TECHNICAL FIELD The present invention relates to a seed of legume having isoflavone enriched and a method for producing the same, more particularly, it relates to a sprouted seed containing high concentration of isoflavone and a method for producing the same, the method comprises treating a sprouted seed of legume selected from the group consisting of mung bean, soybean and red bean with at least one stress-inducing chemicals selected from the group consisting of ethylene, salicylic acid, methyl jasmonic acid and acetyl salicylic acid.

BACKGROUND ART Soybean, as a main food resource for mass-producing high quality protein, is an important crop which is more highlighted as the level of life gets higher because it is a nearly unique vegetable protein source to be able to substitute for animal protein in a situation where the consumption of animal protein source is decreased due to bovine spongiform encephalopathy, avian influenza, foot-and-mouth disease, etc, and at the same time, it contains several health-related functional substances, such as isoflavone, saponin, anthocyanin, etc.

Mung bean sprout is one of the most common vegetables as a vegetable legume together with bean sprout and it is drawing attention as a clean vegetable in USA and Europe, etc. , as well as many countries in Asia including Korea, Japan and

China. Contrary to bean sprout, when it is consumed uncooked or cooked partially, mung bean sprout has a little soybean-characteristic smell caused by volatile substance generated upon degradation of legume fat, such as linoleic acid, etc. , by lipoxigenase in bean sprout, and the high content of moisture and the low content of fiber per unit of weight result in a palatability, thus it is much more consumed vegetable legume than bean sprout in Asian countries including China, Japan, etc, and Western countries including USA and Europe. Mung bean has a carbohydrate and a protein as main ingredients, higher content of carbohydrates, as opposed to soybean, and 34% of starch among carbohydrates. It has been reported that when mung bean is used as mung bean sprout, the contents of protein, lipid, inorganic components, and vitamin are increased, and, in view of efficacy, it has digestion-stimulating effect for having several digestive enzymes, such as amylase, invertase, urease, etc, anti-hypertensive effect, anti-inflammatory effect, pyretic effect, hematopoiesis effect, etc.

Isoflavone, as estrogen analogues mainly comprised in pulse, improves several post-menopausal symptoms when administrated to climacteric women, the target organs of estrogen are reproductive organs of female including breast, uterus and ovary, reproductive organs of male including testis and prostate gland and brain, and estrogen plays an important role physiologically in maintenance of bone and cardiovascular system.

Especially, it is preferable for the family members of patient having the history of disease of breast cancer or uterine cancer to take vegetable isoflavone. Climacteric female shows all kinds of climacteric symptoms for a decrease of about 30% in the concentration of estrogen, however, administration of isoflavone to climacteric female resulted in the improvement of climacteric symptoms such as hot flush, perspiration, nervousness, depression, sleep disturbance, excessive sweating, etc, and shows no side effects caused by estrogen. Also, in respect to prevention and

therapy of premenstrual syndrome (PMS), it has been reported that the administration of isoflavone results in the prevention of PMS and relief of the symptoms by inducing the change of menstrual cycle, and in respect to the prevention and therapy of osteoporosis and senile fracture, isoflavone shows preventive and therapeutic effect against osteoporosis by inhibiting reabsorption of bone for having the similar activity to estrogen in skeletal metabolism as well as increasing the number of osteoblasts functioning in bone formation. In addition, it has been reported that isoflavone shows effects to prevent cardiovascular diseases, such as heart disease, hypertension, arteriosclerosis, etc. , and the aging of female brain, such as a failure of one's memory or depression of attention appearing post- climacteric. Another important advantage of vegetable isoflavone is to inhibit the generation of cancers.

Recently, a study for promoting the production of isoflavone in isoflavone- producing plants has been progressed, and it has been reported that the production of isoflavone in soybean is increased by various difficulties in growth, but a study for stimulating the production of isoflavone in mung bean has not been reported yet. Also, the content of isoflavone in soybean is mainly determined by its accumulated content at the late period of seed development of soybean, on the other hand, the present invention comprises inducing the biosynthesis of isoflavone during the process of sprouting by increasing very low biosynthetic activity of isoflavone during the process of soy bean-sprouting with artificial treatments.

Korea Patent Publication 2003-93025 demonstrates a method for producing sprouted soybean containing high-concentration of isoflavone whose content is increased by 40-70% by sprouting after washed soybean is added into refined water and water-soaked, but the method has a disadvantage that the sprouted soybean has low content of isoflavone.

DISCLOSURE OF INVENTION Therefore, the present inventors have conducted extensive studies to develop a sprouted seed of legume containing high-concentration of isoflavone, and confirmed that the content of isoflavone is increased in a sprouted seed by treating plants with a seed sprouted using stress-related chemicals, thereby perfecting the present invention.

It is an object of the present invention to provide a sprouted seed of legume containing high concentration of isoflavone and a method for producing the same.

Another object of the present invention is to provide a sprouted mung bean and soybean containing high concentration of isoflavone, produced by the method.

In order to accomplish the above objects, the present invention provides a method for producing a sprouted seed containing high concentration of isoflavone, comprising the steps of (a) sprouting a seed of legume selected from the group consisting of mung bean, soybean and red bean at 20-30'C in the dark for 1-7 days; (b) treating the sprouted seed with a plant-specific stress-inducing chemical; and (c) culturing the sprouted seed at 20- 30°C in the dark for 2-24 hours.

In the present invention, the plant-specific stress-inducing chemical is at least one selected from the group consisting of ethylene, salicylic acid, methyl jasmonic acid and acetyl salicylic acid, and the concentration of salicylic acid, methyl jasmonic acid and acetyl salicylic acid is 5~20mM, 0. 1-1. 0% (v/v) and 5~20mM, respectively.

The present invention also provides a sprouted mung bean produced by the method,

wherein the content of isoflavone generated in parts of cotyledon and root is more than 738. 83lig per Ig of sprouted mung bean (dry weight).

The present invention also provides a method for producing a mung bean sprout containing high concentration of isoflavone, the method comprises culturing the sprouted mung bean.

The present invention also provides a health food comprising the sprouted mung bean, the mung bean sprout containing high concentration of isoflavone produced by the method, or processed materials thereof.

The present invention also provides a sprouted soybean produced by the method, wherein the content of isoflavone is more than 8500yg per Ig of root part of sprouted soybean (dry weight).

The present invention also provides a method for producing a bean sprout containing high concentration of isoflavone, characterized by culturing the sprouted soybean.

The present invention also provides a health food comprising the sprouted soybean, the bean sprout containing high concentration of isoflavone produced by the method, or processed materials thereof.

In the present invention, soybean (cultivar Somyoung) and mung bean (cultivar Jangan) were individually sprouted, and then treated with a minimum amount of substances which artificially induce secondary metabolism related to stress tolerance in the plant, thereby artificially increasing the generations of substances of flavonoids, isoflavonoids as well as isoflavone, which are functional substances showing various pharmacological effects on breast cancer, prostatic cancer, heart

disease as well as female menopause. It demonstrated that the production of isoflavone can be increased by simple cultivation method without using a method like gene manipulation, because it is possible to produce soybean and mung bean sprout with about 1.7 fold and 2.3 fold increase in the concentration of isoflavone, respectively, in comparison with the case untreated with stress-inducing chemicals according to the present invention.

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a photograph showing the state after treating soybean sprouted in culture dish with stress-inducing chemicals, and then culturing for 16 hours in the dark (NT: untreated group, SA: lOmM salicylic acid-treated group, MeJA : 0.5% methyl jasmonic acid-treated group, ASA: 12mM acetyl salicylic acid-treated group).

FIG. 2 is a graph indicating the content of isoflavone in the root part of sprouted soybean according to the present invention.

FIG. 3 is a graph indicating the detected amount of 12 isoflavone fractions comprising the total amount of isoflavone in the root part of sprouted soybean (din: daidzin, mdin : malonyldaidzin, acdin: acetyldaidzin, dein: daidzein, gly: glycitin, mgly: malonylglycitin, acgly: acetylglycitin, glein: glycitein, gin: genistin, mgin: malonylgenistin, acgin: acetylgenistin, gein: genistein).

FIG. 4 is a metabolic pathway of phenylpropanoid.

FIG. 5 is a graph indicating the content of isoflavone in the cotyledonal part of sprouted mung bean according to the present invention.

FIG. 6 is a graph indicating the content of isoflavone in the root part of sprouted mung bean (hypocotyl + root) according to the present invention.

FIG. 7 is a graph indicating the content of 12 isoflavone fractions comprising the total amount of isoflavone in the cotyledonal part of sprouted mung bean.

FIG. 8 is a graph indicating the detected amount of 12 isoflavone fractions comprising the total amount of isoflavone in the root part of sprouted mung bean.

DETAILED DESCRIPTION OF THE INVENTION Hereinafter, the present invention will be explained in further detail by the following examples. It will however be obvious to a person skilled in the art that these examples are given for illustrative purpose only, and the scope of the present invention is not limited to or by these examples.

Example 1: Seed disinfection and sprouting condition One hundred soybean seeds (cultivar: Somyoung) were put into each Erlenmeyer flask, and soaked in 95% ethanol for 1 minute, and then drained, followed by being washed three times with sterile water for 20 seconds every time. Next, the soybean seeds were soaked in commercially available ROX solution (Sodium Hypochlorite Solution) (4% of effective content of chloride), and then disinfected using vortex for 5 minutes. At sterile sink, the ROX solution was removed, and then the soybeans were washed three times with sterile water for 20 seconds every time. After the sterile water was removed as completely as possible upon third washing, and then the seeds were distributed on a previously prepared 1% bacto agar plate, the culture plate was wrapped with parafilm and protected from the light

using two layers of aluminum foil, and then the seeds were sprouted in culture room at 25 C (60% humidity) for 8-days. When treated, the cotyledon of the sprouted soybean was yellow and showed no significant differences between the sizes of pre-sprouted seeds and sprouted seeds, and the total length of hypocotyl and root of the soybean averaged 6.5 cm and didn't have remarkable pigment.

(FIG. 1).

As a result of sprouting mung bean (cultivar: Jangna) as the soybean, the sprouted mung bean was hulled, and its cotyledon was yellow. With the development of dicotyledon, the size of the sprouted mung bean seed was 0.3cm wide and 0.2cm long, which shows that there is no significant differences when compared with that of the pre-sprouted seed and the total length of hypocotyl and root of the mung bean averaged 6cm and didn't have remarkable pigment.

Example 2: Secondary metabolism induction and sample extraction After sprouting the soybeans prepared by the method described in Example 1,8 day old root (hypocotyl + root) and total part of cotyledon were soaked in diluted solution of three stress-inducing chemicals [salicylic acid, methyl jasmonic acid and acetyl salicylic acid; aspirin] with the concentration indicated in Table 1 for about 10 seconds, and then the diluted solution of three stress-inducing chemicals remaining on the surface of sprouted soybean was removed as completely as possible.

Table 1: The concentration of diluted solution of stress-related chemicals final concentration Solvent (ethanol) Salicylic acid 10mM 3% Methyl jasmonic acid 0.5% (v/v) 50% Acetyl salicylic acid 12mM 3%

Next, the soybean seeds were transferred to a new plate without 1% bacto agar culture medium, and the plate was completely sealed with parafilm, and then the secondary metabolism by the stress-related chemicals was induced at 25 °C for 16 hours in the dark caused by two layers of aluminum foil. After treatment for secondary metabolism induction, the cotyledon and the root (hypocotyl + root) were carefully separated by cutting the boundary line separated and protruded from cotyledon using an experimental scalpel, to prevent the hilum part of the soybean from being mixed with the root part (hypocotyl + root) of the soybean. Then, the separated cotyledon and root were used as analytic sample while stored at-80 °C after soaked in liquid nitrogen, and then quick refrigeration.

Example 3: Analytic sample preparation and isoflavone extraction The sample in refrigerated storage was transferred using liquid nitrogen to prevent thawing, lyophilized for two days, ground in a mortar and then used as extracting sample of secondary metabolites. Eighty % methanol solution with 500 ul of 0. 1% acetic acid per 0.01 g sample was used for extraction, and the sample was shaken at 150 rpm for 16 hours under the condition of being leaned to the ground at a 25 degree using 15ml disposable tube for centrifugation to make sure that the sample was completely shaken in solution for extraction, and then centrifuged twice, followed by being stored at-80 °C until analysis.

Example 4: Quantitative analysis on isoflavone using HPLC HPLC was performed using HPLC system from JASCO Corp. (Japan), ODS series of YMC AM303 (4. 6x250mm) was used as column. Water with 0.1% acetic acid and acetonitrile with 0. 1% acetic acid were used as mobile phase, gradient elution wherein the concentration of acetonitrile solvent increases from initial 15% to final

35% for 50 minutes was applied. Flow rate was adjusted to I. OmL/min, injected amount of extracted solution was 20pu, and the analysis was preformed with 254nm detection wavelength and 0.32 detection sensitivity.

HPLC analysis was performed using standard solution with concentration of 0. 1-25} jg/mL prepared by dissolving standard substances for isoflavone in methanol, and then calibration curve was obtained from peak area. Standard substances for isoflavone used in the analysis were total 12 types, 4 types from daidzeins, 4 types from genisteins and 4 types from glyciteins were purchased and used (Fluca Co. , Japan).

Example 5: The content of isoflavone after treating with stress-related chemicals (1) Sprouted soybean In the case of treating root part of sprouted soybean (hypocotyl + root) with 3 stress-related chemicals, as shown in FIG. 2, total generated amount of isoflavone was 7914 yg/g, 13492 yg/g, 2360, ug/g and 8667 ltg/g, in the distilled water-treated group (NT), salicylic acid-treated group (SA), methyl jasmonic acid-treated group (MeJA) and acetyl salicylic acid-treated group (ASA), respectively.

The contents of total 9 derivatives (conjugated forms) conjugated with glucose, malonylated glucose or acetylated glucose, besides 3 fundamental forming substances, genistein, daidzein and glycitein as aglycone comprising total amount of isoflavone in the root part of the sprouted soybean were compared (FIG. 3). As a result, the contents of genistein, daidzein and glycitein, as aglycone, showed very low level in all treated group. In salicylic acid-treated group, the content of malonyldaidzein showed particularly high increase.

On the other hand, the content of malonyldaidzein was very low, showing 70% decrease in methyl jasmonic acid-treated group, when compared with distilled water-treated group. It was supposed that the result was caused by an increased activity of phenylalanine ammonia lyase, chalcone reductase, chalcone synthase, isoflavone synthase, etc, by the treatment with salicylic acid in the biosynthetic pathway of isoflavone as shown in FIG. 4, and the result was consistent with prior result that the activity of chalcone synthase was decreased to 1/4 level by the treatment with methyl jasmonic acid in the case of methyl jasmonic acid-treated group and the activity of isoflavone reductase involved in the metabolism of isoflavone degradation was increased more than threefold.

(2) Sprouted mung bean When the cotyledonal part of sprouted mung bean was treated with 3 stress-related chemicals, total generated amounts of isoflavone were 832. 5, ug/g, 1407. 0, ug/g, 393.7 yg/g and 1372.2 yg/g, in the non-treated group (NT), salicylic acid-treated group (SA), methyl jasmonic acid-treated group (MeJA) and acetyl salicylic acid- treated group (ASA), respectively, as shown in FIG. 5. When compared with the total amount of isoflavone from cotyledon of sprouted mung bean in non-treated group (NT), methyl jasmonic acid-treated group (MeJA) showed 47% decreased total amount of isoflavone, while salicylic acid-treated group (SA) and acetyl salicylic acid-treated group (ASA) showed 169% and 165% increased total amounts of isoflavone, respectively.

On the other hand, when the root part of sprouted mung bean (hypocotyl + root) was treated with 3 stress-related chemicals, the total generated amounts of isoflavone were 284.8 yg/g, 1194. 0 yg/g, 345. 1/g/g and 1142. 2/. g/g, in the non- treated group (NT), salicylic acid-treated group (SA), methyl jasmonic acid-treated group (MeJA) and acetyl salicylic acid-treated group (ASA), respectively, as shown in FIG. 6.

As oppose to the cotyledonal part, significant difference was observed from all of the 3 treated groups as compared with non-treated group in the root part of the sprouted mung bean, and salicylic acid-treated group (SA), methyl jasmonic acid- treated group (MeJA) and acetyl salicylic acid-treated group (ASA) showed 419%, 121% and 401% increased total amounts of isoflavone, respectively. In methyl jasmonic acid-treated group, it was observed that the total amount of isoflavone showed no significant difference in the lower part of cotyledon, while it was decreased in cotyledon as compared with non-treated group. Acetyl salicylic acid-treated group showed that the increasing rate of the total amount of isoflavone was significantly different in cotyledon and lower part thereof. On the basis of these results, it was suggested that the mechanism for the production of isoflavone was regulated by different processes from each other in cotyledon and the lower part thereof.

Finally, as a result of measuring the total generated amount of isoflavone in cotyledon and root parts per 1 g of sprouted mung bean (dry weight) in each treated group, the total generated amounts were 1117. 3, ug/g, 2601. 0 yg/g, 738. 8 yg/g and 2514. 4, ug/g, in non-treated group (NT), salicylic acid-treated group (SA), methyl jasmonic acid-treated group (MeJA) and acetyl salicylic acid-treated group (ASA), respectively. When compared on the basis of 1117. 3, ug per lg dry weight of non- treated group, salicylic acid-treated group and acetyl salicylic acid-treated group showed 233% and 225% increases per Ig dry weight, while methyl jasmonic acid- treated group showed a decrease to 66%.

Also, the contents of total 9 derivatives (conjugated forms) conjugated with glucose, malonylated glucose or acetylated glucose, besides 3 fundamental substances, genistein, daidzein and glycitein as aglycone comprising the total amount of isoflavone in the cotyledonal part of the sprouted mung bean were

compared, and the result was shown in FIG. 7. In each group, the contents of daidzein, glycitein and genistein, which are aglycone were small. In cotyledon, salicylic acid-treated group and acetyl salicylic acid-treated group, which showed an increased total amount of isoflavone as compared with non-treated group, showed a significant increase in the amount of malonyldaidzine and malonylglycitin. Methyl jasmonic acid-treated group, which showed a decreased total amount of isoflavone in cotyledon as compared with non-treated group, showed a decrease in all glycoside except a little increase in the amount of glycitein and daidzein.

The contents of total 9 derivatives (conjugated forms) conjugated with glucose, malonylated glucose or acetylated glucose, besides 3 fundamental substances, genistein, daidzein and glycitein as aglycone comprising the total amount of isoflavone in the root part of the sprouted mung bean were compared and the result was shown in FIG. 8. The contents of daidzein, glycitein and genistein which are aglycone were low in non-treated group and salicylic acid-treated group, but, high in methyl jasmonic acid-treated group and acetyl salicylic acid-treated group, especially the content of genistein was high. In salicylic acid-treated group and acetyl salicylic acid-treated group, the content of acetyldaidzein showed especially significant increase. In salicylic acid-treated group, an increase of glycitein was specifically observed.

The contents of total 9 derivatives (conjugated forms) conjugated with glucose, malonylated glucose or acetylated glucose, besides 3 fundamental forming substances, genistein, daidzein and glycitein as aglycone comprising the total amount of isoflavone in the lower part of cotyledon of the sprouted mung bean were compared between non-treated group and each treated group, and the result was shown in FIG. 8. In the lower part of cotyledon which showed an increased total amount of isoflavone in all 3 treated groups as compared with non-treated

group (NT), salicylic acid-treated group and acetyl salicylic acid-treated group showed a significant increase in the content of malonylglycitin, and the generations of malonyldaidzine and malonylglycitin were increased in methyl jasmonic acid- treated group.

While the present invention has been described with reference to the particular illustrative embodiments, it is not to be restricted by the embodiments but only by the appended claims. It is to be appreciated that those skilled in the art can change or modify the embodiments without departing from the scope and spirit of the present invention.

INDUSTRIAL APPLICABILITY The present invention has effects to provide a method for producing a sprouted seed of legume containing high concentration of isoflavone, a sprouted seed containing high concentration of isoflavone produced by the method and a method for producing mung bean sprouts or bean sprouts containing high concentration of isoflavone produced by culturing the sprouted seed. Also, the present invention has effect to provide a health food comprising the sprouted seed, mung bean sprouts, bean sprouts or processed materials thereof.

According to the present invention, it is possible to produce a harmless and environment-friendly soybean or mung bean ingredients containing high content of isoflavone without using genetically modified organism (GMO), so that they can be applied to various kinds of food.