USOO8829205B2
(12) United States Patent (10) Patent No.: US 8,829,205 B2 Jeon et al. (45) Date of Patent: Sep. 9, 2014
(54) METHOD FOR PREPARING COUMESTROL (58) Field of Classification Search AND COUMESTROL PREPARED BY SAME USPC ...... 549/279; 435/119 See application file for complete search history. (75) Inventors: Hee Young Jeon, Yongin-si (KR); Dae Bang Seo, Yongin-si (KR); Si Young (56) References Cited Cho, Seoul (KR); Sang Jun Lee, U.S. PATENT DOCUMENTS Seongnam-si (KR) 3,564,019 A * 2/1971 Holmlund et al...... 549,279 (73) Assignee: Amorepacific Corporation, Seoul (KR) 6,129,937 A 10/2000 Zurbriggen et al. (*) Notice: Subject to any disclaimer, the term of this FOREIGN PATENT DOCUMENTS patent is extended or adjusted under 35 EP 1357,807 B1 3, 2007 U.S.C. 154(b) by 226 days. JP 2008-120729. A 5/2008 KR 10-0360306 B1 11, 2002 (21) Appl. No.: 13/575,267 KR 10-0706279 B1 4/2007 KR 10-0778.938 B1 11, 2007 (22) PCT Filed: Jan. 31, 2011 WO WOO1f97769 A1 12/2001 OTHER PUBLICATIONS (86). PCT No.: PCT/KR2O11AOOO673 S. M. Boué et al., “Induction of the Soybean Phytoalexins S371 (c)(1), Coumestrol and Glyceollin by Aspergillus,” J. Agric. Food Chem. (2), (4) Date: Jul. 25, 2012 vol. 48, No. 6, pp. 2167-2172, 2000. X. Hao et a... "Analysis of Coumestrol Content in Soybean.” Journal (87) PCT Pub. No.: WO2011/093686 of Beijing University of Agriculture, vol. 23, No. 3, pp. 7-9, 2008. PCT Pub. Date: Aug. 4, 2011 * cited by examiner (65) Prior Publication Data Primary Examiner — Nizal Chandrakumar US 2012/O2897 14 A1 Nov. 15, 2012 (74) Attorney, Agent, or Firm - Dickstein Shapiro LLP (51) Int. Cl (57) ABSTRACT cod 193/00 (2006.01) The present invention relates to a method for preparing CI2P 17/18 (200 6. 01) coumestrol, comprising a step in which beans are germinated and a step in which the beans are fermented, and to (52) is: C. CI2P 17/181 (2013.01) coumestrol prepared by the method. USPC ...... 549/279; 435/119 6 Claims, 9 Drawing Sheets U.S. Patent Sep. 9, 2014 Sheet 1 of 9 US 8,829,205 B2
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Non-treated 1% sucrose US 8,829,205 B2 1. 2 METHOD FOR PREPARING COUMESTROL FIG. 8 shows the content of coumestrol depending on the AND COUMESTROL PREPARED BY SAME concentration of fungus; and FIG. 9 shows the content of coumestrol under the opti TECHNICAL FIELD mized condition. The present disclosure relates to a method for producing MODE FOR INVENTION coumestrol and coumestrol produced thereby. Coumestrol (CMS: 3,9-dihydroxy-6H-benzofuro(3.2-c) BACKGROUND ART (1)benzopyran-6-one CHOs, MW: 268.2) is a coumarin 10 like compound structurally similar to the natural estrogen Bean is of great nutritional value and contains various estradiol or stilbestrol. It has a structure of the chemical physiologically active and functional Substances. Especially, formula 1: since the phytoestrogens contained in bean are similar to the estrogen of mammals including human in structure, they have the effect of preventing chronic diseases such as hormonal 15 chemical formula 1 disorders. The phytoestrogens include in general isoflavone, coumestan and lignan. DISCLOSURE
Technical Problem HO The present disclosure is directed to providing a method for producing coumestrol and coumestrol produced thereby. 25 Since the estrogenic activity of coumestrol is 30-100 times Technical Solution higher as compared to isoflavone which is known to have estrogenic activity, it is very useful in alleviating the Symp In one general aspect, the present disclosure provides a toms of uterine cancer, bone marrow cancer, breast cancer, method for producing coumestrol, including: germinating brain cancer or menopausal disorders. The estrogenic activity bean; and fermenting the bean. 30 of coumestrol is thought to be attributable to its structure. In another general aspect, the present disclosure provides Coumestrol is known to be present in very Small amounts coumestrol produced by the above method. in bean or other plants in the family Fabaceae. Accordingly, it is necessary to effectively produce the coumestrol which is Advantageous Effects very useful but is contained in bean in trace amount. 35 Hereinafter, the present disclosure will be described in The method for producing coumestrol according to the more detail. present disclosure enables production of a large amount of In an aspect, the present disclosure provides a method for coumestrol from bean at high yield by increasing the content producing coumestrol, comprising: a germinating process of of coumestrol in the bean. The method can produce a large germinating bean; and a fermenting process offermenting the amount of coumestrol which is very expensive. The produced 40 bean. coumestrol may be used usefully in the fields of pharmaceu In an aspect of the present disclosure, the bean may be any ticals, foods or cosmetics. plant in the family Fabaceae that synthesizes coumestrol. For DESCRIPTION OF DRAWINGS example, the bean that can be used in the present disclosure 45 includes those for paste, bean curd, bean sprouts, bean-mixed FIG. 1 shows the content of coumestrol (mg/kg dry weight) rice or unripe bean. The bean species for paste(Jang) or bean extracted from bean germinated in an automatic watering curd include daepung, hojang, jangwon, daehwang, Sodam, apparatus at 30°C.; Songhak, daewon, jinpum, danbaek, duyu, sinpaldal, taeg FIG.2 shows the content of coumestrol (mg/kg dry weight) Wang, manri, jangsu, muhan, baekun, saeal. hwanggeum and extracted from bean germinated after contacting with 50 jangyeop. The species for bean sprouts include sinhwa, Aspergillus niger, SOWon, anpyeong, Seonam, dachae, Sorok, Soho. Somyeong, FIG.3 shows the content of coumestrol (mg/kg dry weight) dawon, pungsan-namul, iksan-namul, Sobaek-namul, gwan extracted from bean germinated in liquid medium (potato gan, danyeop and eunha. The species for bean-mixed rice dextrose broth) at 30° C. after contacting with Aspergillus include cheongja, heukcheong, galmi, Seonheuk, geomjeong niger, 55 kong and ilpumgeomjeong-kong. Also, the species for unripe FIG. 4 shows the content of coumestrol (mg/kg dry weight) bean include daol, sinrok, Saeeul, geomjeongeul, Seokryang extracted from bean germinated in liquid medium (potato putkong, hwaeom-putkong and keuneul. dextrose broth) at 30° C. after inoculating with Aspergillus In another aspect of the present disclosure, the bean may be Sojae, one that can germinate and is resistant to damage from disease FIG.5 shows the content of coumestrol (mg/kg dry weight) 60 and harmful insects. Examples may include Sinhwa-kong, extracted from bean germinated in liquid medium (potato SOWon-kong, anpyeong, Seonam, dachae, Sorok, Soho, Somy dextrose broth) at 30° C. after inoculating with Bifidobacte eong, dawon, pungsan-kongnamul, iksan-kongnamul, rium infantis, Sobaek-kongnamul, gwangan, danyeop and eunha. FIG. 6 shows the content of coumestrol in different variet The method for producing coumestrol according to the ies of bean: 65 present disclosure comprises a germinating process of germi FIG. 7 shows the content of coumestrol depending on nating bean. The germinating process may be conducted by medium additives; contacting the bean at least partly with oxygen or air. US 8,829,205 B2 3 4 The method for producing coumestrol according to the on the total weight of the medium. The saccharides include present disclosure also comprises a fermenting process of common monosaccharides, disaccharides or polysaccha fermenting the bean. Columestrol is known as a phytoalexin rides, specifically include Sucrose, glucose or SRT. synthesized by the plants in the family Fabaceae to protect In an aspect of the present disclosure, the process may be themselves from attack of fungi or other pathogenic micro conducted by adding 1-50 vol%, specifically 5-20 vol%. organisms. Thus, when bean is contacted with microorgan more specifically 6-12 vol% of bean to the reactor based on isms such as fungi, yeasts or lactic acid bacteria, they may the total Volume of the reactor. During the process. Some of increase the content of coumestrol in the bean by acting as the bean may be immersed in the medium and the remainder coumestrol biosynthesis elicitors. In an aspect of the present may not be immersed in the medium. When the bean is disclosure, the content of coumestrol in the bean may be 10 increased via the fermenting process of contacting a the bean completely exposed to air without being immersed in the at least partly with microorganisms. In another aspect of the medium, the microorganisms contacted to ferment the bean present disclosure, the fermenting process may be conducted may be washed off during watering. Conversely, when the by inoculating the bean with microorganisms 1-10 times, bean is completely immersed in the medium, the germinated specifically 1-5 times, more specifically 1-3 times. Through 15 bean may rot. this process, the microorganisms may be better contacted In an aspect of the present disclosure, the process may be with the bean. conducted while Supplying oxygen or air as much as possible. In a method for producing coumestrol according to an For example, when the process is conducted in a 5000 mL aspect of the present disclosure, the germinating process and reactor, oxygen or air may be Supplied at a rate of 2,500 the fermenting process may be conducted simultaneously or VVm/m. Also, when the process is conducted in a 3 L reactor, Successively. In another aspect of the present disclosure, oxygen or air may be supplied at a rate of 15,000 VVm/m. The when the germinating process and the fermenting process are Sufficient Supply of oxygen or air may lead to effective ger conducted simultaneously, the bean may be contacted with mination. oxygen or air in the state where the bean is contacted with In an aspect of the present disclosure, the process may be microorganisms. Specifically, the bean may be contacted 25 conducted at 20 to 35° C., specifically 20 to 30°C. In another with oxygen or air in the State where the bean is immersed in aspect of the present disclosure, the process may be con a medium containing the microorganisms. ducted in the dark. In another aspect of the present disclosure, In an aspect of the present disclosure, the microorganisms the process may be conducted for 2 to 10 days. For example, may be any one that can act as coumestrol biosynthesis elici the germinating process may be conducted for 2 days and then tor. In another aspect of the present disclosure, the microor 30 the germinating process and the fermenting process may be ganism may be fungi, yeasts or lactic acid bacteria. Specifi cally, the fungus may belong to the genus Aspergillus, conducted simultaneously for 6 to 8 days. Penicillium or Monascus. More specifically, the microorgan Through the germinating process and the fermenting pro ism may be at least one selected from a group consisting of cess conducted under the above-described conditions, the Aspergillus niger, Aspergillus sojae, Aspergillus Oryzae and 35 content of coumestrol in the bean may be increased effec Bifidobacterium infantis. The above-listed microorganisms tively. are adequate for promoting the production of coumestrol by A method for producing coumestrol according to an aspect the bean. of the present disclosure may further comprise, prior to the In an aspect of the present disclosure, the microorganisms germinating process, a sterilizing process of sterilizing the may be contacted with the bean by applying a Suspension of 40 bean with at least one of sterilized water, ethanol and sodium the spores of the microorganisms on the Surface of the coty hypochlorite, specifically with sterilized water. ledon of the bean. In another aspect of the present disclosure, A method for producing coumestrol according to an aspect the microorganisms may be contacted with the beanby apply of the present disclosure may further comprise, after the ing a powder of the microorganisms to a medium containing germinating process and the fermenting process, an extract the bean. In another aspect of the present disclosure, 0.05-2 45 ing process of extracting coumestrol from the germinated and wt %, specifically 0.75-2 wt %, more specifically 1.0-2.0 wt fermented bean. The extraction may be conducted by a %, of the microorganisms may be contacted based on the total method commonly employed in the art, specifically by alco weight of the medium. hol extraction, more specifically by ethanol extraction. In an aspect of the present disclosure, the condition of at In another aspect, the present disclosure provides least one of the germinating process and the fermenting pro 50 coumestrol produced by the method for producing cess is not particularly limited as long as germination and coumestrol. The coumestrol may be used usefully in the fields fermentation can be achieved consistently. An exemplary of pharmaceuticals, foods or cosmetics. In another aspect, the condition of the germinating or fermenting process is as fol present disclosure provides a pharmaceutical, food or cos lows. metic composition comprising the coumestrol as an active In an aspect of the present disclosure, at least one of the 55 ingredient. germinating process and the fermenting process may be con The features and effects of the present disclosure will now ducted in a reactor. In another aspect of the present disclosure, be described in detail through examples. The following the process may be conducted in a reactor containing 20-80 examples are for illustrative purposes only and not intended vol%, specifically 40-60 vol%, more specifically 45-55 vol to limit the scope of this disclosure. %, of a medium based on the total volume of the reactor. 60 In an aspect of the present disclosure, the medium is not EXAMPLES 1-6 particularly as long as the germination and fermentation can be achieved effectively. Specifically, a liquid nutrient Germination and Fermentation of Bean medium, more specifically a potato dextrose broth (PDB) medium may be used. In another aspect of the present disclo 65 Glycine max (L.) Merrill two varieties; variety 1 (herein sure, the medium may contain 0.001-10 wt %, specifically after, variety 1) and variety 2 (hereinafter, variety 2). 0.1-5 wt %, more specifically 0.5-2 wt % of saccharides based which is a bean for bean sprouts, was used. After germination, US 8,829,205 B2 5 6 the bean was contacted with microorganisms and it was inves Column: NOVA-PAK RP185um (3.9x150 mm) tigated whether the production of coumestrol is enhanced. Column temperature: 27°C. The microorganisms used were the fungi Aspergillus niger Detector: UVD (260 nm) (KCCM 11240, Korean Culture Center of Microorganisms) Solvent: 1% acetic acid in water (acetonitrile gradient) and Aspergillus sojae (KCCM 60354, Korean Culture Center 5
No. Treatment condition Example 1 The bean was germinated for 8 days in an automatic watering apparatus without inoculation of microorganisms (see FIG. 1). Example 2 OOIL of the spore Suspension was inoculated at the seedling of the bean that germinated for 2 days in an automatic watering apparatus, ollowed by one more inoculation 4 hours later. After the inoculation of Aspergilius niger, the bean was further germinated for 2, 4 and 6 days in an automatic watering apparatus (see FIG. 2). Example 3 00LL of the spore suspension (10 spores/mL, Aspergillus niger) and 4 surface-sterilized germinated beans were added together in a glass bottle (030 mm x h 75 mm) containing 1 mL of a liquid medium (potato dextrose broth, Difco TM, BD Diagnostics, Sparks, MD, USA). Then, he bean was further germinated for 2, 4 and 6 days at 30° C. (see FIG. 3). Example 4 Experiment was performed in the same manner as in Example 3 except or culturing the germinated bean together with Aspergilius Soiae (see FIG. 4). Example 5 Experiment was performed in the same manner as in Example 3 except or culturing the germinated bean together with Bifidobacterium infantis (see FIG. 5). Example 6 4 surface-sterilized germinated beans were completely immersed in a glass bottle (030 mm x h 75 mm) containing 5 mL of a liquid medium (potato dextrose broth, Difco TM, BD Diagnostics, Sparks, MD, USA). Then, after adding 100 L of the spore suspension (10° spores/mL), the bean was further germinated for 2, 4 and 6 days at 30° C.