proteins. The fact that impurities are denatured and adsorbed and removed has a great influence. The polyamine composition was desalted with an electrodialyzer (Acylizer S3, manufactured by Astom Corp.) and concentrated.

From the above results, it was revealed that a very high content of polyamine or polyamine composition can be recovered from soybean germ (soybean embryo). In particular, there has been no report that this amount of polyamine or polyamine composition has been recovered from soybean germ, and it was also the first finding that the polyamine content in soybean germ was extremely high. In the past, when polyamines or polyamine compositions were extracted from plants and used industrially, the content of polyamines contained in plants was extremely low and the extraction efficiency was low, but 1 kg of soybean germ Since about 0.5 g of polyamine or polyamine composition was recovered from (soybean bud), the production cost was greatly improved. Since it was confirmed that the polyamine or the polyamine composition can be recovered from the plant with 1N hydrochloric acid, the polyamine or the polyamine composition excellent in safety can be provided.

Example 4 Preparation of Polyamine Composition from Wheat Germ (Wheat Embryo) Diluted internal standard solution (1,7-diaminoheptane, internal standard amount) in 100 g of wheat germ (cultured and roasted type, manufactured by Nisshin Pharma) = 1200 nmol), 500 ml of 5% perchloric acid aqueous solution was added, and the mixture was allowed to stand at room temperature for 1 hour. Thereafter, 16 g of polyclar VT (ISP), which is a polyphenol adsorbent, was added, and the soybean germ was sufficiently crushed with a blender mixer, and then left at room temperature for 30 minutes to extract a polyamine composition under acidic conditions. The crushed material was centrifuged at 2 ° C. and 22,000 × g for 20 minutes to collect a liquid fraction, which was neutralized with a 30% sodium hydroxide solution to obtain a polyamine composition. The polyamine composition contained 6.4 mg of putrescine, 23.9 mg of spermidine and 14.3 mg of spermine as polyamines, for a total of 44.6 mg. Further, the recovered polyamine composition was passed through a column packed with a cation exchange resin (AG 50W-X4, 200-400 mesh, H + type, manufactured by Bio-Rad), and the polyamine was adsorbed on the resin. The column was washed by flowing 0.7N NaCl / 0.1M sodium phosphate solution (pH 8.0), water and 1N hydrochloric acid sequentially. After removing the impurities, the polyamine was eluted with 6N hydrochloric acid and neutralized with 30% sodium hydroxide to obtain a purified polyamine crude product. The purified polyamine composition contained 5.7 mg of putrescine, 21.5 mg of spermidine and 12.9 mg of spermine as polyamines, for a total of 40.1 mg. The purified polyamine composition was desalted with an electrodialyzer (Acylizer S3, manufactured by Astom Corp.) and concentrated.

Diluted internal standard solution (1,7-diaminoheptane, internal standard amount = 10 μmol) and 5 L of 1N hydrochloric acid solution are added to 1 kg of wheat germ (cultured and roasted type, manufactured by Nisshin Pharma) and left at room temperature for 1 hour. did. Thereafter, 80 g of polyphenol VT (manufactured by ISP), which is a polyphenol adsorbent, was added and the wheat germ was sufficiently crushed with a homogenizer, and then left at room temperature for 1 hour to extract a polyamine composition under acidic conditions. The crushed material was centrifuged at 2 ° C. and 22,000 × g for 30 minutes to collect a liquid fraction, which was neutralized with a 30% sodium hydroxide solution to obtain a polyamine composition. The polyamine composition contained 75.1 mg of putrescine, 294.0 mg of spermidine, and 119.4 mg of spermine as polyamines, for a total of 488.5 mg. A chart of the HPLC analysis of the polyamine composition is shown in FIG. Sharp single peaks of putrescine (detection time: 10.110 minutes), spermidine (detection time: 15.892 minutes), and spermine (detection time: 20.19 minutes) were detected. The single peak at 12.994 minutes is a diluted internal standard solution (1,7-diaminoheptane). Impurity peaks other than polyamine and diluted internal standard were hardly detected, and it was clearly confirmed that the polyamine composition was highly pure. The reason for the high purity of the polyamine composition is that the use of the germ (embryo) part reduces the contamination of seed proteins and the like, and the addition of polyphenol adsorbent and extraction under acidic conditions removes impurities such as proteins. Degeneration or adsorption removal has a great influence. The polyamine composition was desalted with an electrodialyzer (Acylizer S3, manufactured by Astom Corp.) and concentrated.

From the above results, it was possible to recover a polyamine or a polyamine composition that can be used industrially or practically from wheat germ (wheat embryo). Since wheat germ contains many effective components in addition to polyamines, the recovered polyamine composition may contain active components in addition to the main component polyamines. By mixing the effects and actions of active ingredients other than polyamines with the effects and actions of polyamines, more excellent effects and actions can be expected. Since it was confirmed that the polyamine or the polyamine composition can be recovered from the plant with 1N hydrochloric acid, the polyamine or the polyamine composition excellent in safety can be provided.

(Example 5) Preparation of polyamine composition from soymilk 100 ml of soymilk (variety 'Fukuyutaka') diluted internal standard solution (1,7-diaminoheptane, internal standard amount = 1200 nmol), 100 ml of 5% perchloric acid aqueous solution added. Then, after sufficiently mixing with a mixer, the polyamine composition was extracted under acidic conditions by being left at room temperature for 30 minutes. The mixture was centrifuged at 4 ° C., 27,700 × g for 20 minutes to collect a liquid fraction, and neutralized with a 30% sodium hydroxide solution to obtain a polyamine composition. In the polyamine composition, 0.7 mg of putrescine, 1.9 mg of spermidine and 0.7 mg of spermine were contained as polyamines, and the total amount was 3.3 mg. The recovered polyamine composition was neutralized with 30% sodium hydroxide, desalted with an electrodialyzer (Acylizer S3, manufactured by Astom Corp.) and concentrated.

100 ml of soymilk (variety 'Fukuyutaka') diluted internal standard solution (1,7-diaminoheptane, internal standard amount = 960 nmol), 10% perchloric acid aqueous solution 2.5 ml (final concentration 0.25%), 5 ml (final) 10 ml (final concentration 1%), 20 ml (final concentration 2%), and 40 ml (final concentration 4%) were added. Then, after sufficiently mixing with a mixer, the polyamine composition was extracted under acidic conditions by being left at room temperature for 30 minutes. The mixture was centrifuged at 2 ° C., 27,700 × g for 20 minutes, and a liquid fraction was collected. The liquid fraction was white at a perchloric acid concentration of 0.25%. It is considered that because the perchloric acid concentration was low, the soy protein was not sufficiently denatured due to a decrease in pH and was mixed into the liquid fraction. Slight white color was exhibited even at 0.5% and 1% perchloric acid concentrations. Polyamine recovery amount is 0.25% perchloric acid concentration polyamine composition shows peaks other than polyamine and other components are mixed. Polyamine recovery amount is lower than other perchloric acid concentration did. On the other hand, polyamine compositions with a perchloric acid concentration of 0.5% or more contain the same amount of recovered polyamines containing 1.8 mg of putrescine, 2.8 mg of spermidine, and 1.0 mg of spermine. It was 5.6 mg. It was confirmed that the polyamine or polyamine composition could be recovered even at a perchloric acid concentration of 0.5%, and an excellent preparation method was found in terms of safety.

A diluted internal standard solution (1,7-diaminoheptane, internal standard amount = 800 nmol) was added to 100 ml of soymilk (variety 'Fukuyutaka'), and final concentrations of 0.0625N, 0.125N, 0.25N using 6N hydrochloric acid were added. 0.5N and 1N were added. Then, after sufficiently mixing with a mixer, the polyamine composition was extracted under acidic conditions by being left at room temperature for 30 minutes. The mixture was centrifuged at 2 ° C., 27,700 × g for 20 minutes, and a liquid fraction was collected. The liquid fraction was slightly white at hydrochloric acid concentrations of 0.0625N, 0.125N, 0.25N, and 0.5N. It is considered that the soy protein was not sufficiently denatured due to a decrease in pH due to the low concentration of hydrochloric acid and mixed into the liquid fraction. The recovered amount of polyamine was almost the same at all hydrochloric acid concentrations, including 1.1 mg of putrescine, 2.3 mg of spermidine, and 0.6 mg of spermine as a polyamine, for a total of 4.0 mg. It was confirmed that the polyamine or the polyamine composition could be recovered even with hydrochloric acid of 1N or less, and an excellent preparation method was found in terms of safety. The chart of the HPLC analysis of the polyamine composition recovered with 1N hydrochloric acid is shown in FIG. Sharp single peaks of putrescine (detection time: 10.124 minutes), spermidine (detection time: 15.929 minutes), and spermine (detection time: 20.040 minutes) were detected. Impurity peaks other than polyamine were hardly detected, and it was clearly confirmed that the polyamine composition had high purity. The reason for the high purity of the polyamine composition is that most of the seed protein mixed in the soymilk was denatured by extraction under acid conditions, and the precipitate was removed by centrifugation.

From the above results, it was possible to recover a polyamine or a polyamine composition that can be used industrially or practically from soy milk. When recovering a polyamine or a polyamine composition from soymilk, crushing or pulverization is not required, and the preparation process is small and the merit is great. Since soy milk contains many effective components other than polyamines, the recovered polyamine composition may contain active components in addition to the polyamines as the main components. By mixing the effects and actions of active ingredients other than polyamines with the effects and actions of polyamines, more excellent effects and actions can be expected. In particular, by reducing the concentration of perchloric acid and hydrochloric acid, it has been confirmed that the amount of soy protein mixed in the polyamine composition is increased, so if the concentration of perchloric acid and hydrochloric acid is reduced depending on the application, Good. Since soy milk is generally eaten and consumed, the safety of polyamines or polyamine compositions prepared from soy milk is extremely high.

(Example 6) Preparation of polyamine composition from okara To 1 kg of okara, diluted internal standard solution (1,7-diaminoheptane, internal standard amount = 10 μmol), 5 L of 5% aqueous perchloric acid solution was added, and 1 at room temperature. Left for hours. Thereafter, the okara was sufficiently crushed with a homogenizer, and allowed to stand at room temperature for 1 hour to extract a polyamine composition under acidic conditions. The crushed material was centrifuged at 2 ° C. and 22,000 × g for 30 minutes to collect a liquid fraction, which was neutralized with a 30% sodium hydroxide solution to obtain a polyamine composition. The polyamine composition contained 5.4 mg of putrescine, 17.8 mg of spermidine and 6.5 mg of spermine as the polyamine, and the total amount was 29.7 mg. The polyamine composition was desalted with an electrodialyzer (Acylizer S3, manufactured by Astom Corp.) and concentrated.

From the above results, polyamines or polyamine compositions that can be used industrially or practically from Okara could be recovered. Okara was a residue after collection of soymilk and was thought to contain little polyamine or polyamine composition, but was confirmed to contain a lot of polyamine, although less than soymilk. Since Okara is a residue after collecting soymilk, it has also been clarified that the content of soybean protein and the like is low, the polyamine purity in the polyamine composition is high, and purification with a cation exchange resin is unnecessary. Since most of okara is discarded, a great industrial advantage is expected if polyamine or polyamine composition is recovered using okara as a production material.

According to the present invention, it is possible to provide a method for preparing a polyamine or a polyamine composition having a high safety in a large yield with a high yield. Therefore, it becomes possible to provide a safe polyamine or polyamine composition that can be used for health, cosmetics, food, and pharmaceutical applications, and is expected to contribute greatly to industrial development.

Purity confirmation of polyamine composition (left) and purified polyamine composition (right) prepared from soybean seeds Purity confirmation of polyamine composition prepared from soybean germ Purity confirmation of polyamine composition prepared from wheat germ Purity confirmation of polyamine composition prepared from soy milk

Claims (7)

(1 ) a step of treating the wheat germ and / or the plant extract of wheat germ under acidic conditions at pH 6.0 or less by adding an acid solution containing a mineral acid and / or an organic acid ; and (2) (1 ) And a step of separating and recovering the liquid fraction produced after the step of) by centrifugal separation and / or filtration separation, and a method for preparing a polyamine composition. Mineral acid and / or organic acid is hydrochloric acid, sulfuric acid, nitric acid, acetic acid, phosphoric acid, citric acid, lactic acid, propionic acid, butyric acid, succinic acid, malonic acid, succinic acid, fumaric acid, maleic acid, malic acid, tartaric acid, benzoic acid 2. The method for preparing a polyamine composition according to claim 1 , wherein the polyamine composition is at least one acid selected from the group consisting of acids, sulfosalicylic acid and formic acid. The method for preparing a polyamine composition according to claim 2 , wherein the mineral acid and / or organic acid is hydrochloric acid and / or citric acid. At the same time or after the wheat germ and / or the wheat germ plant extract is subjected to acidic conditions of pH 6.0 or less , a polyphenol adsorbent is added, and the resulting liquid fraction is separated by centrifugation and / or filtration. method of adjusting the polyamine composition according to any one of claims 1 to 3, characterized by recovering. The polyamine composition is 1,3-diaminopropane, putrescine, cadaverine, cardine, spermidine, homospermidine, aminopropyl cadaverine, theremin, spermine, thermospermine, canabalmine, aminopentylnorspermidine, N, N-bis (aminopropyl) cadaverine, Homosuperumin, caldopentamine, homo caldopentamine, according to any one of claims 1-4, characterized in that it comprises at least one compound selected from the group consisting of cardo hexamine and homo cardo hexamine A method for preparing a polyamine composition. The method for preparing a polyamine composition according to any one of claims 1 to 5 , wherein the polyamine composition comprises at least one compound selected from the group consisting of putrescine, cadaverine, spermidine and spermine. The polyamine composition recovered by the preparation method according to any one of claims 1 to 6 , further comprising at least one selected from the group consisting of an ion exchange method, a gel filtration method, a membrane fractionation method, and an electrodialysis method. A method for preparing a polyamine composition, which is purified by treatment.

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