KR20180134702A - Preparation method of Fabaton soybean leaves composition having high in genistein and daidzein content and a composition having effective components to alleviate climacteric symptoms prepared by the same method - Google Patents

Abstract

The present invention provides: a method for producing bean leaves composition containing 70% or more of non-glycosylated isoflavones such as daidzein and genistein in isoflavone derivatives by fermenting fabaton bean leaves, which contain a high content of isoflavone derivatives, with certain lactic acid bacteria; a bean leaves composition having enhanced menopausal syndrome alleviating efficacy components produced therefrom; and foods cosmetics and the like containing the same. The content of the non-glycoside isoflavones (daidzein and genistein) in the composition of the present invention is remarkably enhanced, and the antioxidative activity and digestive enzyme inhibitory activity, which are physiological activities, are enhanced, so that the composition is effective for prevention and alleviation of menopausal syndrome due to female hormone imbalance and is useful as food, medicine and cosmetic materials.

Description

The present invention relates to a method for producing a parvon bean curd composition comprising a high level of daidzein and genistein, and a method for preparing a soybean curd derivative having a high content of a genistein having effective components to alleviate climacteric symptoms prepared by the same method}

The present invention relates to a method for highly efficiently converting an isoflavone derivative of parvaton bean curd, which is a high content of isoflavone derivative, into an unglycosylated isoflavone, and a composition for enhancing an effective ingredient for the improvement of menopausal syndrome produced therefrom, and more particularly, The present invention relates to a method for producing a canola composition comprising 70% or more of non-glycosylated isoflavones such as daidzein and genistein in an isoflavone derivative by fermenting a high content of isoflavone derivative with a specific lactic acid bacterium, The present invention relates to a composition for bean curd.

The canola leaves are 3 or 5 small leaves and are covered with short hairs. In the form of bean leaf pickle and bean leaf kimchi, the bean leaf is consumed as a local food which is mainly used in Gyeongsang area. It is picked up in the summer by soft young lentil leaves with watery kimchi, or in the fall, yellow leaves deciduous leaves are eaten and then eaten by salted fish. At this time, it is considered that there is a difference in the physiological activity between the blue and the deciduous yellow cane leaves. It is suggested that isoflavone, which is a representative functional ingredient of soybean, is contained in the soybean leaf, and the activity of inhibiting the hydrolysis enzyme is different according to the stage of growth of the soybean leaf, and it can be utilized as an excellent material for the development of functional foods such as obesity and diabetes (Yuk et al., 2011, Food Chem.).

Isoflavone derivatives are flavonoids with excellent pharmacological efficacy, and a great deal of capital is invested in the development of functional plants with high content of them worldwide. Isoflavones are non-steroidal estrogens, termed phytoestrogenic estrogens, because they have chemical structural and physiological characteristics similar to those of estrogen. These are similar to estrogen, a female hormone, and show useful physiological activities expected in estrogen. It has been reported that it reduces the risk of osteoporosis especially after menopause and lowers plasma cholesterol. Also, antioxidant activity and antioxidant activity have been reported to lower the risk of coronary heart disease.

Isoflavones exist in the form of a glycoside linked to a beta-glycoside or in the form of a non-glycosylated sugar. Examples of aglycon isoflavones include genistein, daidzein and glycitein. The glycoside isoflavones include genistin, daidzin and glycine. Glycitin, and the like. In particular, various physiological activities have been reported to be due to aglycone type non-glycoside isoflavones. Among non-glycoside isoflavones, genistein is effective for antioxidant activity as well as for breast cancer and prostate cancer, and daidzein is known to be effective for osteoporosis in postmenopausal women. The Food and Drug Administration recommends isoflavones as a raw material for health functional foods, which can contribute to bone health after menopause and about 24 ~ 27 mg of total non-glycosylated isoflavone derivatives as recommended daily intake.

Recently, a technique for cultivating a high content of isoflavone derivative soybean leaves (patent pending Patent Application No. 10-1451298) has been developed using molecular farming technology. However, the isoflavones of this soybean leaf have been reported to contain glycoside isoflavones such as? -Glycoside and malonyl- The non-glycosylated isoflavones, which contain about 98% of β-glycoside and have excellent physiological activity, exist only in about 2% or less, which is insufficient in terms of utilization as a functional food (Yuk et al., 2016, J. Agric. .).

Therefore, it is required to develop a technology for converting a high content of isoflavone derivatives contained in the parvaton bean curd to an unglycosylated isoflavone, and to develop a processed food of the parvaton bean curd with the improved effectiveness.

It is an object of the present invention to provide a method for producing a composition of a canola composition containing 70% or more of non-glycosylated isoflavones such as daidzein and genistein in an isoflavone derivative by fermenting a high content of isoflavone derivative with a specific lactic acid bacterium.

It is still another object of the present invention to provide an unglycosylated isoflavone-enriched bean curd composition prepared by the above method.

Yet another object of the present invention is to provide health functional foods and cosmetics for improving menopausal symptoms comprising the above unglycosylated isoflavone-enriched bean curd composition.

In order to achieve the above object, the present invention provides a method for producing an isoflavone derivative, comprising the step of fermenting a high content of isoflavone derivative soybean leaves as a bacterium of Lactobacillus paracasei and / or Lactobacillus amylovorus , Of at least 70%.

I) Isoflavone derivatives High content  Carrot leaf

In the present invention, a high content of isoflavone derivatives is treated with ethylene, ethylene donor (ethylene oxide), or ethylene generator, which is a plant growth hormone, before or after cultivation, to produce a soybean leaf containing a high content of isoflavone derivatives , &Quot; parvaton bean leaf ") is preferably used.

Parvaton bean leaf is commercially available.

The content of isoflavone is about 5,000 ~ 15,000 ㎍ / g in the parvalbumin.

The parvon canola leaves can be harvested, washed and then directly used in a juice, or dried and pulverized.

Ii)

In the present invention, the 'seed bacterium' uses a bacterium containing at least one strain selected from the group consisting of Lactobacillus paracasei and Lactobacillus amylovorus .

Surprisingly, a high content of isoflavone derivatives was converted to non-glycosylated isoflavones at a high efficiency (over 70%, Table 2) when ferbatonose leaves were fermented using these strains as seeds. In addition, antioxidant activity, antidiabetic activity, and anti-obesity activity were also enhanced (Figs. 3A to 4B).

Iii) Fermentation

Ferbaton iso-fructose is inoculated on the canola leaves and fermented to convert the isoflavone derivatives contained in the parvaton bean leaves into non-glycosylated isoflavones.

For smooth fermentation, fermentation can be done by adding saccharin and water to the parvalbumin. It is preferable to add 1 to 5 parts by weight and 500 to 1,000 parts by weight of the glycogen and water to 100 parts by weight of the persimmon leaves. If desired, the mixture of persimmon leaves, sugar sources and water may be sterilized prior to inoculation. The sterilization can be carried out according to a conventional method.

Inoculation is carried out with 1-10% (v / v) of seed culture and fermentation is carried out at 20 ~ 40 ℃ for more than 12 hours.

If the inoculation is carried out below 1% (v / v), the fermentation rate may be delayed and if it is carried out above 10% (v / v) If the fermentation temperature is lower than 20 ° C, the fermentation rate is retarded. If the temperature is higher than 40 ° C, the fermentation rate is retarded and abnormal fermentation may occur. When the fermentation time is less than 12 hours, the conversion rate is less than 50%.

According to the conversion method of the present invention, it is possible to obtain a composition of a soybean curd containing not less than 70% of nonisaccharide isoflavone, especially daidzein and genistein, in the contained isoflavone derivative.

According to another object of the present invention, there is provided a non-glycosylated isoflavone-enriched soybean curd composition prepared by the above method.

In the non-glycosylated isoflavone-enriched bean curd composition according to the present invention, more than 70% of the high content of isoflavone derivatives is converted to the non-glycosylated isoflavone, and the content of the daidzein and the genistein is remarkably increased (Table 2).

In addition, the composition of the present invention has excellent radical scavenging ability and reducing power, and thus has an enhanced antioxidant activity, excellent alpha-glucosidase inhibiting ability, enhanced anti-diabetic activity and excellent pancreatic-lipase inhibitory activity And enhanced physiological activity such as enhanced anti-obesity activity (Figs. 3A to 4B).

Therefore, the bean curd composition according to the present invention can be used as a food, medicine, and / or cosmetic material as estrogen substitute which is a female hormone due to a high content of non-glycoside isoflavone.

According to another object of the present invention, there is provided a food for improving menopausal symptoms comprising the above non-glycoside isoflavone-enriched bean curd composition.

According to yet another object of the present invention, there is provided a cosmetic comprising the non-glycosylated isoflavone-enriched bean curd composition.

The food or cosmetic of the present invention can be prepared by adding the composition of the present invention or its extract directly or mixed with other food ingredients, and can be suitably prepared according to a conventional method.

In the present invention, the type of the food is not particularly limited, and examples of such foods include soybean products (pills, tablets, capsules), fermented foods, fermented foods (kimchi, pickles, etc.), fermented beverages (pouches, . The types of cosmetics may be mask packs, skins, lotions, creams, and the like.

Menopausal syndromes that can be prevented or improved by the foods according to the present invention (due to high levels of non-glycosylated isoflavones and enhanced physiological activity) include facial flushing, obesity, insomnia, osteoporosis, diabetes and skin aging .

The content of the non-glycosylated isoflavones (daidzein and genistein) in the composition of the present invention is remarkably enhanced and the antioxidative activity and the inhibitory activity of digestive enzymes (carbohydrate and lipid hydrolyzing enzyme), which are physiological activities, It is effective for the improvement of the menopausal syndrome according to the hormone imbalance, namely, the weight control, the cholesterol lowering, the improvement of the hyperlipidemia, the alleviation of the arteriosclerosis, the diabetic relief, the improvement of visceral obesity, the improvement of immunity, the improvement of facial flushing, the skin aging prevention and osteoporosis improvement. Therefore, the composition of the present invention is useful as a medicament and a cosmetic material for preventing and improving the menopausal syndrome.

The production method of the present invention produces an unglycosylated isoflavone fortified (containing a high content) bean curd composition by converting at least 70% of a high content isoflavone derivative of the parvaton bean curd into an unglycosyl isoflavone. In addition, the method according to the present invention can greatly improve the utility of the parvaton bean leaf as a processed food, which has not been developed in advance for effective ingredient enhancement.

The food according to the present invention has a high content of non-glycosylated isoflavone and enhanced physiological activity and can prevent or ameliorate menopausal syndromes such as facial flushing, obesity, insomnia, osteoporosis, diabetes and skin aging.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a photograph showing a fermentation composition of parvaton bean curd (Isoflavone derivative high content bean curd) prepared by the method according to the present invention.
2 shows the HPLC chromatogram of the fermented composition of the parvaton bean curd. 2 (a) and 2 (b) show HPLC chromatograms of Comparative Examples 1 to 6, respectively.
Fig. 3 shows antioxidant activities of the fermented bean curd compositions (Examples 1 and 2; Comparative Examples 1 to 6, Reference Example). FIG. 3A shows the DPPH radical scavenging activity of the parvaton bean curd composition, FIG. 3B shows the ABTS radical scavenging activity of the parvaton bean curd fermented composition, and FIG. 3C shows the hydroxyl radical scavenging activity of the parvaton bean curd fermented composition And Fig. 3d shows the FRAP reducing power of the parvaton bean curd fermentation composition.
4 shows digestive enzyme inhibitory activity of the fermented bean curd composition (Examples 1 and 2; Comparative Examples 1 to 6, Reference Example). Fig. 4a shows the alpha-glucosidase inhibitory activity of the fermented bean curd plant, and Fig. 4b shows the pancreatic-lipase inhibitory activity of the fermented bean curd plant.

The present invention will be described in more detail by the following examples. These examples are provided for illustrating the present invention, and the scope of the present invention should not be limited thereby.

Manufacturing example : Farbaton  Preparation of fermented soybean curd composition

The strains for fermentation were selected from two strains of lactic acid bacteria suitable for seedling according to the present invention (KCTC) (Table 1, Examples 1 and 2). For comparison, six strains of lactic acid bacteria Comparative Examples 1 to 6) were used together.

Strain Example 1 Lactobacillus paracasei KCTC 3166 < RTI ID = 0.0 > Example 2 Lactobacillus amylovorus KCTC 3597 < RTI ID = 0.0 > Comparative Example 1 Lactobacillus casei (KCTC 3109) Comparative Example 2 Lactobacillus fermentum KCTC 3112 Comparative Example 3 Lactobacillus delbrueckii KCTC 3635) Comparative Example 4 Lactobacillus eight Simi Nice (Lactobaillus farciminis KCTC 3681) Comparative Example 5 Lactobacillus brevis KCTC 3320 < RTI ID = 0.0 > Comparative Example 6 Lactobacillus plantarum KCTC 13093)

Each lactic acid bacterium was cultured for 24 hours in an MRS liquid medium (Difco, USA), and 5 g of purified water was added to 100 g of germinated malt extract. The extract was then extracted at 100 ° C for 6 hours and filtered through gauze. And cultured for 48 hours to prepare seed culture for inoculation.

The parvaton cannabis was cultivated and treated with espe - phon in August 2015 at the natural chemical laboratory of Gyeongsang National University.

100 g of the parvaton carnauba wax (powder) and 10 g of sugar were mixed with 500 ml of distilled water, and then 9 fermentation bases sterilized at 121 ° C for 15 minutes were prepared. Eight of the fermentation bases were added to the seed culture Were each inoculated at 5% (v / v) and fermented at 37 ° C for 72 hours to complete the fermented soybean curd composition (FIG. 1). The remaining one was kept under the same conditions for 72 hours without inoculation (non-inoculated group; reference example).

Reference example : Physiological activity assay preparation

Each of the parvaton bean curd fermented composition prepared in the Production Example was lyophilized at -70 ° C and powdered. 200 ml of 80% fermented alcohol was added to 5 g of the lyophilized powder sample, and the mixture was extracted at 20 ° C. and 300 rpm for 12 hours, followed by primary filtration through a filter paper, followed by secondary filtration through a 0.45 μm filtration filter. The test sample was used to determine the isoflavone content.

To 20 g of the lyophilized powder sample, 200 ml of 50% methanol was added, and the mixture was stirred at room temperature for 12 hours for extraction. Then, the supernatant was collected by centrifugation and then collected by filtration with a filter paper (No. 2, Whatman, Tokyo Roshi Co.), and the filtrate was concentrated under reduced pressure at 60 ° C using a vacuum concentrator (EYELA, Tokyo, Rikakikai). Concentrated samples were dissolved in extraction solvent to prepare analytical samples at concentrations of 0.1, 0.25, 0.50 and 1.0 mg / ml, respectively. Antioxidative and digestive enzyme inhibitory activities were evaluated in the test.

Test Example  1: Analysis of isoflavone content

Isoflavone content was analyzed by HPLC chromatogram. Specifically, each of the filtrate samples obtained by secondary filtration using a 0.45 μm filter prepared in Reference Example was used. The analytical column was a Lichrophore 100 RP C18 column (4.6 × 250 mm, 5 μm, Merck, Germany) . The mobile phase solvent was analyzed with 0.1% glacial acetic acid in water (solution A) and glacial acetic acid in acetonitrile (solution B) in 100% acetonitrile. 20%, 25% and 35% for 20, 10, 10 and 10 min. 20 μl of the sample was injected and the flow rate of the mobile phase was maintained at 1 mL / min at 30 ° C. The isoflavones were quantified at 254 nm absorbance of the diode array UV detector (Agilent 1200 series, Agilent, USA) and the content was calculated by comparing with the calibration curve of the standard product. The results are shown in Figs. 2A to 2C and Table 2.

As shown in the chromatograms of Figs. 2 (a) to 2 (c), six kinds of isoflavone derivatives (daidzin, genistin, malonyldaidzin, , Malonylgenistin, daidzein and genistein) were detected. The major derivatives detected in the reference example (uninoculated area) were the high content of glycoside isoflavones, daidzin and genistin (peaks 1 and 2), but the fermentation compositions of Examples 1 and 2 Showed that the non-glycoside isoflavones daidzein and genistein (peaks 5 and 6) were greatly increased. In the fermentation compositions of Comparative Examples 1 to 6, the increase of the non-glycoside isoflavone was little or insufficient.

Isoflavone content (μg / g) NIM
Reference Example 3320
Comparative Example 5 3109
Comparative Example 1 3112
Comparative Example 2 3166
Example 1 3597
Example 2 3635
Comparative Example 3 3681
Comparative Example 4 13093
Comparative Example 6 β-Glycosides (glycoside isoflavones) Daidzin 4024.56 1468.66 3146.01 4424.15 585.69 473.46 4684.74 3762.25 1316.57 Genistin 2256.53 540.23 779.70 2231.14 297.89 231.35 2547.31 1263.16 1315.72 Sum 6281.09 2008.90 3925.71 6655.29 883.58 704.81 7232.05 5025.40 2632.29 ratio(%) 73.8 30.9 52.8 76.4 13.9 11.4 78.0 63.1 36.5 Malonyl-β-glycosides (glycoside isoflavones) Daidzin 507.31 505.67 607.20 583.68 517.16 486.13 556.83 600.23 354.76 Genistin 394.15 403.27 364.74 466.59 454.30 214.05 587.04 191.62 147.69 Sum 901.46 908.94 971.94 1050.27 971.46 700.18 1143.86 791.85 502.45 ratio(%) 10.6 13.9 13.1 12.1 15.3 11.3 12.3 9.9 7.0 Aglycones (non-glycoside isoflavones) Daidzein 1055.18 2510.55 1563.21 743.57 3252.06 3464.46 656.48 1357.95 3255.67 Genistein 273.03 1078.28 970.44 264.73 1229.50 1307.62 240.03 783.62 829.15 Sum 1328.21 3588.83 2533.65 1008.30 4481.55 4772.08 896.51 2141.57 4084.81 ratio(%) 15.6 55.2 34.1 11.5 70.8 77.3 9.7 27.0 56.5 total 8510.75 6506.66 7431.30 8713.86 6336.59 6177.07 9272.42 7958.83 7219.55 NIM (non-contact ball); 3320, Lac . brevis KCTC 3320; 3019, Lac . casei KCTC 3109; 3112, Lac . fermentation KCTC 3112; 3166, Lac . paracasei KCTC 3166 ; 3597, Lac . amylovorus KCTC 3597 ; 3635, Lac. delbrueckii KCTC 3635; 3681, Lac . farciminis KCTC 3681; 13093, Lac . plantarum KCTC 13093

As shown in Table 2, the ferbaton bean curd fermentation compositions of Examples 1 and 2 (fermentation with lactobacillus paracase and lactobacillus amyloborus strains, respectively) contained unglycosylated isoflavones of isoflavone derivatives (daidzein and zenithin ) Was contained in 70% or more (occupancy). Therefore, the fermentation composition of the present invention showed 70.8% and 77.3% conversion of glycoside isoflavone to unglycosylated isoflavone, respectively, and about 5 times as much non-glycoside isoform as that of the fermented composition of the reference example It is confirmed that flavon is promoted.

On the other hand, the parvaton bean curd fermented compositions of Comparative Examples 1 to 4 showed a decrease or slightly increased occupancy rate of the unglycosylated isoflavones (daidzein and genistein) in the isoflavone derivatives compared to the reference examples, This was insufficient.

Accordingly, it can be seen that the content of the non-glycoside isoflavones (daidzein and genistein) is significantly enhanced in the parvaton bean curd composition of the present invention fermented with at least one strain comprising Lactobacillus paracasei and Lactobacillus amyloborus.

Test Example  2: Antioxidant activity test

The antioxidative activity of the parvaton bean curd composition according to the present invention was measured by four analytical methods of DPPH radical scavenging activity, ABTS radical scavenging activity and hydroxyl radical scavenging activity, and reducing power (FRAP) analysis.

≪ DPPH radical scavenging activity >

DPPH (1,1-diphenyl-2- picrylhydrazyl, Sigma D9132, FW 393.4, C1 8 H 12 N 5 O 6) is a purple compound showing a specific absorption at 525 nm as an extremely stable radical. DPPH radical scavenging activity was measured to confirm antioxidant activity.

Specifically, 0.8 ml of DPPH solution and 0.2 ml of each analytical sample prepared in Reference Example were added, and the mixture was allowed to stand in a dark room for 30 minutes. After the reaction, the absorbance was measured at 525 nm using a spectrophotometer. For the negative control experiment, PBS buffer (NaCl 8.76 g, NaH ₂ PO ₄ 0.11 g, Na ₂ HPO ₄ 0.596 g) was used instead of the sample, and the radical scavenging activity was determined by the absorbance value between the addition of the analytical sample and the non- (%), And the results are shown in Figure 3a: < RTI ID = 0.0 >

DPPH radical scavenging activity (%) =

    [1- (negative control absorbance / experimental absorbance)] × 100

As shown in FIG. 3A, the DPPH radical scavenging activity of the fermentation compositions of Examples 1 and 2 (fermentation with Lactobacillus paracasei and lactobacillus amyloborus strains, respectively) was greatly enhanced compared to the reference strain (uninoculated strain) , Showing higher activity than the comparative examples.

In particular, the fermented composition of Example 2 showed the highest activity as 74.21%.

<ABTS radical scavenging activity>

The ABTS radical scavenging activity is a method for examining the antioxidant activity of 2-azino-bis according to the degree of reduction of colored radicals. This method is a method to measure the antioxidative activity by comparing with the values of sample and reference material (Trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) (ABTS +) is discarded and discolored in cyan. At this time, the degree of ABTS cations (ABTS +) removal can be known by measuring the absorbance, and the discoloration reaction is completed within 1 minute.

Specifically, ABTS ⁺ radicals were formed by mixing 5 ml of 7 mM ABTS ⁺ 5 ml of 2.45 mM K ₂ S ₂ O _{8 in a} dark room for 12 to 16 hours. After incubation at 732 nm, the absorbance of ABTS ⁺ 0.9 ml was adjusted to 0.7 ± 0.02, and 1.0 ml of each sample prepared in Reference Example was added for 3 minutes to measure the absorbance at 732 nm. In the negative control experiment, PBS buffer (NaCl 8.76 g, NaH ₂ PO ₄ 0.11 g, Na ₂ HPO ₄ 0.596 g) was used instead of the sample, and the radical scavenging activity was determined by the absorbance value between the addition of the sample solution and the no addition, (%), And the results are shown in FIG. 3B.

 ABTS radical scavenging activity (%) =

   [1- (negative control absorbance / experimental absorbance)] × 100

As shown in Fig. 3B, the ABTS radical scavenging activity of the fermentation compositions of Examples 1 and 2 (fermentation with lactobacillus paracase and lactobacillus amyloborus strains, respectively) was greatly enhanced as compared with the reference strain (uninoculated strain) , Showing higher activity than the comparative examples.

In particular, the fermented composition of Example 2 showed the highest activity as 77.56%.

<Hydroxyl radical scavenging activity>

To determine the hydroxyl radical scavenging activity, 0.2 ml of 10 mM FeSO ₄ · 7H ₂ O-EDTA, 0.2 ml of 10 mM 2-deoxyribose, 0.2 ml of 10 mM H ₂ O ₂ and 1.4 ml of each analytical sample prepared in Reference Example The mixture was reacted at 37 DEG C for 4 hours. 1 ml of 1% thiobarbituric acid / distilled water and 2.8% trichloroaceric acid / distilled water were added to the mixture, and the mixture was heated at 100 ° C for 20 minutes to develop color. After cooling appropriately, absorbance at 520 nm Were measured. In the negative control experiment, the PBS buffer solution (8.76 g of NaCl, 0.11 g of NaH ₂ PO ₄ , and 0.596 g of Na ₂ HPO ₄ ) was used, and the hydroxyl radical scavenging activity was determined by subtracting the absorbance difference between the addition of the analytical sample and the non- (%), And the results are shown in FIG. 3C.

Hydroxyl radical scavenging activity (%) =

  1- (negative control absorbance / experiment absorbance)] × 100

As shown in Fig. 3C, the hydroxyl radical scavenging activity of the fermentation compositions of Examples 1 and 2 (fermentation with lactobacillus paracase and lactobacillus amyloborus strains, respectively) was greatly enhanced compared to the reference strain (uninoculated strain) And showed higher activity than the comparative examples.

In particular, the fermented composition of Example 2 showed the highest activity as 56.2%.

<FRAP reducing power>

Ferric reducing antioxidant power (FRAP) is a method of measuring the reducing power of Fe3 + to Fe2 + by measuring the reducing power of a compound. FeII-TPTZ (ferric tripyridyl triazine) To determine the antioxidant activity by measuring the absorbance of TPTZ (ferrous tripyridyl triazine).

Specifically, the reaction solution is 30 mM acetate buffer (pH 3.6), 40 mM 10 mM dissolved in hydrochloric acid, 2,4,6-pyridyl -s- triazine _{(TPTZ, T1253, C 18 H} 12 in the reducing power FRAP analysis N _6, MW312.33) and _{20mM FeCl 3 (F7134, MW 162.20} , the preparation was the in DW), acetate buffer, TPTZ solution and FeCl ₃ solution is 10: 1 were mixed in a 1 (v / v / v) 37 Lt; 0 &gt; C for 15 minutes. 50 μl of each analytical sample prepared in Reference Example and 950 μl of FRAP reagent were dispensed in a test tube and allowed to react for about 15 minutes. Absorbance was measured at 593 nm using a microplate reader (Biorad 3055, Sweden) Respectively.

As shown in FIG. 3D, the hydroxyl radical scavenging activity of the fermentation compositions of Examples 1 and 2 (fermentation with Lactobacillus paracase and Lactobacillus amyloborus strain, respectively) was greatly enhanced compared to the reference strain (uninoculated strain) And showed higher activity than the comparative examples.

In particular, the fermented composition of Example 2 showed the highest activity at 2,270.

From the above results, it can be seen that the antioxidant activity of the parvaton bean curd composition according to the present invention is greatly enhanced.

Test Example 3: Digestive enzyme inhibition assay

The ferbatonobacillus fermentation composition according to the present invention was tested for alpha-glucosidase inhibitory activity, which is an index of anti-diabetic (diabetic) effect, and pancreatic lipase inhibitory activity, which is an index for anti-obesity (improvement of obesity). Alpha-glucosidase inhibitory activity and pancreatic lipase inhibitory activity were assayed by the para-nitrophenol method.

&Lt; Alpha-Glucosidase Inhibitory Activity >

50 μl of each of the analytical samples prepared in Reference Example, 50 μl of α-glucosidase (0.5 U / ml) enzyme solution and 50 μl of 200 mM sodium phosphate buffer solution (pH 6.8) were mixed and preliminarily reacted at 37 ° C. for 10 minutes . After that, 100 μl of p-NPG (5 mM) dissolved in sodium phosphate buffer solution (pH 6.8) was added and reacted at 37 ° C for 10 minutes. After the final reaction was stopped by adding 0.75 ml of Na 2 CO 3 (100 mM) nm were measured using a spectrophotometer. The negative control was taken as the extraction solvent instead of the sample, and the difference in absorbance between the sample with and without the analytical sample was expressed as a percentage (%), and the results are shown in FIG. 4A.

 As shown in Fig. 4A, the alpha-glucosidase inhibitory activity of the fermentation compositions of Examples 1 and 2 (fermentation with lactobacillus paracase and lactobacillus amyloborus strains, respectively) was higher than that of the reference example 2-fold, and exhibited higher activity than Comparative Examples 1 to 4 and 6, respectively.

In particular, the fermented composition of Example 2 showed the highest activity as 39.15%.

Therefore, it can be seen that the fermentation composition according to the present invention significantly enhances the alpha-glucosidase inhibitory activity and thus has an excellent blood glucose lowering activity, thereby improving the diabetic improvement effect.

<Pancreatic-lipase inhibitory activity>

50 μl of each analytical sample prepared in Reference Example, 50 μl of pancreatic lipase (1.0 U / ml) enzyme solution and 50 μl of 200 mM sodium phosphate buffer solution (pH 6.8) were mixed and preliminarily reacted at 37 ° C. for 10 minutes. After the reaction, 100 μl of p-NPB (5 mM) dissolved in sodium phosphate buffer solution was added and reacted for 10 minutes in the same manner. Then, 0.75 ml of 100 mM Na ₂ CO ₃ was added to terminate the reaction and the absorbance was measured at 420 nm. As a negative control, the extraction solvent was used instead of the sample. The difference in absorbance between the addition of the sample solution and the non-addition solution was expressed as a percentage (%), and the results are shown in FIG.

 As shown in Fig. 4B, the pancreatic lipase inhibitory activity of the fermentation compositions of Examples 1 and 2 (fermentation with lactobacillus paracase and lactobacillus amyloborus strains, respectively) was remarkably (approx. 3 times), and showed higher activity than the comparative examples.

In particular, the fermented composition of Example 2 showed the highest activity as 28.54%.

Therefore, it can be seen that the fermentation composition according to the present invention significantly enhances the pancreatic lipase inhibitory activity, thereby enhancing the effect of improving obesity.

Test Example 4: Physicochemical properties and viable cell count

The physicochemical properties (pH, acidity and viable cell count) of the fermented Parvaton bean curd composition according to the present invention prepared in Preparation Example were analyzed and shown in Table 3.

The pH was measured using a pH meter. The acid number was determined by measuring the consumption ml of 0.1 N NaOH required to neutralize 1 ml of the fermented composition to pH 8.4 ± 2 with 0.1 N NaOH solution, and the amount of lactic acid Expressed as% in total. The viable cell count was determined by counting viable cells (log cfu / g) by measuring lactic acid bacteria-specific colonies after 1 ml of the fermented composition was diluted in sterilized physiological saline stepwise and plated on MRSA medium and cultured at 30 ° C for 48 hours.

pH Acidity Viable cell count Reference example (non-vaccinated) 6.30 0.36 - Example 1 ( L. paracasei KCTC 3166) 4.62 1.37 9.82 Example 2 ( L. amylovorus KCTC 3597) 4.31 1.75 10.32 Comparative Example 1 ( L. casei KCTC 3109) 4.81 1.28 9.80 Comparative Example 2 ( L. fermentum KCTC 3112) 4.55 1.53 10.11 Comparative Example 3 ( L. delbrueckii KCTC 3635) 4.28 1.71 10.69 Comparative Example 4 ( L. farciminis KCTC 3681) 4.51 1.64 10.34 Comparative Example 5 ( L. brevis KCTC 3320) 5.06 1.06 9.80 Comparative Example 6 ( L. plantarum KCTC 13093) 4.07 2.21 9.99

As shown in Table 3, the fermentation composition according to the present invention has a reduced pH and an increased acidity and total number of lactic acid bacteria, thereby improving the shelf life of the food. In addition, the lactic acid bacteria product (lactic acid bacterial number is defined as 9.0 log cfu / It is also possible to use as a.

Claims (7)

Isoflavone derivatives High content Soybean leaves are fermented with at least one seed bacterium selected from the group consisting of Lactobacillus paracasei and Lactobacillus amylovorus to produce an isoflavone derivative having 70% Or more of the composition of the present invention,
Characterized in that the high content of the isoflavone derivative is a soya bean having a high content of isoflavone derivatives of 5,000 ~ 15,000 占 퐂 / g by treating the soybean leaves with ethylene or an espefen before and after cultivation.
9. A composition according to claim 1, wherein the non-glycoside isoflavone is fortified.
The bean curd composition of claim 2, wherein the non-glycoside isoflavone is a daidzein and a genistein.
[Claim 4] The composition of claim 3, wherein the composition for bean curd refuse is an estrogen substitute, which is contained in a pharmaceutical or cosmetic material.
A food comprising the non-glycosylated isoflavone-fortified bean curd composition according to claim 2.
A cosmetic comprising the non-glycosylated isoflavone-fortified bean curd composition according to claim 2.
6. The food according to claim 5, wherein the food is effective for preventing or improving the menopausal syndrome.

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