A METHOD FOR PREPARING A FERMENTATION PRODUCT OF SOYBEAN H AVING ENRICHED y-AMINOBUTYRIC ACID AND PHYTOESTROGENS USING THE BIFIDOBACTERIUMBREVE K-110 OF KOREAN-TYPE LACTIC ACID BACTERIA Technical Field The present invention relates to a method for preparing a fermentation product of soybean having enriched y-aminobutyric acid'and phytoestrogens using the Bifidobacterium breve K-110 of Korean-type lactic acid bacteria. More specifically, it relates to a method for preparing a fermentation product of soybean, said method comprising steps of soaking soybean in water for 4-6 hours, admixing said soaked soybean with water in a ratio of 1: 2 to 1: 8 (W/V), grinding them to obtain the ground materials of soybean, heating them to a temperature of 60 °C-75 °C and allowing them to cool, and then fermenting them for 12-48 hours, when performing of a soybean fermentation using the Bifzdobacterium breve K-110 of Korean-type lactic acid bacteria; and a method for preparing a freeze- dried fermentation product of soybean comprising the steps of adding ascorbic acid to said ground materials of soybean in concentration of 0. 025-0. 2%, heating and allowing them to cool, inoculating the Bif dobacterium breve K-110 of Korean-type lactic acid bacteria into them, fermenting them for 12-48 hours and freeze-drying them.

Further, it also relates to a method for preparing a fermentation product of soybean in which lactic acid bacteria was fermented, comprising the steps of grinding germinated soybean to obtain the ground materials of soybean, adding 0.05% of ascorbic acid thereto, inoculating 1% of the Bifidobacterium breve K-110 of Korean-type lactic acid bacteria (1. 5

- 2. 0 X 102/z) into them and then fermenting them for 12-48 hours at 37 °C.

Background Art The Bifidobacterium breve K-110 of Korean-type lactic acid bacteria has a property which can improve intestinal climate and also inhibit productivity of harmful enzymes by intestinal bacteria. Referring to a change of intestinal bacteria with age of man, Bifidobacteriufn appears on day 3-4 after his birth, and become the most dominant bacteria on day 5 after his birth. Also, upon completing of weaning period, anaerobic bacteria and Bif dobacterium are dominant, while other lactic acid bacteria, Lactobacillus, etc. are much less.

When getting older, bacterial type of the intestinal bacteria becomes aged and then the influence of dominant Bif dobacterìum becomes weak, so the influence of Lactobacillus becomes stronger than that of the Bifidobacterium. Thus, as man is getting old, taking of Bifìdobacterium will be able to make up an intestinal flora similar to that of infancy or adolescence-manhood. A difference between Lactobacillus and Bifdobacterium is in that Lactobacillus are classified into two groups, one group which does homofermentation and the other group which does heterofermentation, respectively while Bifidobacterium does heterofermentation only.

Important characteristics of Bifidobacterium breve K-110 of Korean-type lactic acid bacteria are in that it has an therapeutic effect of treating colon cancer, bladder cancer and ascites cancer (J. Microbiol. Biotechnol. 9 (3), 368 (1999) ) and a preventive effect of gastric ulcer by inhibiting the growth of Helicobacter pylori which is a causative agent of gastric ulcer, thus being capable of using for treating gastric diseases (J. Microbiol. Biotechnol. 10 (4), 532 (2000) ). Further, it was found

that it improves an immunity by increasing production of the secretory antibody IgA, alleviates diarrhoea and constipation (Journal of the Korean Society for Food Science and Technology, 31 (2), 547 (1999)), and has an anti-mutagenesis effect against carcinogenic substance (Arch Pharm Res. 23 (4), 482 (2000) ). In addition to, it was shown to have hepatoprotective effect against hepatotoxicity (Journal of the Korean Society for Food Science and Technology, 31 (4), 1096 (1999)).

Generally, examples of the physiological activity of lactic acid bacteria include inhibition of the growth of harmful bacteria, alleviation of lactose intolerance, reduction of serum cholesterol, anti-cancer action, immunological action, relief of diarrhoea and constipation, vitamin synthesis, etc. Bif dobacteria begin to appear from about day 3-4 after one's birth, then grow to be the most dominant bacteria on about day 5 and existing E. coli or other lactic acid bacteria, Lactobacillus, Streptococcus, etc. decrease to one hundredth. This is believed due to a factor which is present only in breast milk and promotes the growth of the Bifidobactef°ia and thus we can see that among the intestinal flora Bifidobacteria are the most important bacteria, compared with the other lactic acid bacteria.

The prior art is a generalized method comprising the steps of inoculating lactic acid bacteria into milk and adding powdered skim milk thereto to proliferate lactic acid bacteria so as to thereby prepare lactic acid beverage. Examples of patents for producing lactic acid beverage or lactic acid using cereals include a vegetable and herbal materials-derived lactic acid fermentation beverage (Korean Patent Publication Laid-Open No. 2000-0055832) and a rice sprout beverage (Korean Patent Publication Laid-Open No. 1998-0023072) using Lactobacillus planta7-UM, a process for preparing a lactic acid fermentation product using rice in the form of grains (Korean Patent Publication Laid-Open No. 1992-16030), a

process for preparing a liquid lactic acid fermentation product using powdered cereals (Korean Patent Publication Laid-Open No. 1997-14592), a process for producing lactic acid using soybean-derived ingredients (Korean Patent Publication Laid-Open No. 2000-73826), etc.

However, these methods comprise steps of mixing milk, or adding saccharides such as glucose, which is considerably different from the present method, and bacterial strain used therein is also different from the present invention using the Bifidobacteriuyn breve K-110 of Korean-type lactic acid bacteria.

Disclosure of Invention The object of the present invention is to provide a method for preparing a fermentation product of soybean by using Korean-type lactic acid bacteria, said method comprising the steps of soaking soybean in water for 4-6 hours, adding water to said soaked soybean in a predetermined ratio, grinding them to obtain the ground materials of soybean, heating and allowing them to cool, inoculating said Bifidobacterium breve K-110 into them, and then fermenting them, when performing of a soybean fermentation using the Bifidobacteriurn breve K-110 of Korean-type lactic acid bacteria.

Another object of the present invention is to provide a method for preparing a fermentation product of soybean comprising the steps of adding ascorbic acid to the ground materials of soybean in concentration of 0. 025-0. 2% and fermenting them to thereby promote the growth of the Bifìdobacterium breve K-110 of Korean-type lactic acid bacteria, when performing of a soybean fermentation using the Bifidobacterium breve K- 110 of Korean-type lactic acid bacteria.

A further object of the present invention is to provide a method for preparing a powdered fermentation product of soybean having viable lactic acid bacteria, comprising

the step of freeze-drying a fermentation product of soybean obtained by inoculating the Bifidobacterium breve K-110 of Korean-type lactic acid bacteria into the ground materials of soybean and fermenting them for 12-48 hours.

A further object of the present invention is to provide a method for preparing a fermentation product of germinated soybean, comprising the steps of adding ascorbic acid to ground materials of the germinated soybean and fermenting them with the Bifidobacterium breve K-110 of Korean-type lactic acid bacteria.

A further object of the present invention is to provide a method for enhancing content of y-aminobutyric acid, one of the physiologically active substance and phytoestrogens comprising step of fermenting soybean and germinated soybean using the Bifidobacterium breve K-110 of Korean-type lactic acid bacteria.

Best mode for Carrying Out the Invention The following examples are provided to better elucidate the practice of the present invention. It will be readily apparent to those skilled in the art that various changes and modifications of an obvious nature may be made without departing from the spirit of the invention, and all such changes and modifications are considered to fall within the scope of the invention as defined by the appended claims.

EXAMPLES Example 1 : Determination of the ratio of water in performing of soybean fermentation using Korean-type lactic acid bacteria Commercially available soybean was soaked in water for 4-6 hours and water was drained. Said soaked soybean was mixed with water in a ratio of 1: 2 (W/V), 1: 4 (W/V), 1: 6 (W/V) and 1: 8 (W/V), respectively. They were finely ground with a mixer or colloidal

mill for 5-10 minutes to prepare ground materials of soybean. Said materials were heated and boiled in a double pot or heating vessel for further 5 minutes from when it reached a maximum temperature as shown in Table 1. The maximum temperature showed 60 ~ 75 °C although it was slightly different with a ratio of water.

Table 1. A maximum temperature with a mixing ratio of soybean to water, upon heating soybean: water (W/V) Maximum Temperature (°C) 1 : 2 60 1 : 4 75 1 : 6 75 1 : 8 75 The ground materials of soaked soybean, which were heated under the condition of temperature in Table 1 as above, were allowed to cool, and thereto was inoculated 1% of Bifidobacterium breve K-110 (bacterial number of 1. 5-2. 0 X 102/lnQ) grown on TS medium for 24 hours. Then, they were fermented at 37°C for 24 hours and observed for a change in pH. The result measured was shown in Table 2.

When performing of fermentation with a different ratio of water, there was no significant difference in the value of pH measured, but the pH value for a fermentation product of soaked soybean to water (1: 4, W/V) was lowest with pH 4.2. However, in characters of the ground materials, if amount of water is excessively less, i. e. 1: 2 (W/V) of soybean to water ratio, compounded condition was very thick, with a result of difficulty in homogeneous fermentation. After the lowest pH value appeared at 1: 4 (W/V) of soaked soybean to water ratio, the pH value showed a tendency to rise gradually from a ratio of more than 1: 6.

In other words, the pH value has showed 4. 2-0. 07 for 1: 4 (W/V) of soaked soybean to water ratio, 4. 4~0. 07 for 1: 6 (W/V) of soaked soybean to water ratio and 4. 6t0. 21 for 1: 8 (W/V) of soaked soybean to water ratio, respectively. The optimum

addition ratio of water was 4 times as the weight of soaked soybean, when performing of fermentation using Korean-type lactic acid bacteria, based on the characters and pH values of the ground materials of soybean.

Table 2. Change in pH value after 24 h-fermentation with a mixing ratio of soybean and water Soybean* : Water(W/V) pH Character and Property 1:2 4.3~0.07 very thick and so difficulty in homogeneous fermentation 1 : 4 4. 20. 07 Homogeneous fermentation 1 : 6 4. 40. 07 Homogeneous fermentation h84. 60. 21Homogeneous fermentation * represents soybean soaked in water for 4 hours.

Example 2: Determination of the addition amount of ascorbic acid in performing of soybean fermentation using Korean-type lactic acid bacteria Water-soaked soybean was mixed with water in a ratio of 1: 4 (W/V) and then finely ground with a mixer or colloidal mill for 5 to 10 minutes to prepare ground materials. Said materials were heated and boiled in a double-pot or heating vessel for further 5 minutes from when it reached a maximum temperature of 75'C and then allowed to cool.

Thereafter, to said materials was inoculated 1% of Bifidobacteriunl breve K-110 of Korean-type lactic acid bacteria (grown on TS medium for 24 hours). At that time, to examine the effect of ascorbic acid, there was added ascorbic acid with concentrations of 0%, 0.025%, 0. 05%, 0. 1% and 0.2%, respectively, prior to heating of said ground materials. After addition of ascorbic acid with the above concentrations and fermentation for 24 hours, the bacterial number was determined using pH and BL agar plate. Table 3 shows the result.

The pH value prior to fermenting was the value of 6. 6-5. 7 which was decreased with increasing of the concentration of ascorbic acid, but the pH value after 24 h- fermenting was the value of 4. 4 # 4. 2 which was lowered by order of 2. 4-1. 4 compared with prior to fermenting, so it can be seen that lactic acid produced by fermentation process have induced reduction of pH value.

After fermentation, the number of Bifidobacteria has showed 4.40 X 109 for the group to which 0% of ascorbic acid was added, 2.02 X 1012 for the group to which 0.025% of ascorbic acid was added, 9.21 X 1013 for the group to which 0.05% of ascorbic acid was added, 2. 34 X 108 for the group to which 0.1% of ascorbic acid was added and 1. 01 X to8 for the group to which 0.2% of ascorbic acid was added, respectively.

The number of Bifidobacteria increased until addition amount of ascorbic acid increased by 0.05%, while at concentration of ascorbic acid after 0. 05% the number of Bifidobacteria rather showed a tendency to decrease. Thus, a suitable addition concentration of ascorbic acid was 0.05% at which the number of Bifidobacteria was maximum.

Table 3. pH value and the number of Bifidobacterium breve K-110 with concentration of ascorbic acid added, in soybean fermentation. Number of Concentration of pH Bifidobacteria after 24 ascorbic acid (%) h-fermenting After 24 h- Prior to fermenting fermenting 0 6.6 4.3 4.40 X 10 0. 025 6. 5 4. 3 2. 02 X 10'2 0. 05 6.3 4.2 9.21 X 10 0.1 6.1 4.4 2.34 X 10 .. 0. 2 5. 7 4. 3 1. 01 Example 3: Determination of fermentation period

Water-soaked soybean was mixed with water in a ratio of 1: 4 (W/V) and then finely ground with a mixer or colloidal mill for 5 # 10 minutes to prepare ground materials of soybean. 0.05% of ascorbic acid was added thereto, heated and boiled in a double pot or heating vessel for further 5 minutes from when it reached a maximum temperature of 75 °C and then allowed to cool. Thereafter, to said materials was inoculated 1% of Bifidobacterium breve K-110 of Korean-type lactic acid bacteria and they were fermented at 37°C for 0,2, 12,24, 36 and 48 hours to obtain fermentation product of soybean. pH and the number of Bifidobacterium breve K-110 for said fermentation product were measured and the result was shown in Table 4. The number of lactic acid bacteria prior to fermenting was 0, but it was 4.65 X 104 for 2 h-fermenting, 5.70 X 1013 for 12 h- fermenting and 6.01 X 10l4 for 24 h-fermenting with showing maximum number of bacteria, respectively. After that, the bacterial number was 4.18 X 1013 for 36 h- fermenting in which it was decreased, compared to 24 h-fermenting, and 4.13 X 1012 for 48 h-fermenting with further decreasing.

Therefore, the maximum number of bacteria can be obtained by 24 h-fermenting the ground materials of soybean using Bifidobacterium breve K-110 of Korean-type lactic acid bacteria.

Table 4. Determination of the number of lactic acid bacteria with fermentation period of ground soybean materials. Fermentation period pH. Number of Bifidobacterium (hour) (per gram) 0 6.3 0 2 6.3 4.65 X 104 12 4. 6 5. 70 X 10" 24 4.2 6.01 X 10 36 4. 2 4. 18 X 10 484, 24. 13X10" Example 4: Number of lactic acid bacteria in freeze-drving fermentation

product of soybean.

When 24 h-fermentation product of soybean which showed maximum number of lactic acid bacteria was frozen at a temperature of-20 to -30°C and then freeze-dried for 24 hours, the number of viable lactic acid bacteria measured was 7.79 X 1012/g.

Prior to freeze-drying by freezing, the number of lactic acid bacteria was 6.01 X 1014/g, while upon freeze-drying it was 7.79 X 1012/g, with a decrease of 102. Meanwhile, for using as preparations of lactic acid bacteria according to the Korean Food Code, bacterial number should be 108/g, while a fermentation product of soybean according to the present invention has 7.79 X 1012/g which is much larger than 108/g, thus being capable of using as preparations of lactic acid bacteria.

Example 5: Preparation of fermentation product of the Korean-type lactic acid bacteria using germinated soybean Water-soaked soybean for 4 h was germinated at a temperature of 25-30°C for 0, 12,24 and 36 hours, respectively. Said germinated soybean was mixed with water in a ratio of 1: 4 (W/V) and then finely ground with a mixer or colloidal mill for 5 # 10 minutes to prepare ground materials of soybean. 0.05% of ascorbic acid was added thereto, heated and boiled in a double pot or heating vessel for further 5 minutes from when it reached a maximum temperature of 75 oC and then allowed to cool. Thereafter, to said materials was inoculated 1% of Bifidobacterium breve K-110 of Korean-type lactic acid bacteria (1. 5 # 2.0 X 102/lue) and they were fermented at 37°C for 24 hours to obtain fermentation product of soybean. The pH and number of BiC ? dobacte)-ia for said fermentation product were measured and the result was shown in Table 5.

When fermenting for 24 hours using ungerminated (before germination) soybean,

the number of lactic acid bacteria was 8. 50 X 1014. When fermenting for 24 hours using 12 h-germinated soybean, the number of lactic acid bacteria was 1.00 X 1014 which was not significantly different from the case of using ungerminated soybean. However, the number of lactic acid bacteria was 7.07 X 1012 for the case of using 24 h-germinated soybean and 1.00 X 1012 for the case of using 36 h-germinated soybean, respectively, with resulting in decrease of bacterial number by using soybean having a long germination period. Thus, when using germinated soybean, it is preferred to use less than 12 h- germinated soybean.

Table 5. Changes in pH and the number of lactic acid bacteria for fermentation product using soybean having different germination period Germination period Ph Bifidobacterium (hour) (per gram) 0 4. 4 8. 50 X 10 12 4.4 1.00 X 10 24-4. 6 7. 07 X 10tZ 36 4.6 1.00 X 10 Example 6 : Content of Y-aminobutyric acid in a fermentation product of soybean using Korean-type lactic acid bacteria It was examined for a change in content of y-aminobutyric acid contained in a fermentation product of soybean fermented according to procedures of Examples 3 and 5.

The result showed from Table 6 that the Bifidobacteriuii7 breve K-110 of Korean-type lactic acid bacteria increased a content of y-aminobutyric acid. A content of y- aminobutyric acid in soybean prior to fermenting was 20. 850. 70 mg%, but it was 54. 50~3. 54 mg% with an increase of about 2.6 times upon 24 h-fermenting with Korean- type lactic acid bacteria. The content of y-aminobutyric acid was 89. 66~5. 97 mg% for 12 h-germinated soybean, but it was 133. 24~13. 05 mg% for fermenting of 12 h-germinated

soybean by using the Bifìdobacterium breve K-110 of Korean-type lactic acid bacteria, with an increase of about 1.5 times, compared to before fermenting.

Thus, it was believed that the Bifidobacterium breve K-110 also had a function of synthesizing y-aminobutyric acid. y-aminobutyric acid (GABA) is one of the neurotransmitters present in cerebrum, etc. and reported to have functions of improving cerebral blood flow and enhancing cerebral metabolism, thereby having effects in cerebral apoplexy, brain trauma and cerebral arterial sequela (Arch. Neurol. 37,352 (1980) ). Besides, due to having a hypotensive effect, it is one of the substances using for prevention of hypertension (Japan Society for Bioscience, Biotechnology, and Agrochemistry (JSBA), 61, I449 (1987)).- aminobutyric acid, which is one of the neurotransmitters present in cerebrum, is used as preparations for intravenous injection for improving cerebral blood flow and enhancing cerebral metabolism in cerebral apoplexy, brain trauma and cerebral arterial sequela, and is commercially available as oral preparations in the form of tablet containing 5-10 mg of y-aminobutyric acid in Japan (Journal of the Japanese Society for Food Science and Technology 46, 323 (1999) ). It was examined for the improving effects of climacteric disorders and senile mental disorders using y-aminobutyric acid and the result showed that when taking 26.5 mg of y-aminobutyric acid/day, about 75% of the psychic diseases such as headache and depression or the climacteric disorders accompanying various symptoms were reported to be cured (Journal of the Japanese Society for Food Science and Technology 47,596 (2000) ). Thus, the Bifidobacterium breve K-110 of Korean-type lactic acid bacteria which is capable of increasing content of y-aminobutyric acid can be utilized as a new y-aminobutyric acid-enriched food material or a functional food material. yric acid before and after fermenting of soybean using Bifidobacterium breve K-110 Samples Content of y-aminobutyric acid (mg%) Soybean prior to fermenting 20. 85A0. 70 24 h-fermented soybean54. 503. 54 _ 12 h-gerrninated soybean 89. 66i5. 97 24 h-fermented product of 133. 2413. 05 12 h-germinated soybean

Example 7: Increase in content of isoflavone, phytoestrogen of soybean fermentation product by usine the Korean-type lactic acid bacteria It was examined for change in content of isoflavone contained in a fermentation product of soybean fermented according to procedures of Examples 3 and 5. The result showed from Table 7 that the Bifdobacterium breve K-110 of Korean-type lactic acid bacteria increased content of phytoestrogens such as diadzein, glycitein and genistein. Further, content of isoflavonoids diadzin, glycitin and genistin decreased as a fermentation process progressed. In other words, during the course of fermentation using the Korean- type lactic acid bacteria, contents of diadzin, glycitin and genistin decreased, while contents of diadzein, glycitein and genistein having strong activity of phytoestrogen increased. Thus, it could be seen that diadzin, glycitin and genistin had been converted to diadzein, glycitein and genistein. Generally, isoflavonoids diadzin, glycitin and genistin which are present in soybean don't have effects like phytoestrogens by themselves, but it was reported that diadzein, glycitein and genistein in the form of aglycon in which sugar residues bound to diadzin, glycitin and genistin were removed, have function as phytoestrogens. Therefore, it could be seen that flavonoids (such as diadzin, glycitin and genistin) having no activity of phytoestrogens prior to fermenting were converted to the form of flavonoids (such as diadzein, glycitein and genistein) having strong activity of phytoestrogens, when fermenting of soybean using lactic acid bacteria.

Prior to fermenting, contents of diadzein, glycitein and genistein in soybean were

5. 38 mg/lOOg, 1.11 mg/lOOg and 9.91 mg/lOOg, respectively, but when 24 h-fermenting using the Korean-type lactic acid bacteria, they were 25.06 mg/lOOg, 3. 70 mg/lOOg and 52.41 mg/lOOg with an increasing rate of 4.66, 3.33 and 5.23 times, respectively.

Meanwhile, when fermenting 12 h-germinated soybean for 24 hours by using the Bifidobacterium breve K-110 of Korean-type lactic acid bacteria, contents of diadzein, glycitein and genistein were 19.89 mg/lOOg, 3.40 mg/lOOg and 41.36 mg/lOOg with an increasing rate of 3. 97,3. 06 and 4.17 times, respectively, compared to before germinating of soybean. Thus, the Bifìdobacterium breve K-110 was considered to have a function of synthesizing phytoestrogens as well.

Because phytoestrogens diadzein, glycitein and genistein have an action to increase a metabolism of carcinogenic substances in cell cultivation experiment, it was reported that they have the effects to prevent breast cancer and prostate cancer and also various kinds of cancers such as gastric cancer, colon cancer, etc. (The Korean Society for the Research of Soybean, Academic Publication 2001), and serve to reduce a risk of cardiovascular diseases and cancers (The Food Industry, Nov. 1998).

They say that these phytoestrogens diadzein, glycitein and genistein have an anti- oxidative effect, and among them an anti-oxidative effect of glycitein is greatest (The Korean Society for the Research of Soybean, 14: 91 (1997) ). Further, genistein is reported to block the growth of tumor (The Korean Society for the Research of Soybean, 14: 91 (1997) ), and it inhibits the lipid oxidation caused by lipid peroxides (J. Nutr. Biochem., 6: 481 (1995)).

Meanwhile, They say that in countries of East Asia such as Korea, China and Japan, taking a large amount-of soybean in one's diet, the reason why the mortality caused by breast cancer or prostate cancer is lower than in U. S. A. is due to the actions of various ingredients including phytoestrogen substances and also soybean peptide, etc. contained in soybean (The Korean Society for the Research of Soybean, 14: 91 (1997)). Thus, it is

important to have a technology for preparing soybean products containing a large amount of phytoestrogen substances.

Therefore, the Bifidobactei-iuiii breve K-110 of Korean-type lactic acid bacteria, capable of enhancing the contents of phytoestrogens diadzein, glycitein and genistein, can be used as a new phytoestrogens-enriched food material or a functional food material.

Table 7. Changes in contents of phytoestrogens diadzein, glycitein and genistein before and after fermenting of soybean using the Bifidobacterium breve K-110 (unit: mg/100g) Samples diadzin glycitin genistin Diadzein glycitein genistein soybean prior to 18.97 3.38 42.30 5.38 1.11 9.91 fermenting 24 h-fermented not found not found 1.99 25.06 3.70 52.41 soybean 24 h-fermented product of 12 h-3. 93 not found 5.37 19.89 3.40 41.36 germinated soybean Industrial Applicability As above-mentioned, based on said technical constitution, the the present invention can provide a lactic acid bacteria-fermentation product of soybean using the Bif dobacterium breve K-110 of Korean-type lactic acid bacteria.

The fermentation products of soybean and germinated soybean containing more than lOg of the Bifidobacterium breve K-110 of Korean-type lactic acid bacteria obtained according to the present invention can be used as preparations of lactic acid bacteria, as well as materials for various functional foods such as materials for health food and materials for eating uncooked food and specified raw food due to high contents of the y- aminobutyric acid, one of the physiologically active substances and the diadzein, glycitein and genistein functioning as phytoestrogens.