KR102386296B1 - Complex-fermented composition of Fabaton soybean leaves and Schisandra chinensis having enhanced anti-diabetic and anti-obesity effects and preparation method thereof - Google Patents

Abstract

In the present invention, it is prepared by complex fermentation of a mixture of Pabaton soybean leaves and Schisandra with a specific complex seed to enhance digestive enzyme inhibitory activity, non-saccharide isoflavones, omega-6 fatty acids and antioxidant activity, and food processing aptitude (taste, flavor, color) Provided are the enhanced Pabaton soybean leaf and Schisandra complex fermented composition and a method for manufacturing the same.
Fabaton bean leaf and Schisandra complex fermented composition according to the present invention have excellent antidiabetic and obesity improvement effects as alpha-glucosidase inhibitory activity and pancreatic lipase inhibitory activity are significantly improved, and the unique flavor and taste of pabaton bean leaf are improved. It also has excellent palatability. In addition, the compound fermented composition of Fabaton bean leaf and Schisandra Schisandra according to the present invention is fortified with anti-diabetic and anti-obesity activity, antioxidant activity, omega-6 fatty acids, total phenolics and total flavonoids, and non-saccharide isoflavone derivatives to improve diabetes and obesity. It can be usefully used as food, as a food for improving metabolic syndrome and menopausal syndrome.

Description

Complex-fermented composition of Fabaton soybean leaves and Schisandra chinensis having enhanced anti-diabetic and anti-obesity effects and preparation method thereof

The present invention relates to a composite fermented composition of Pabaton soybean leaves and Schisandra Schisandra having enhanced antidiabetic and antiobesity activity, and a method for preparing the same. In more detail, it is manufactured by complex fermentation of a mixture of Pabaton bean leaves and Schisandra Schisandra with a specific complex spawn to enhance digestive enzyme inhibitory activity, non-saccharide isoflavones, omega-6 fatty acids and antioxidant activity, and food processing aptitude (taste, flavor, color ) relates to a composite fermented composition of Pabaton soybean leaves and Schisandra Schisandra, and a method for manufacturing the same.

Compared to soybean (Glycine max. Merr L), soybean leaf is known to be rich in vitamins, although its utilization is somewhat lower. There is no known country that consumes soybean leaves as food, and in Korea, it is mainly used for pickles, side dishes, and ssamjang only in Gyeongsang-do and Jeju-do. Research conducted on soybean leaves is mainly related to bean leaf kimchi or pickled radish, and studies on physiological activity have been conducted on flavonols and polyphenols contained in soybean leaves. As such, studies on various processing aptitudes of soybean leaves are still insufficient.

Isoflavones are flavonol compounds that have already been proven worldwide to be effective for menopausal or bone health. Isoflavone derivatives show several physiological properties comparable to existing phytoestrogens in forming a structure very similar to that of phytoestrogens in chemical structure. In particular, it has been reported to be effective in diabetes and obesity due to the alleviation of menopausal symptoms and the reduction of the risk of osteoporosis after menopause or the reduction of cholesterol, and it is also known to have excellent antioxidant activity. On the other hand, isoflavones present in soybean leaves exist in the form of glucose-bound glycosides and non-saccharides independent of glucose. Unlike soybeans, only daidzein and genistein exist as non-saccharide isoflavones in soybean leaves, and daidzin, malonyl daidzin and genistine exist as glycoside isoflavones. (genistin) and malonyl genistin only exist. These isoflavone derivatives are known not only for their antioxidant activity, but also for genistein to have excellent anticancer effects such as breast cancer, and daidzein to improve osteoporosis.

Recently, a technology for cultivating soybean leaves (Pabaton bean leaves) with a high content of isoflavone derivatives has been developed using molecular farming technology (Patent Registration No. 10-1451298). was not sufficient in Accordingly, there has been a demand for the development of materials and technologies that can be used as health functional foods by supplementing the disadvantages of processing aptitude and enhancing the functionality of Pabaton bean leaves.

Schisandra ( Schisandra ) chinensis ) is a round shape, about 1 cm in diameter, and a dark red fruit containing red juice and 1 to 2 reddish-brown seeds. Omija, so named because it tastes sweet, sour, bitter, salty, and spicy, has six effects, including bronchial improvement, liver function improvement, brain disease prevention, kidney function strengthening, skin improvement, and energy recovery. , In particular, it has been registered as a functional ingredient for relieving symptoms of menopausal women from the Ministry of Food and Drug Safety in December 2016. There are North Schisandra, South Schisandra, and Black Schisandra, which are mainly grown in the Taebaeksan area. It has been reported in academia that South Schisandra grows well in the southern island region and Black Schisandra grows well in Jeju Island. The main active ingredient of Schisandra is a lignan compound, and its derivatives can be classified into schisandrin, schisantherin, schisandrol, and gomisin series. As a result of the study using these functional ingredients of Schisandra, Gochujang with Schisandra (Kim et al. 2003. Korean J. Food Sci. Technol.), tofu (Kim et al. 2008. Korean J. Food Nutr.), and yogurt (Hong) et al. 2004. Korean J. Food Nutr.) has one form, but the complex fermentation technique with Pabaton bean leaf and the remarkable effect thereof are not known.

It is an object of the present invention to provide a complex fermented composition of Pabaton soybean leaf and Schisandra Schisandra with improved digestive enzyme inhibitory activity, antioxidant activity and food processing aptitude (taste, flavor, color),

Another object of the present invention is a method for producing a complex fermented composition of Pabaton soybean leaves and Schisandra with improved digestive enzyme inhibitory activity, non-saccharide isoflavones, omega-6 fatty acids, antioxidant activity and food processing aptitude (taste, flavor, color) is to provide

Another object of the present invention is to provide a functional health food for improving diabetes and/or obesity comprising the complex fermented composition of Pabaton soybean leaves and Schisandra Schisandra.

In order to achieve the above object, Pabaton bean leaf and Schisandra complex with improved digestive enzyme inhibitory activity, non-saccharide isoflavones, omega-6 fatty acids, antioxidant activity and food processing aptitude (taste, flavor, color), including the following steps A method for preparing a fermentation composition is provided:

i) preparing a mixture of pabaton bean leaf and omija in a weight ratio of 7: 3, and

ii) After sterilizing the mixture, inoculating a complex seed of Lactobacillus plantarum P1201 and Lactobacillus brevis WCP02 and performing complex fermentation.

Step i) Pavaton Preparation of bean leaf and Schisandra mixture

Prepare a mixture of pabaton bean leaf and omija in a weight ratio of 7:3.

In the present invention, Pavaton soybean leaves refer to soybean leaves containing high isoflavone derivatives by treating plant growth hormone ethylene, an ethylene donor (Esepone), or an ethylene generator before and after cultivation. Fabaton soybean leaves are commercially available, and the average isoflavone content in Pabaton soybean leaves is about 2,000 to 15,000 μg/g in high concentration.

Pabaton soybean leaves can be harvested and washed and then used immediately after squeezing, or dried and powdered.

In the present invention, omija may be used by juicing immediately after washing, or dried and powdered.

According to the present invention, food processing aptitude (taste, aroma, color) is most appropriately supplemented when omija is mixed with pabaton bean leaf in a weight ratio of 7: 3 (FIG. 1 and Table 1).

ii) Complex Fermentation

After the mixture is sterilized, a complex seed of Lactobacillus plantarum P1201 and Lactobacillus brevis WCP02 is inoculated and subjected to complex fermentation.

The complex spawn used in the complex fermentation in the present invention is Lactobacillus plantarum P1201 deposited under the accession number KACC91848P ( Lactobacillus plantarum ) P1201) strain and Lactobacillus brevis WCP02 deposited with accession number KACC92159P ( Lactobacillus brevis WCP02) strains.

For smooth fermentation, sugar and purified water can be added to the mixture of Pabaton soybean leaves and Schisandra for fermentation. The sugar and purified water are preferably added in an amount of 1 to 50 parts by weight and 500 to 2,000 parts by weight, respectively, based on 100 parts by weight of the mixture of Pabaton bean leaf and Schisandra.

Sterilization can be performed according to a conventional method.

For inoculation, 1 to 10% (v/v) of the complex seed culture medium is inoculated, preferably 5% (v/v). Fermentation is carried out at 20-40° C. for more than 12 hours.

If the inoculation is performed at less than 1% (v/v), the fermentation rate may be delayed, and if it is performed at more than 10% (v/v), the economical effectiveness is lowered. If the fermentation temperature is less than 20℃, the fermentation rate is delayed, and if it exceeds 40℃, the growth of bacteria is inhibited, thereby delaying the fermentation rate and abnormal fermentation may occur. If the fermentation time is less than 12 hours, the bioconversion rate is less than 50%.

When the Pabaton bean leaf and Schisandra Schisandra mixture mixed in a specific weight ratio using the above strains as a complex seed strain were subjected to complex fermentation, it was surprisingly alpha-glucosidase inhibitory activity (anti-diabetic ) and pancreatic lipase inhibitory activity (anti-obesity) were significantly enhanced (FIGS. 2a, 2b).

According to another object of the present invention, there is provided a composite fermented composition of Pabaton bean leaf and Schisandra Schisandra, prepared by the above manufacturing method.

Fabaton bean leaf and Schisandra complex fermented composition according to the present invention has alpha-glucosidase inhibitory activity of 75% or more and pancreatic lipase inhibitory activity of 46% or more, so it is useful as a food material for improving diabetes and obesity (Fig. 2a, 2b).

In addition, in the Pabaton bean leaf and Schisandra complex fermented composition according to the present invention, oleic acid and linoleic acid, which are omega-6 fatty acids, were significantly improved to 337 mg/100 g or more and 1720 mg/100 g or more, respectively (Table 2).

The Fabaton bean leaf and Schisandra complex fermented composition according to the present invention has improved reducing sugar and protein content (Table 3), GABA (gamma-aminobutyric acid) and essential amino acid content (Table 4), total phenolics and total flavonoids The content was also increased (Table 5).

The manufacturing method of the Pabaton bean leaf and Schisandra complex fermented composition according to the present invention is a non-saccharide isoflavone derivative, i.e., daid, by performing complex fermentation using the specific complex strain as described above. More than 40% conversion to zein and genistein (Table 6).

Fabaton bean leaf and Schisandra complex fermented composition according to the present invention contained 348 μg/g or more of daidzein and 119 μg/g or more of genistein, so that non-saccharide isoflavone derivatives were remarkably strengthened (Table 6).

Fabaton bean leaf and Schisandra complex fermented composition according to the present invention also enhanced antioxidant activity (Table 7).

According to another object of the present invention, it is an object of the present invention to provide a food for improving diabetes and/or obesity comprising the Pabaton bean leaf and Schisandra complex fermented composition.

The food according to the present invention may be prepared by adding the Pabaton bean leaf and Schisandra complex fermented composition as it is or by using an extract thereof and mixing it with other food ingredients, and may be appropriately prepared according to a conventional method.

In the present invention, the type of the food is not particularly limited, and may be soybean leaf products (pills, tablets, capsules, etc.), fermented tea, powders and granules, fermented beverages (pouches, drinks, etc.).

The food according to the present invention contains a very high content of non-saccharide isoflavone derivatives, including the complex fermented composition of Pabaton soybean leaves and Schisandra, so it can be used as a food for relieving menopause.

Fabaton bean leaf and Schisandra complex fermented composition of the present invention is excellent in the improvement of diabetes and obesity as the alpha-glucosidase inhibitory activity and pancreatic lipase inhibitory activity are significantly enhanced. In addition, the Pabaton bean leaf and Schisandra complex fermented composition according to the present invention has improved palatability and flavor unique to the Pabaton bean leaf.

In addition, the compound fermented composition of Fabaton bean leaf and Schisandra Schisandra according to the present invention has anti-diabetic, anti-obesity activity, non-saccharide isoflavone derivatives, omega-6 fatty acids, antioxidant activity, total phenolics and total flavonoids for improving diabetes and obesity. It can be usefully used as a health food, as a health functional food for improving metabolic syndrome and menopausal syndrome.

1 is a photograph showing the process of selecting the optimal mixing ratio of soybean leaves and Schisandra for the preparation of the Pabaton bean leaf and Schisandra complex fermented composition of the present invention.
Figure 2 is a graph showing the alpha-glucosidase inhibitory activity (Fig. 2a) and the pancreatic lipase inhibitory activity (Fig. 2b) of the combination fermented composition of Fabaton bean leaf and Schisandra of the present invention.
Figure 3 shows the HPLC chromatogram of the mixture of Pabaton soybean leaves and Schisandra omija before complex fermentation.
Figure 4 shows the HPLC chromatogram of the composition of Pabaton soybean leaf and Schisandra Schisandra complex fermented according to the present invention.

The present invention is further illustrated by the following examples. These examples are for the purpose of illustrating the present invention, and the scope of the present invention should not be limited thereto.

production example : Pavaton Manufacture of composite fermented composition of bean leaf and Schisandra Schisandra

The seed strains Lactobacillus plantarum P1201 or Lactobacillus brevis WCP02 used for fermentation were purchased from the Korea Genetic Bank (KCTC) and used individually or in combination as a seed strain in the present invention.

Pabaton soybean leaves were grown and harvested by Esepone treatment at Gyeongsang National University's Natural Product Chemistry Lab, and dried with hot air and powdered were used. Omija was grown in Geochang-gun, Gyeongsangnam-do, dried, and powdered was used.

Prior to the preparation of the Pabaton bean leaf and Schisandra mixed fermentation composition, each Pabaton bean leaf and Schisandra leaf were mixed in a ratio of 9:1, 8:2, 7:3, 6:4, respectively, and divided into tea bags and brewed in hot water ( Fig. 1). This sensory evaluation was conducted on 30 undergraduate students at Kyungnam University of Science and Technology, and as a result, in the case of 9:1 and 8:2 ratios, the amount of Pabaton soybean leaf added was high and the amount of Schisandra Schisandra was low. It obtained a low score and was excluded from the optimal mixing ratio due to its strong astringent taste. In the case of 7:3 ratio of Pabaton bean leaf and Schisandra material, the sour taste of Omija could be felt moderately, and the astringent taste could not be felt. When mixing at this mixing ratio, the unique aroma and astringency of Pabaton bean leaves were recalled and a suitable acidity and flavor could be produced. The correct mixing ratio for food processing aptitude was finally selected as a 7:3 ratio of Pabaton bean leaves and Schisandra (Table 1). ).

Sensory evaluation according to the mixing ratio of Pabaton soybean leaves and Schisandra Schisandra Item ^{1 )} Mixing ratio (Pabaton bean leaf: Omija) 9:1 8:2 7:3 6:4 Sour taste 0 0 3 3 bitterness 0 0 0 0 color 0 0 2 2 incense One 2 3 2 bitter taste 5 5 0 0 total score 6 7 8 7 ¹⁾ It was conducted for 30 students.

Based on the final selected mixing ratio (7:3), a fermented composition of Pabaton bean leaf and Schisandra Schisandra mixture was prepared.

Specifically, a mixture (5 g) of Pabaton soybean leaves and omija (5 g) and sugar (2 g) are placed in a 250 ml glass bottle, 95 ml of purified water is added and mixed, and the sterilization process is performed at 121°C for 15 minutes, followed by Lactobacillus Plantarium P1201 strain and Lactobacillus brevis WCP02 strain were combined and cultured for 48 hours in MRS liquid medium (Difco, USA), inoculated with 5% (v/v), and then fermented for 72 hours at 37 ° C. A composite fermentation composition according to the invention (Example 1) was prepared.

For comparison, each, put a mixture (5 g) of Pabaton bean leaf and Schisandra leaf and sugar (2 g) in a 250 ml glass bottle, add and mix 95 ml of purified water, and perform a sterilization process at 121°C for 15 minutes , Lactobacillus plantarum P1201 strain alone or Lactobacillus brevis WCP02 strain alone was cultured for 48 hours in MRS liquid medium (Difco, USA), 5% (v/v) after inoculation at 37°C for 72 hours By stationary fermentation, Lactobacillus plantarum P1201 single fermentation composition (Comparative Example 1) and Lactobacillus brevis WCP02 strain single fermentation composition (Comparative Example 2) were prepared.

In addition, the samples of each of Example 1, Comparative Example 1 and Comparative Example 2 fermentation 0 hours (non-inoculated) were maintained at 37° C. for 72 hours (Reference Example).

Reference Example: Preparation of samples for analysis of anti-diabetic and anti-obesity efficacy

Each of the Pabaton bean leaves and Schisandra fermented compositions (Example 1, Comparative Example 1, Comparative Example 2, Reference Examples) prepared in Preparation Example were freeze-dried and then powdered. After adding 40 ml of 80% fermented alcohol to 1 g of the freeze-dried powder sample, extraction was performed at 20° C. and 300 rpm for 12 hours, centrifuged, and the filtrate obtained by filtering the supernatant through a 0.45 μm filter was used as an analysis sample.

test example One: antidiabetic and anti-obesity Efficacy measurement

Anti-diabetic and anti-obesity efficacy was conducted based on the enzyme model. The anti-diabetic enzyme model is alpha-glucosidase and the anti-obesity enzyme model is pancreatic lipase. These anti-diabetic and anti-obesity effects were tested by the para-nitrophenol method.

<alpha-glucosidase inhibitory activity>

50 μl of each analysis sample prepared in Reference Example, 50 μl of alpha-glucosidase (0.5 U/ml) enzyme solution, and 50 μl of 200 mM sodium phosphate buffer (pH 6.8) were mixed and pre-reacted at 37° C. for 10 minutes. . After that, 100 μl of p-NPG (5 mM) dissolved in sodium phosphate buffer (pH 6.8) was added and reacted at 37° C. for 10 minutes, and 0.75 ml of Na ₂ CO ₃ (100 mM) was added to the reaction solution to stop the final reaction. Then, the absorbance was measured at 420 nm using a spectrophotometer. For the negative control, distilled water was taken instead of the sample, and the difference in absorbance between the group with and without the addition of the analysis sample was expressed as a percentage (%), and the results are shown in FIG. 2A.

As shown in Fig. 2a, the alpha-glucosidase inhibitory activity of the fermentation compositions of Comparative Examples 1 and 2 (fermented with Lactobacillus plantarium P1201 and Lactobacillus brevis WCP02 single strains, respectively) was a reference example (no inoculation). Although slightly increased compared to the previous), in the Pabaton bean leaf and Schisandra complex fermented composition of Example 1 (fermented with Lactobacillus plantarium P1201 and Lactobacillus brevis WCP02 complex strains), it was significantly improved by about 2 times (75% or more).

Therefore, it can be seen that the Fabaton bean leaf and Schisandra fermented composition according to the present invention significantly improved the alpha-glucosidase inhibitory activity and thus had an excellent diabetes improvement effect.

<pancreas lipase inhibitory activity>

50 μl of each assay 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 (pH 6.8) were mixed and pre-reacted at 37° C. for 10 minutes. After the reaction, 100 μl of p-NPB (5 mM) dissolved in sodium phosphate buffer was added and reacted for 10 minutes. Then, 0.75 ml of 100 mM Na ₂ CO ₃ was added to terminate the reaction, and absorbance was measured at 420 nm. For the negative control, distilled water was taken instead of the sample, and the difference in absorbance between the addition and the non-addition group of the sample solution was expressed as a percentage (%), and the results are shown in FIG. 2B.

As shown in FIG. 2b , the pancreatic lipase inhibitory activity of the fermentation compositions of Comparative Examples 1 and 2 was 37% and 35%, respectively, but in the case of the complex fermentation composition of Example 1, inhibition of 46% or more activity was markedly improved.

Therefore, it can be seen that the Fabaton soybean leaf and Schisandra complex fermented composition according to the present invention has an effect of improving obesity as the pancreatic lipase inhibitory activity is enhanced.

test example 2: Pavaton Fatty acid composition of soybean leaf and Schisandra complex fermented composition

The Pabaton bean leaf and Schisandra complex fermented composition (Example 2) and the Pabaton bean leaf and Schisandra mixture raw material (Comparative Example 3), which were prepared by complex fermentation of 1 kg of Pabaton bean leaf and Schisandra mixture in the same manner as in Preparation Example A fatty acid content comparative analysis process was performed.

Fatty acid analysis was carried out by methyl esterification of fatty acids using 12 N HCl. Each of the freeze-dried powders of Comparative Example 3 and Example 2 was hydrolyzed at 100° C. for 10 minutes by adding 5 ml of 6 N HCl hydrochloric acid to 100 mg of each. Then, 2 ml of boron trifluoride (BF ₃ ) was added, stirred and heated again for 30 minutes to proceed with fatty acid methyl esterification. After completion of the reaction, 1 ml of isooctane was added to cause layer separation, dehydrated with anhydrous sodium sulfite, filtered through a 0.45 μm filter, and analyzed by gas chromatography according to Food Standards. The results are shown in Table 2.

Unsaturated fatty acids Fatty acid content (mg/100 g) ^{1 )} Comparative Example 3 Example 2 Oleic acid (C18:1) 295.10 337.90 Linoleic acid (C18:2) 1573.70 1720.00 Total 2170.80 2363.10 ¹⁾ All items were repeated 3 times.

As shown in Table 2, the omega-6 fatty acids oleic acid and linoleic acid in Example (complex fermentation composition) were significantly increased to 337 mg or more and 1720 mg/100 g or more, respectively, compared to Comparative Example 3 (raw material).

test example 3: Pavaton Evaluation of Physicochemical Characteristics of Soybean Leaf and Schisandra Schisandra Complex Fermentation Composition

Physicochemical properties of each composition prepared in Test Example 2 (Comparative Examples 3 and 2) were evaluated by measuring pH, acidity, reducing sugar, protein content, and number of viable cells.

The pH was recorded by contacting each sample with an electrode using a pH meter (MP 220, London, UK). The acidity was calculated as the number of ml required to neutralize to pH 8.3 with 0.1 N NaOH solution for 10 ml of the sample, and converted into the final amount of lactic acid and expressed as a percentage (%). For reducing sugar, a certain amount of each sample is centrifuged, 1 ml of DNS reagent is added to 0.1 ml of an appropriately diluted solution, color developed at 100° C. for 30 minutes, rapidly cooled by immersion in ice water, and absorbance measured at 570 nm. was substituted into the calibration curve prepared using glucose and expressed as g/L. For protein content, 1 ml of distilled water was added to 0.1 g of lyophilized powder, mixed, and 4 ml of biuret reagent was added, followed by reaction at 37° C. for 20 minutes. After the reaction was completed, centrifugation was carried out appropriately, and the absorbance value of only the supernatant was measured at 540 nm. At this time, it was substituted into the standard calibration curve prepared using bovine serum albumin and expressed as mg/g. The number of viable cells was obtained by diluting each sample step-by-step with sterile physiological saline, plated on a MRS lactic acid bacteria-only detection medium, and the colonies that appeared after incubation at 37°C for 24 hours were measured with a counter and expressed as cfu. Each measurement result is shown in Table 3.

Sample Item ^{1 )} Number of viable cells (log cfu/g) pH total acidity
(lactic acid, %) reducing sugar
(g/L) protein
(mg/g) P1201 WCP02 total number of bacteria Comparative Example 3 4.00 0.17 1.82 7.31 nd ^{2 )} nd nd Example 2 3.82 0.19 2.75 7.00 4.59 6.58 11.17 ¹⁾ All items were repeated 3 times. ²⁾ nd: not detected.

The pH decreased to 3.82 in Example 2, the fermentation composition, and the acidity slightly increased. The reducing sugar increased to 2.75 g/L in Example 2 than in Comparative Example 3 (1.82 g/L). The protein content showed a similar content to Comparative Example 3 without any difference according to the fermentation process, and the number of viable cells was increased.

test example 4: Pavaton Soybean leaf and Schisandra complex fermented composition free amino acids compositional analysis

Free amino acid analysis was performed with an automatic amino acid analyzer. Specifically, each of the powder of Comparative Example 3 prepared in Test Example 2 and 100 mg of the powder of Example 2 were precisely weighed in a test tube, 4 ml of distilled water was added thereto, and a hydrolysis process was performed at 60° C. for 1 hour. To this, 2 ml of 5-sulfosalicylic acid was added, and the protein was precipitated by allowing it to stand for 2 hours. After centrifugation for about 3 minutes, only the supernatant was completely evaporated under reduced pressure at 60°C. To the remaining filtrate, 2 ml of lithium citrate buffer (pH 2.2) was added and dissolved, and the filtrate filtered through a 0.45 μm filter was injected into an automatic amino acid analyzer to analyze free amino acids, and the results are shown in Table 4.

As shown in Table 4, 5 types of non-essential amino acids and 5 types of essential amino acids were detected, and the amino acid content of Example 2 was increased compared to Comparative Example 3. In non-essential amino acids, GABA (γ-aminobutyric acid) was increased to 126.01 mg/100 g in Example 2, and among essential amino acids, valine was similarly increased in Example 2 (80.06 mg/100 g) compared to Comparative Example 3 has been greatly improved in

Content (mg/100 g) ^{1 )} Sample Comparative Example 3 Example 2 Non-essential amino acids Proline 113.92 144.41 Aspartic acid 99.04 394.85 Serine 38.64 50.56 Glycine 9.03 19.89 γ-aminobutyric acid 112.84 126.01 Total 373.47 735.72 essential amino acids Threonine 26.35 40.95 Valine 66.00 80.06 Isoleucine 41.73 54.27 Leucine 36.39 45.75 Phenylalanine 39.44 53.12 Total 209.91 274.15 Total amino acids 583.38 1009.87 ¹⁾ All items were repeated 3 times.

test example 5: Pavaton Analysis of physiologically active substances of soybean leaf and Schisandra complex fermented composition

For the analysis of physiologically active substances, the analytical samples of Comparative Examples 3 and 2 were prepared based on the method prepared in Reference Example. Total phenolics and total flavonoid content were measured through a colorimetric method of measuring absorbance values at 750 and 420 nm for each of the analytes.

<Gun phenolics and total flavonoids>

Determination of total phenolics content is widely known, and the Folin-Denis method is slightly modified. 0.5 ml of each analysis sample was dispensed into a test tube, 0.5 ml of a 25% Na ₂ CO ₃ solution was added, and after reaction for 3 minutes, 0.25 ml of 2 N Folin-Ciocalteu phenol solution was dispensed and the color reaction was performed at 37° C. for 1 hour. The absorbance of each colored sample was measured at 750 nm, and the value was calculated in mg/g by substituting it into a standard calibration curve prepared from gallic acid, and the results are shown in Table 5.

Comparative Example 3 Example 2 total phenolics 2.21 2.74 total flavonoids 2.18 2.87 ¹⁾ All items were repeated 3 times.

As shown in Table 5, the total phenolics content was increased to 2.74 mg/g in Example 2 compared to Comparative Example 3.

To measure the total flavonoid content, 0.5 ml of each assay sample was taken in a test tube, 1.0 ml of diethylene glycol solution was added thereto, and 0.01 ml of 1 N NaOH was dispensed thereto and reacted at 37° C. for 1 hour. Thereafter, absorbance was measured at 420 nm, and the value was calculated in mg/g by substituting it into a standard calibration curve prepared from rutin, and the results are shown in Table 5.

As shown in Table 5, it can be seen that the total flavonoid content is also significantly increased to 2.87 mg/g in Example 2 compared to Comparative Example 3.

test example 6: Pavaton Analysis of Isoflavone Content of Soybean Leaf and Schisandra Schisandra Complex Fermentation Composition

Isoflavone content analysis was analyzed using HPLC chromatogram. Specifically, the analytical samples of Comparative Examples 3 and 2 prepared based on Reference Examples were used, and the Lichrophore 100 RP C18 column (4.6×250 mm, 5 μm, Merck, Germany) was used as the analytical column. Mobile phase solvents were analyzed with HPLC grade H ₂ 0 (A) and acetonitrile (B), and mobile phase conditions were maintained at 10%, 20%, 25% and 35% for 20, 10, 10 and 10 minutes, respectively, on B basis. made it 20 μl of each assay sample was injected and the mobile phase was applied at a flow rate of 1 ml per minute at 30°C. Isoflavones were quantified at absorbance at 254 nm of a diode array UV detector (Agilent 1200 series, Agilent, USA), and the content (㎍/g) was calculated by comparing with the calibration curve of the standard product. Each result is shown in Table 6 and FIG. 3 (Comparative Example 3) and FIG. 4 (Example 2).

Comparative Example 3 Example 2 glycosides (glycosides) Dairied Jeans 746.30 601.18 genistine 439.03 296.30 content 1,185.33 897.48 Glycoside (malonylglycoside) Dairied Jeans 731.49 558.16 genistine 557.31 418.81 content 1,288.80 976.97 Aglycone (aglycone) Daidzein 100.14 348.00 Genistein 64.68 119.68 content 164.82 467.68 ¹⁾ All items were repeated 3 times.

As shown in Table 6. The glycoside content of the glycoside of Comparative Example 3 was 1,185.33 μg/g, but decreased to 897.48 μg/g in Example 2. The glycoside malonylglycoside was also reduced to 976.97 μg/g in Example 2.

On the other hand, it is confirmed that the aglycone content of the aglycone form is increased by about 2.8 times or more, from 164.82 μg/g (Comparative Example 3) to 467.68 μg/g. In particular, Example 2 contained 348 µg/g or more of daidzein and 119 µg/g or more of genistein.

As shown in the chromatograms of FIGS. 3 and 4 , in Comparative Example 3 ( FIG. 3 ) and Example 2 ( FIG. 4 ), four types of glycoside isoflavones (daidzine, genistine, malonyldaidzine) , malonylgenistine) and two non-saccharide isoflavone (daidzein, genistein) derivatives were detected. In the complex fermentation composition of Example 2, it can be seen that the peaks 5 and 6, daidzein and genistein in the form of non-saccharides, were significantly increased.

Therefore, the Pabaton soybean leaf and Schisandra complex fermented composition according to the present invention, which is complex fermented with two complex strains consisting of Lactobacillus plantarum P1201 and Lactobacillus brevis WCP02, has a higher content of non-saccharide isoflavones (daidzein and genistein). It can be seen that it is significantly strengthened.

test example 7: Pavaton Analysis of antioxidant activity of soybean leaf and omija complex fermented composition

Antioxidant activity analysis was performed by methods of DPPH, ABTS, hydroxyl radical scavenging activity and FRAP reducing power.

< DPPH Radical scavenging activity>

DPPH (1,1-diphenyl-2-picrylhydrazyl) is a very stable radical and is a purple compound exhibiting maximum absorbance at 517-525 nm.

In more detail, 0.8 ml of the DPPH solution and 0.2 ml of the analysis samples of Comparative Example 3 and Example 2 prepared as in Reference Example were added, respectively, and reacted in the dark for 30 minutes. Absorbance was measured at 525 nm using a spectrophotometer. Distilled water was used as a negative control, and the difference in absorbance was obtained and expressed as a percentage (%) by substituting it in the following formula, and the results are shown in Table 7:

Radical scavenging activity (%) = [1-(absorbance of negative control ÷ absorbance of experimental group)] × 100

Comparative Example 3 Example 2 DPPH scavenging activity 38.14 55.67 ABTS scavenging activity 43.64 67.99 Hydroxyl scavenging activity 37.90 46.66 FRAP (OD 593 nm) 1.36 1.83

As a result, it can be seen that the DPPH radical scavenging activity of Comparative Example 3 was 38% and that of Example 2 was significantly increased to 55%.

< ABTS Radical scavenging activity>

ABTS radical scavenging activity is a method of examining antioxidant activity according to the degree of reduction of the colored radical of 2-azino-bis. This method is a method of measuring the antioxidant activity by comparing the value of the sample and the standard material (Trlox, 6-hydroxyl -2,5,7,8-tetramethylchroman-2-carboxylic acid). (ABTS+) is removed and the color is decolorized into cyan. At this time, the degree of removal of the ABTS cation (ABTS+) can be known by measuring the absorbance.

Specifically, 7 mM ABTS ⁺ 5 ml and 2.45 mM K2S2O8 5 ml were mixed and left in the dark for 12 to 16 hours to form ABTS ⁺ radicals. Thereafter, ABTS ⁺ 0.9 ml adjusted so that the absorbance value of the control was 0.7±0.02 at 732 nm and 1.0 ml of each analysis sample were added and reacted for 3 minutes to measure the absorbance at 732 nm. Distilled water was used as a negative control, and the difference in absorbance was obtained and expressed as a percentage (%) by substituting it in the above formula, and the results are shown in Table 7.

As shown in Table 7, it can be seen that the ABTS radical scavenging activity of Example 2 is greatly increased to 67% compared to Comparative Example 3.

< hydroxyl Radical scavenging activity>

Hydroxyl radical scavenging activity was measured by mixing 0.2 ml of 10 mM FeSO4.7H20-EDTA, 0.2 ml of 10 mM 2-deoxyribose, 0.2 ml of 10 mM H2O2, and 1.4 ml of each assay sample, followed by reaction at 37° C. for 4 hours. To this mixture, 1 ml of each of 1% thiobarbituric acid (TBA) and 2.8% trichloroaceric acid (TCA) was added, heated at 100° C. for 20 minutes to develop color, and after cooling appropriately, absorbance at 520 nm was measured. Distilled water was used as a negative control, and the difference in absorbance was obtained and expressed as a percentage (%) by substituting it in the above formula, and the results are shown in Table 7.

As shown in Table 7, the hydroxy radical scavenging activity was also enhanced in Example 2.

<FRAP (ferric reducing antioxidant power) reducing power>

FRAP reducing power was measured in 30 mM acetate buffer (pH 3.6), 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ, T1253, C18H12N6, MW312.33) and 20 mM FeCl3 (F7134) dissolved in 40 mM hydrochloric acid. , MW 162.20, in DW) was prepared first, and an acetate buffer, TPTZ solution, and FeCl3 solution were mixed at a ratio of 10:1:1 (v/v/v), and a preliminary reaction was performed at 37° C. for 15 minutes. After dispensing 50 μl of each assay sample and 950 μl of FRAP reagent into a test tube, the reaction was performed at 37° C. for 15 minutes, and the absorbance value was measured at 593 nm using a spectrophotometer as in antioxidant activity. Distilled water was used as a negative control, and the difference in absorbance was obtained and expressed as a percentage (%) by substituting it in the above formula, and the results are shown in Table 7.

As shown in Table 7, compared to Comparative Example 3 (1.36), Example 2 (1.83) also improved reducing power.

From the above results, it can be seen that the complex fermented composition of Pabaton bean leaf and Schisandra Schisandra according to the present invention also enhances antioxidant activity.

Combining the above results, the Pabaton bean leaf and Schisandra complex fermented composition according to the present invention have physiologically active substances (total phenolics, total flavonoids, non-saccharide isoflavones), antioxidant activity and digestive enzyme inhibitory activity. It can be usefully used as a food for preventing metabolic syndrome.

Agricultural Biotechnology Research Institute KACC91848P 20130719 Agricultural Biotechnology Research Institute KACC92159P 20161212

Claims (7)

A method for producing a complex fermented composition of Pabaton soybean leaves and Schisandra, the method comprising:
i) preparing a mixture of pabaton bean leaf and omija in a weight ratio of 7: 3, and
ii) After sterilizing the mixture, inoculating a complex seed of Lactobacillus plantarum P1201 strain deposited with accession number KACC91848P and Lactobacillus brevis WCP02 strain deposited with accession number KACC92159P and performing complex fermentation,
The complex fermentation composition has 75% alpha-glucosidase inhibitory activity and 46% pancreatic-lipase inhibitory activity, and contains 337 mg/100 g of oleic acid and 1720 mg/100 g of linoleic acid. .
The method of claim 1, wherein the glycoside isoflavones contained in Fabaton soybean leaves are converted to 40% non-saccharide isoflavones.
A food comprising the complex fermented composition of Pabaton soybean leaves and Schisandra Schisandra produced by the method according to claim 1 .
The food according to claim 3, characterized in that it contains 348 μg/g of daidzein and 119 μg/g of genistein. delete delete delete

Images