KR20190020381A - Preparation method for soybean-fermented composition having enhanced GABA, Conjugated linoleic acid and Aglycone isoflavones, combined probiotics for its fermentation and functional food of anti-diabetic and anti-obesity effects containing the soybean-fermented composition - Google Patents

Abstract

The present invention provides a method for producing a Glycine max fermented composition fortified with GABA, conjugated linoleic acids and isoflavone aglycones; complex probiotics used therefor; and anti-diabetic and anti-obesity functional foods containing the Glycine max fermented composition as an active ingredient. The Glycine max fermented composition according to the present invention ensures significantly increased content of GABA, conjugated linoleic acids, and isoflavone aglycones (daidzein and genistein), has an excellent total content of phenolics, antioxidant activity, and digestive enzyme (hydrolyzing enzymes of carbohydrate and lipid) inhibitory activity, thereby having excellent anti-diabetic and anti-obesity effects and capable of being used as a material for functional foods and pharmaceuticals.

Description

The present invention relates to a method for preparing a soybean fermented composition enhanced with soybean, ganoderma, conjugated linoleic acid and non-glycosylated isoflavone, a combined prophylactic agent used therefor, and an antidiabetic and anti-obesity functional food containing the fermented soybean composition as an active ingredient. composition having enhanced GABA, conjugated linoleic acid and aglycone isoflavones, combined probiotics for fermentation and functional food of anti-diabetic and anti-obesity effects containing the soybean-fermented composition,

The present invention relates to a process for producing a soybean fermentation composition in which GABA, conjugated linoleic acid and non-glycosylated isoflavone are promoted, a combined prophylactic agent used therefor and an antidiabetic and anti-obesity functional food containing the fermented soybean composition as an active ingredient. More specifically, the present invention relates to a method for producing a soybean fermentation composition in which both Gabon, conjugated linoleic acid and non-glycosylated isoflavone are enhanced by using a complex prophylactic agent of Lactobacillus brevis WCP02 and Lactobacillus plantarum P1201 as a seed, and a method for producing a soybean fermented composition comprising Lactobacillus brevis WCP02 and lactose A complex prophylactic agent of Bacillus plantarum P1201, and an enhanced fermented soybean composition containing enhanced gabas, conjugated linoleic acid and non-glycosylated isoflavones as an active ingredient, and to an excellent antidiabetic and anti-obesity functional food.

As the diet has become westernized, the amount of calories consumed by adults has increased steadily, and metabolic diseases such as stroke, atherosclerosis, hypertension, diabetes and obesity have increased along with excess calories. In general, obesity is caused by accumulation of adipose tissue by fat cell formation process such as differentiation. As a result, it induces various hormone resistance such as insulin, which is considered as a cause of type 2 diabetes or cardiovascular disease.

Probiotics refers to 'active live bacteria culture', meaning 'live bacteria', ie live bacteria, which keep the host's gastrointestinal tract healthy, such as humans and animals. It also means microorganisms that live competitively from top to bottom in the most barren environment of the human body. These probiotics are commonly known as lactic acid bacteria and have been used in a variety of food fermentations. It is a microorganism that is generally recognized as safe through long history of intake. It is known that antimicrobial substances secreted by the probiotics by ingestion thereof have a great effect on the prevention of harmful bacteria, stabilization of intestinal flora, intolerance of lactose, and improvement of colon diseases such as irritable bowel syndrome.

Gamma-aminobutyric acid or GABA is a non-monoclonal amino acid composed of four carbon atoms, which is converted by glutamate decarboxylase (GADase) to glutamic acid (GA) Together with the release of carbon dioxide, it is converted to GABA. In addition to its role as a neurotransmitter in the brain, GABA has a wide variety of physiological functions such as brain function promotion, mental stabilization action, blood pressure lowering action, diuretic action, liver function improvement action, obesity prevention action, alcohol metabolism acceleration action, It is also used as a medicine to treat headaches, tinnitus, and decreased motivation due to stroke or cerebral artery sequelae. GABA is the first grade of health functional food raw material with a daily intake of 20 mg. Generally, it causes the generation of GABA in the body from aging due to excessive intake of inappropriate food or additives, insufficient intake of vitamins and minerals, and so on.

Conjugated Linoleic Acid (CLA) is an isomer of linoleic acid and has a conjugated double bond structure. It has anticancer activity, reduction of body fat, prevention and alleviation of arteriosclerosis, blood total- and LDL-cholesterol and TG (triglyceride; triglyceride) (PARK YK et al., 1997 Lipids 32: 853-858; McCarty MF 2000. Medical hypotheses 55: 187-188). The conjugated linoleic acid (CLA) is a raw material of the first grade of health functional food of the Ministry of Health, Food and Drug Administration 'helping reduce body fat' physiological activity, and the daily intake is set to 1.5 to 3 g. The conjugated linoleic acid is chemically synthesized or produced by microorganisms. When chemically synthesized, expensive equipments are required, or the process takes too much time, and there is a possibility that all the isomers of conjugated linoleic acid (CLA) are produced, which is inefficient. Therefore, it has been attempted to produce foods in which conjugated linoleic acid is increased through the process of converting linoleic acid into conjugated linoleic acid by microorganisms (Patent No. 10-1201184).

Isoflavones exist in the form of the glycoside isoflavone glycosides in their natural state. The glyoxylated isoflavones are not absorbed in the body because they are not decomposed by the gastric acid, and they are hydrolyzed by enzymes secreted by microorganisms existing in the large intestine The absorption rate is low. On the other hand, isoflavones in the form of non-glycosides in which sugar is removed by microbial fermentation or enzymatic reaction are not only directly absorbed in stomach and small intestine, but also have a remarkably high absorption rate (Patent No. 10-1498919). The Ministry of Health, Welfare and Food and Drug Administration has set up 24 ~ 27 mg daily intake of non-glycoside isoflavones (total amount of daidzein, glycitein and genistein) .

Therefore, the necessity and usefulness of the probiotic agent and the fermented food prepared by using the active ingredient of food, which are capable of simultaneously promoting gaba, conjugated linoleic acid and nonisaccharide isoflavone, have been greatly increased.

Accordingly, the present inventors have found that soybeans contain more than 15% of protein in glutamic acid (GA), more than 50% of fat in linoleic acid (LA) and more than 95% of isoflavones in glycoside isoflavone The present inventors have developed a probiotic agent capable of simultaneously converting glutamic acid, linoleic acid and glycoside isoflavones of soybean into gabas, conjugated linoleic acid and non-glycosylated isoflavones, and by using the thus-obtained probucol, conjugated linoleic acid and non-glycoside isoflavones To prepare a fermented soybean composition, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a process for producing a soybean fermentation composition in which all of gaba, conjugated linoleic acid and nonisaccharide isoflavone are enhanced.

Another object of the present invention is to provide a fermented soybean composition having improved contents of gabas, conjugated linoleic acid and nonisaccharide isoflavones prepared by the above method.

It is still another object of the present invention to provide a complex prophylactic agent composition of Lactobacillus brevis WCP02 and Lactobacillus plantarum P1201 which can be used as a fermentation seed of the above production method.

It is still another object of the present invention to provide an antidiabetic and anti-obesity functional food comprising the fermented soybean composition containing enhanced gabas, conjugated linoleic acid and non-glycoside isoflavones as an active ingredient.

In order to accomplish the above object, the present invention provides a soybean fermentation composition containing enhanced gabas, conjugated linoleic acid and non-glycosylated isoflavones by fermenting soybean with a combination prodrug of Lactobacillus brevis WCP02 and Lactobacillus plantarum P1201 . ≪ / RTI >

I) Soybean

In the production method of the present invention, soybean, germinated soybeans, expanded soybeans and germinated soybeans may be used as the soybeans, but germinated soybean ginseng having been grown at 100 to 120 ° C for 30 to 60 minutes is most preferable.

In addition, the soybean may be subjected to fermentation after the juice is processed into soybean hydrolyzate before fermentation. The throughput of the juice is preferably 1% to 10% (v / v).

The fruit juice is preferably a fruit juice of kiwi, radish, pineapple or papain which has excellent proteolytic ability. Such fruit juice treatment can further increase the productivity of physiologically active substances (Gabar, CLA, and non-glycoside isoflavones).

Ii)

In the production method of the present invention, the probiotics are a combination of Lactobacillus brevis WCP02 and Lactobacillus plantai P1201.

The Lactobacillus brevis WCP02 strain is a novel strain isolated and identified from Kimchi by the present inventors and has excellent probiotic properties (resistance to stomach acid, resistance to succulent acidity) On Dec. 12, 2016, and granted accession number KACC92159P (patent application no. 2017-0102863).

The Lactobacillus brevis WCP02 strain has a white oval shape and can grow to a pH of 3 to 11, a salt concentration of 6% and a temperature of 10 to 50 ° C., and 12 species including L-arabinose can be used And 32.37% of palmitic acid (C16: 0) as a saturated fatty acid, which has a fatty acid composition in the lactobacillus (Figs. 1 to 3).

The Lactobacillus brevis WCP02 showed a survival rate of 39% or more after 4 hours of culture at pH 2, and survival rate of 86% or more after 48 hours of culture in bile acid at pH 3.0, and 0.1% (w / v) monosodium glutamic acid (monosodium glutatmate, MSG) (Table 1 and Table 2).

The Lactobacillus plantarum P1201 strain is also a novel strain obtained by isolating / identifying a strain having excellent conjugate linoleic acid productivity and excellent antibiotic resistance properties such as acid resistance and bile acid resistance from fermented foods, (KACC) on July 19, 2013, and received the accession number KACC91848P (registered patent No. 10-154418).

 Lactobacillus plantai P1201 strain is a yellow round shape and can grow from pH 4 to 11, salt concentration up to 4%, and temperature 10 ℃ to 45 ℃, and 24 kinds including lactose can be used And palmitic acid (C16: 0) in saturated fatty acid account for 37.81%, which is a fatty acid composition of lactobacillus (Figs. 1 to 3).

Lactobacillus plantai P1201 showed a survival rate of 62.22% or more after 4 hours of culture at pH 2.5, and a survival rate of 89.62% or more after 48 hours of culture at pH 3.0 of bile acid. (44.97 to 93.66 μg / ml at the concentration of linoleic acid) (Table 1, Table 2, Fig. 4).

In the present invention, the combined prophylactic agent as an inoculum may be added as a separate culture medium for the Lactobacillus brevis WCP02 strain and the Lactobacillus plantai P1201 strain, respectively, or may be added as a mixed cultured mixed culture.

The mixing ratio of the two strains in the combined probiotic agent may be 3: 1 to 1: 3.

Iii) Fermentation

The fermentation can be carried out by inoculating the soybean culture with the seed culture and fermenting at 20 to 40 DEG C for 12 hours or more.

The inoculation is preferably 1.0% (v / v) to 10.0% (v / v).

The fermentation characteristics of the two strains are complemented and synergized during the fermentation to increase the content of conjugated linoleic acid, guar and non-glycoside isoflavone compounds, daidzein and genistein, as well as effective physiological activity such as total phenolics The content of the substance is enhanced, and both the antioxidative activity and the digestive enzyme (carbohydrate and lipid hydrolyzing enzyme) inhibitory activity are enhanced (Example 3).

According to another object of the present invention, there is provided a soybean fermentation composition produced by the above-described method and having enhanced content of Gabas, conjugated linoleic acid and nonisaccharide isoflavones.

The soybean fermentation composition according to the present invention has an improved content of GABA, conjugated linoleic acid and non-glycosylated isoflavones (in particular, daidzein and genistein), and also has excellent total phenolic content, antioxidant activity, digestive enzymes Decomposing enzyme) inhibitory activity, and is excellent in antidiabetic and anti-obesity effects (Example 4).

According to still another object of the present invention, there is provided a combined prophylactic agent for Lactobacillus brevis WCP02 strain and Lactobacillus plantarum P1201 strain which can be used as a fermentation broth of the above production method.

The compound probiotic agent according to the present invention can also be used for edible use of animals including humans, and specifically, it can also be used as a food or pharmaceutical material. The probiotics can be supplied in various forms such as dried cell forms or fermented products.

According to another object of the present invention, there is provided an anti-diabetic and anti-obesity functional food comprising the soybean fermentation composition as an active ingredient.

In the present invention, the functional food may be in the form of soy milk, tofu, soybean meat, soybean snack, soybean cake, liquid yogurt, yogurt yogurt, soy sauce, seasoning, sauce, beverage, but is not limited thereto.

The production method of the present invention produces a soybean fermentation composition in which Gabava, conjugated linoleic acid and nonisaccharide isoflavone are significantly enhanced compared with the conventional method.

The soybean fermentation composition according to the present invention is characterized in that the content of gaba, conjugated linoleic acid and non-glycoside isoflavones (daidzein and genistein) is remarkably increased and excellent content of total phenolics, antioxidant activity, digestive enzymes (carbohydrate and fat hydrolysis Enzyme) inhibitory activity, and is excellent in anti-diabetic and anti-obesity effects and can be used as a material for functional foods and pharmaceuticals.

The functional food according to the present invention has excellent anti-diabetic and anti-obesity functionalities and is effective in suppressing fat production, controlling body weight, decreasing cholesterol, improving hyperlipidemia, alleviating atherosclerosis, alleviating diabetes, improving blood circulation, improving immunity, And improvement of menopausal symptoms due to female hormone imbalance.

The combined probiotics of the present invention are far more effective in the productivity of both GABA, conjugated linoleic acid and non-glycosylated isoflavones than conventional seed bacteria, and have excellent resistance to gastric acid and bile acid, thus being highly useful as a probiotic agent.

FIG. 1 is a phylogenetic tree of Lactobacillus brevis WCP02, Lactobacillus plantai P1201 and other lactic acid bacteria.
Fig. 2 shows a comparison of morphological, biochemical and biochemical characteristics of Lactobacillus brevis WCP02 and Lactobacillus plantai P1201.
Fig. 3 shows the result of analysis of the composition of the microbial fatty acids of Lactobacillus brevis WCP02 and Lactobacillus plantai P1201.
Figure 4 The graph shows CLA productivity of Lactobacillus plantarum P1201 strain.
Figure 5 is a chromatogram of the gover, conjugated linoleic acid and isoflavone analysis of soy-yogurt according to the present invention. FIG. 5A is a chromatogram of a GABA assay automatic amylase analyzer, FIG. 5B is a conjugated linoleic acid analysis gas (GC) chromatogram, and FIG. 5C is an isoflavone analytical high pressure liquid (HPLC) chromatogram.
6 shows the antioxidative activity of soybean-yogurt according to the present invention. a shows DPPH radical scavenging activity, b shows ABTS radical scavenging activity, and c shows hydroxyl radical scavenging activity.
Fig. 7 shows digestive enzyme inhibitory activity of soybean-yogurt according to the present invention. a shows the alpha-glucosidase inhibitory activity, and b shows the pancreatic-lipase inhibitory activity.
Figure 8 shows the effect of fat reduction in 3T3-L1 adipose precursor cells of bean-yogurt according to the present invention. Fig. 8A shows the result of oil red O staining, and Fig. 8B shows the neutral lipid content.
9 shows the weight loss effect of the soybean-yogurt according to the present invention in a mouse animal model according to a 12-week diet.
FIG. 10 shows the effect of improving the blood glucose level by blood analysis after 12 weeks in a mouse animal model of soybean-yogurt according to the present invention. FIG. 10A shows the blood sugar content, and FIG. 10B shows the c-peptide content.
FIG. 11 shows the effect of improving liver fat as a result of liver tissue analysis after 12 weeks in a mouse animal model of soybean-yogurt according to the present invention. (A) and (B) show the morphology and weight of the liver, and (C) show the results of HNO and oil red O staining of liver tissues.

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

Example

Example 1. Activity analysis of Lactobacillus brevis WCP02 strain and Lactobacillus plantarum P1201 strain

The activity (characteristics) of Lactobacillus brevis WCP02 strain and Lactobacillus plantarum P1201 strain was compared with each of the published strains.

<Comparative analysis of gastric acid tolerance>

by a pepsin (pensin) in the adjusted MRS broth pH (2.0, 2.5, 3.0) ) was added to 1% and then preparing the artificial gastric juice acid, the disclosed strains (L. brevis KCTC 3320, L. plantarum KCTC 3012), WCP02 strain and P2101 strain were inoculated at 5%, respectively, and cultured at 37 ° C for 4 hours. The viable cell counts were confirmed by the stepwise dilution method in the MRS solid medium immediately after culturing and at 2 hours and 4 hours after the culture, respectively. The survival rate was the number of viable cells at the initial stage of culture and the number of colonies formed on the MRS solid medium at 2 hours and 4 hours The survival rate (%) was calculated and shown in Table 1.

 Survival rate (%) = (number of initial live bacteria ÷ number of living cells after cultivation) × 100

Strain Survival rate (%) / incubation time (h) pH 2.0 pH 2.5 pH 3.0 2 4 2 4 2 4 L. brevis   KCTC 3320 15.27 nd 49.51 33.48 94.05 94.86   WCP02 44.43 39.64 50.68 35.18 98.86 102.24 L. plantarum   KCTC 3012 17.65 9.45 53.66 42.31 95.62 86.78   P1201 26.85 15.32 92.56 62.22 112.55 102.79 nd: Not detected.

As shown in Table 1, WCP02 strain and P1201 strain were superior to gastrointestinal acid tolerance strain. At pH 2.0, WCP02 showed a survival rate of 39.64% at 4 hours after culture, and P1201 at pH 2.5 showed a survival rate of 62.22% at 4 hours after culture.

<Comparison of bile acid tolerance>

pH after preparing a 1% pancreatin and bile acids by the addition of bile salts (bile salt) to the (2.0, 2.5, 3.0) adjusted MRS broth, the strains disclosed (L. brevis KCTC 3320, L. plantarum KCTC 3012), WCP02 strain and P2101 strain were inoculated at 2.5%, respectively, and cultured at 37 ° C for 48 hours. The viable cell counts formed on the MRS solid medium were compared with the number of viable cells The results are shown in Table 2.

Strain Survival rate (%) pH 2.0 pH 2.5 pH 3.0 L. brevis   KCTC3320 nd 49.58 79.59   WCP02 nd 51.26 86.25 L. plantarum   KCTC 3012 nd 51.22 64.23   P1201 nd 65.44 89.62 nd: Not detected.

As shown in the results in Table 2, the bile acid tolerance of WCP02 strain and P2101 strain was higher than that of the published strain at pH 2.5 and pH 3.0.

Example  2. Compound Probiotics  Germ Production capacity  analysis

The productivity of each lactic acid bacterium strains constituting the combined probiotics was compared with that of the mixed probiotics (mixed seedlings) fermentation, and the changes of the production ability by juice treatment were analyzed.

A. Comparison of production ability of single seed and mixed seed

&Lt; Preparation of fermentation product &

Lactobacillus brevis WCP02 strain, Lactobacillus plantarum P1201 strain WCP02 strain and publicly available strains ( L. brevis KCTC 3320, L. plantarum KCTC 3012) was inoculated into the MRS liquid medium and cultured at 30 ° C for 2 days to prepare a culture medium.

The water was soaked in water for 2 hours at room temperature for 12 hours, and then germinated in a bean sprout cultivator so as to be twice the amount of the raw bean grains. Lt; 0 &gt; C for 30 minutes, and dried at 55 &lt; 0 &gt; C for 2 to 3 days with hot air and pulverized.

100 g of distilled water was added to 10 g of germinated soybean powder, sterilized at 121 ° C for 15 minutes, cooled at room temperature (25 ° C), and each of the seeds prepared above was inoculated into 5% (v / v), and in case of mixed seed inoculation, 2.5% (v / v) of each of two seeds was inoculated and fermented at 30 ° C for 72 hours.

<Comparison of production capacity of Gabba>

Free amino acid analysis for the determination of glutamic acid and guar gum content was carried out using an automatic amino acid analyzer (Hitachi, L-8900, Japan). Specifically, 1 ml of the fermented sample was precisely weighed in a test tube, and 4 ml of distilled water was added, followed by hydrolysis at 60 ° C for 1 hour. Then, 1 ml of 10% 5-sulfosalicylic acid was added and the mixture was allowed to stand at 4 ° C for 2 hours to precipitate proteins. The resulting filtrate was filtered through a glass filter, and the filtrate was concentrated under reduced pressure at 60 ° C to completely evaporate the water. The concentrated sample was dissolved in 2 ml of lithium citrate buffer (pH 2.2), filtered through a 0.45 μm membrane filter (Dismic-25CS), and analyzed with an automatic amino acid analyzer. The results are shown in Table 3.

The strain / Before and after fermentation Analysis item GA & GABA  (mg / g d.w.) GA GABA L. plantarum  P1201 (5%)  Before fermentation (0 hr) 0.61 0.42  After fermentation (72 hr) 0.30 0.36 L. brevis WCP02  (5%)  Before fermentation (0 hr) 0.61 0.38  After fermentation (72 hr) 0.31 0.72 L. plantarum P1201 (2.5%) + L. brevis WCP02  (2.5%)  Before fermentation (0 hr) 0.69 0.36  After fermentation (72 hr) 0.37 0.72

As shown in Table 3, the inoculum amount of the Lactobacillus brevis WCP02 strain (which is a GABA-producing strain) at the time of mixed seed fermentation exhibited the same level of the ability to produce GABA even when it was only ½ of that of the single seedy fermentation.

<Comparison of production yield of conjugated linoleic acid>

For the analysis of conjugated linoleic acid (CLA), 2 ml of each fermented product before fermentation (0 h) and after fermentation (72 h) was added, and 3 ml of methanolic 0.5 N NaOH was added. After heat treatment at 100 ° C for 10 min, BF ₃ 2 ml was added to extract the fatty acid, and the extracted fatty acid was subjected to saponification and methylation, followed by filtration. The fatty acid was analyzed by GC chromatogram according to the fatty acid analysis method in the general component analysis method of food revolution, and the results are shown in Table 4.

Strain / before and after fermentation Analysis item CLA (mg / g d.w.) c 9, t 11 t 10, c 12 Total L. plantarum  P1201 (5%)  Before fermentation (0 hr) nd nd nd  After fermentation (72 hr) 0.54 0.05 0.59 L. brevis WCP02 (5%)  Before fermentation (0 hr) nd nd nd  After fermentation (72 hr) nd nd nd L. plantarum P1201 (2.5%) + L. brevis  WCP02 (2.5%)  Before fermentation (0 hr) nd nd nd  After fermentation (72 hr) 0.47 0.4 0.51 nd: not detected

As shown in Table 4, when inoculated amount of Lactobacillus plantarum P1201 (conjugated linoleic acid-producing strain) was almost equal to 1/2 as compared with the case of single-seeded fermentation, it showed nearly equivalent conjugated linoleic acid-producing ability .

<Non-glycosylated isoflavone production ability comparison>

For the analysis of isoflavone content, the fermented product was lyophilized, and 1 g of the pulverized powder was added with 10 times 50% methanol. The extract was extracted for 12 hours at room temperature and centrifuged for 30 minutes. The supernatant was filtered with a 0.45 μm filter (Dismic-25CS) And used as a sample.

Isoflavones were analyzed by HPLC. The mobile phase solvent was 0.2% glacial acetic acid (in water) (solution A) and 0.2% acetonitrile (in glacial acetic acid) using a Lichrophore 100 RP C18 column (4.6 × 250 mm, 5 μm, Merck, Germany) (solution B). The mobile phase conditions were maintained at 0 min -100%, 15 min-90%, 25 min-80%, 35 min-75%, 45 min-65% and 50 min-65% on the A solvent basis. 20 μl of the sample prepared above was injected and the mobile phase velocity was maintained at 30 ° C at 1 ml / min. The detector was detected at 254 nm using a diode array detector (DAD) and the results are shown in Table 5

Analysis item
Isoflavones (μg / g) L. plantarum  P1201 (5%) L. brevis WCP02 (5%) L. plantarum P1201 (2.5%) +
L. brevis  WCP02 (2.5%) Before fermentation
(0 hr) After fermentation
(72 hr) Before fermentation
(0 hr) After fermentation
(72 hr) Before fermentation
(0 hr) After fermentation
(72 hr)  Glycosides   Daidzin 303.02 21.37 284.74 119.15 325.27 20.36   Glycitin 207.21 26.12 183.17 189.03 199.08 25.05   Genistin 364.07 5.39 330.05 87.84 379.81 2.03   Total 874.3 52.88 797.96 396.02 904.16 47.44  Aglycones   Daidzein 21.62 296.31 17.62 224.45 22.27 297.74   Glycitein 6.47 87.71 7.01 55.33 8.44 57.54   Genistein 18.02 251.19 14.90 175.95 16.77 246.70

As shown in Table 5, even when the amount of lactobacillus plantarum P1201 inoculated at the time of the mixed seed fermentation was less than 1/2 of that in the single seedy fermentation, the yield of the non-saccharide isoflavone (particularly, daidzein, genistein) Respectively.

As a result, the fermentation characteristics of the two probiotics of the present invention are complemented by the fermentation characteristics of the two strains, so that the conjugate linoleic acid, the gaba and the non-glycoside isoflavone compound, And the content of genistein was significantly improved (in terms of inoculation amount).

B. Analysis of Production Capacity by Juice Treatment

The kiwi was poured into a Hurrom mixer (HUROM HU-400 / HU-400G) to prepare kiwi juice.

After adding and mixing 100 ml of distilled water to 10 g of the germinated soybean powder prepared as described in A. above, the kiwi juice was added at 2.5, 5.0 and 10.0% (v / v) The mixture was sterilized at 121 DEG C for 15 minutes, and the culture broths of Lactobacillus plantai strain P1201 and Lactobacillus brevis WCP02 prepared as described in A. above were inoculated at 2.5% each for 72 hours To prepare a fermented product.

<Change in production ability of GABA and conjugated linoleic acid>

The yield of guar and conjugated linoleic acid was measured in the same manner as described in A. above, and the results are shown in Table 6. &lt; tb &gt; &lt; TABLE &gt;

Kiwifruit throughput ( % ) / Before and after fermentation Analysis item CLA (mg / g d.w.) GA & GABA (mg / g d.w.) c 9, t 11 t 10, c 12 Total GA GABA Total Kiwi juice 0%  Before fermentation (0 hr) nd nd nd 1.07 64.1 0.60  After fermentation (72 hr) 0.61 0.06 0.67 0.32 26.7 0.88 Kiwi juice 2.5%  Before fermentation (0 hr) nd nd nd 1.02 62.6 0.61  After fermentation (72 hr) 0.63 0.06 0.69 1.34 58.0 0.97 Kiwi juice 5.0%  Before fermentation (0 hr) nd nd nd 1.06 62.0 0.65  After fermentation (72 hr) 0.72 0.07 0.79 2.77 68.9 1.25 Kiwi juice 10%  Before fermentation (0 hr) nd nd nd 1.04 60.5 0.68  After fermentation (72 hr) 0.84 0.8 0.92 5.25 76.6 1.60 nd: not detected

As shown in Table 6, the conjugate linoleic acid (CLA) content and the governing content of the fermented product were further increased when the kiwifruit juice was added. Especially, when the kiwifruit juice was added at 5 to 10.0% (v / v), the content of conjugated linoleic acid And the increase in the size of the product is larger.

Example 3. Preparation of Soybean Fermentation Composition and Activity Analysis

<Soybean - Yogurt Production>

To 1,000 g of germinated soybean powder, 10 L of distilled water was added and dissolved. After sterilization treatment at 121 ° C for 15 minutes, the mixture was cooled to room temperature, and the culture broth of Lactobacillus plantai P1201 and Lactobacillus brevis WCP02 was added to 2.5% v / w) and fermented at 30 DEG C for 60 hours to prepare soybean fermented soybean-yogurt according to the present invention.

<Preparation of analytical sample>

Soybean-yogurt was frozen at 70 캜 for 2 days, lyophilized, and pulverized to powder. The extracts were centrifuged for 30 minutes, and the supernatant was filtered with a 0.45 μm filter (Dismic-25CS) to obtain a total phenolix and isoflavone content The remaining filtrate was concentrated under reduced pressure and prepared as lyophilized powder at a concentration of 0.1 to 1 mg / ml and used as a physiological activity (GABA, conjugated linoleic acid, antioxidant activity, functional) measurement.

<Determination of GABA, conjugated linoleic acid and isoflavone content>

The content of Gabas, conjugated linoleic acid and non-glycosylated isoflavones of soybean-yogurt was measured in the same manner as described in Example 2 using the analytical samples prepared above, and the results are shown in Table 7 and FIG.

Analysis item sample Before fermentation After fermentation CLA (mg / g) cis 9, trans 11 CLA nd 0.94 trans 10, cis 12 CLA nd 0.09  Total nd 1.03 Glutamic acid (GA) & GABA content (mg / g)  GA 0.50 0.47  GABA 0.43 1.23 Isoflavones (μg / g)  Glycosides   Daidzin 903.02 27.22   Glycitin 301.77 20.34   Genistin 1,041.38 nd  Aglycones   Daidzein 69.25 779.69   Glycitein 8.71 87.46   Genistein 57.57 546.73 nd: not detected

As shown in Table 7, soybean-yogurt according to the present invention produced GABA in an amount increased about 2.9 times as compared to before fermentation (Fig. 5A)

Conjugated linoleic acid (CLA) was not detected even before fermentation. After fermentation, the total conjugated linoleic acid was significantly increased to 1.03 mg / g, with cis 9, trans 11 CLA 0.94 mg / g, trans 10 and cis 12 CLA 0.09 mg / (Fig. 5B), and

The conversion of the glycoside isoflavone to the non-glycosyl isoflavone was excellent at 96%, and the unglycosylated isoflavone was produced in an amount 10 times or more higher than that of the fermentation before all of the daidzein, glycitein and genistein (FIG. 5c) .

<Total phenolic content measurement>

The total phenolic content was determined by the Folin Denis method (1952). Specifically, 0.5 ml of the sample is dispensed into a test tube, and 25% Na ₂ CO ₃ 0.5 ml was added and allowed to stand for 3 minutes. After addition of 0.25 ml of 2 N Folin-Ciocalteu phenol reagent, the mixture was incubated at 30 ° C for 1 hour to develop color. The color developed blue was measured at 750 nm using a spectrophotometer (Spectronic 2D). The content of total phenolics was determined from a standard curve prepared using gallic acid, and the results are shown in Table 8.

Analysis item sample Before fermentation After fermentation Total phenolics (mg / g) 0.41 0.63

As shown in Table 8, the total phenolic content of soybean-yogurt according to the present invention was increased by about 50% (0.63 mg / g) compared to before fermentation (0.41 mg / g).

<Antioxidant activity>

DPPH radical scavenging activity was determined by adding 0.8 ml of DPPH solution (1.5 × 10 ^-4 M) to 0.2 ml of each of the samples prepared above (0.25 mg / ml, 0.5 mg / ml, 0.75 mg / ml and 1 mg / And the mixture was allowed to stand for 30 minutes. The absorbance was measured at 525 nm. The negative control of the DPPH radical scavenging activity proceeded in the same manner using distilled water instead of the sample, and the difference in absorbance was calculated as a percentage (%) according to the following equation, and the results are shown in FIG.

Radical scavenging activity (%) = [1- (negative control absorbance / experiment absorbance)] × 100

The ABTS radical scavenging activity was obtained by mixing 5 ml of 7 mM ABTS reagent and 5 ml of 140 mM K ₂ S ₂ O ₈ (FW 270.3, Sigma 9392) in a dark place for 14 to 16 hours to generate cation radicals. At 732 nm, the absorbance of the control was adjusted to 0.7 ± 0.02. 0.1 ml of each sample (4 kinds of 0.25 mg / ml, 0.5 mg / ml, 0.75 mg / ml and 1 mg / ml concentration) and 0.9 ml of ABTS solution were mixed and reacted for 3 minutes and absorbance was measured at 732 nm. The ABTS radical inhibitory activity was also measured by the same method using distilled water instead of the sample as the negative control, and the difference in absorbance was calculated as a percentage (%) according to the above equation. The results are shown in FIG. 6B.

Hydroxyl radical scavenging activity was measured 10 mM FeSO _{4. 7H 2 0-EDTA 0.2ml, 10} mM 2- deoxyribose _{0.2 ml, 10 mM H 2 O} 2 0.2 ml, samples (each 0.25 mg / ml, 0.5 mg / ml, 0.75 mg / ml, 1 mg / ml concentration (In DW) and 2.8% trichloroacetic acid (in DW) were added to the mixture, and the mixture was added to the mixture at 100 ° C For 20 minutes, cooled, and then absorbance was measured at 520 nm. As a negative control, PBS (8.76 g of NaCl based on 1 L, 0.11 g of NaH ₂ PO _{4 and} 0.596 g of Na ₂ HPO ₄ ) was used instead of the sample. The hydroxyl radical scavenging activity was calculated by the above equation using the above equation, and the result is shown in FIG. 6 (c).

As shown in FIG. 6, the antioxidative activity of soybean-yogurt according to the present invention was enhanced compared to that before fermentation, and the enhancement width was increased in a concentration-dependent manner.

<Digestive enzyme inhibitory activity>

Alpha-glucosidase inhibitory activity to evaluate antidiabetic effect was measured, and pancreatic-lipase inhibitory activity was evaluated to evaluate anti-obesity effect.

50 μl of 0.5 U / ml alpha-glucosidase enzyme solution was added to 50 μl of each sample (0.25 mg / ml, 0.5 mg / ml, 0.75 mg / ml, and 1 mg / ml concentration) 50 μl of a 200 mM sodium phosphate buffer solution (pH 6.8) was mixed and pre-incubated at 37 ° C for 10 minutes. 100 μl of sodium phosphate buffer (pH 6.8) was added and reacted at 37 ° C for 10 minutes. The reaction was stopped by adding 0.75 ml of 100 mM Na ₂ CO ₃ to the reaction solution, and the absorbance at 420 nm was measured. Negative control was carried out in the same manner using distilled water instead of the sample, and the difference in absorbance was expressed as a percentage (% The results are shown in Fig.

Alpha-glucosidase inhibitory activity (%) =

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

The inhibitory activity of alpha-glucosidase, which is a carbohydrate enzyme of soybean-yogurt according to the present invention, was enhanced before fermentation, and the enhancement width was increased in a concentration-dependent manner (FIG.

50 μl of 1.0 U / ml pancreatic lipase enzyme solution, 50 μl of a 200 mM sodium phosphate buffer solution (pH 7.0, (pH 6.8) were mixed and preliminarily cultured at 37 ° C for 10 minutes. Then, 100 μl of sodium phosphate buffer (pH 6.8) was added and reacted at 37 ° C for 10 minutes. The reaction was stopped by adding 0.75 ml of 100 mM Na ₂ CO ₃ to the reaction solution, and the absorbance at 420 nm was measured. Negative control was carried out in the same manner using distilled water, and the difference in absorbance was expressed as a percentage The results are shown in Fig.

Pancreatic-lipase inhibitory activity (%) =

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

The pancreatic-lipase inhibitory activity of soybean-yogurt according to the present invention, which is a lipolytic enzyme of soybean-yogurt, was also enhanced compared with that before fermentation, and the enhancement width was increased in a concentration-dependent manner B).

Example 4. Functional Assay of Soybean Fermentation Composition

<Fat reduction functional test>

3T3-L1 adipose precursor cells (ATCC) were used to measure the effect of fat reduction of soybean-yogurt according to the present invention.

3T3-L1 adipose precursor cells were cultured in DMEM medium supplemented with 10% fetal calf serum and 1% penicillin-streptomycin (Dulbecco's modified Eagle's medium) at 37 ° C and 5% CO ₂ . To induce differentiation of 3T3-L1 adipose precursor cells into adipocytes, the cells were plated at 5 × 10 ⁵ cells / μl on a 60 cm plate, and on the first day, 1.5 μg / ml insulin, 1 μM dexamethasone, 500 μM IBMX and 1 μM rosiglitazone (3T3- L1 differentiation kit, Biovision).

Three days after induction of adipocyte differentiation, soybean-yogurt extract samples and DMEM containing 1.5 μg / ml insulin were cultured for 14 days while exchanging them every other day. The medium was removed from the cultured 3T3-L1 adipose precursor cells, washed three times with PBS, stained with oil red O, and 3T3-L1 prepared in the same manner to measure the amount of triglyceride (TG) The medium was removed from L1 adipose precursor cells and washed three times with PBS. The cells were collected using a rubber policeman and observed with a microscope. The results are shown in FIG. 8A.

The collected cells were suspended in an appropriate amount of TG standard diluent (Triglyceride colorimetric assay kit, Cayman chemicals), treated with ultrasonication (1 second, 20x), and centrifuged at 12,000 rpm for 15 minutes. After centrifugation, TG in the supernatant was measured by the Triglyceride colorimetric assay kit. The results are shown in FIG. 8B, with the control adjusted to 100.

As shown in FIG. 8A, as a result of the oil red O staining, no neutral lipid was produced (treatment not treated) when 3T3-L1 lipid precursor cells were not treated with the differentiation inducer, and when the differentiation inducing substance was treated, A red line of neutral lipid was produced (treatment). Addition of soybean - yoghurt before fermentation to neutral lipid - producing cells by adding differentiation inducing substances to lipid precursor cells resulted in a similar effect to that of treatment with differentiation inducing substances, When soybean - yoghurt samples were treated after fermentation, the neutral lipid was reduced significantly and the neutral lipid production inhibitory effect was confirmed.

As shown in FIG. 8B, when the neutral lipid content was measured, the addition of the soybean-yogurt sample after fermentation showed a significant decrease in the neutral lipid.

<Weight loss functional test>

The weight loss effect of the soybean-yogurt of the present invention was examined using the mouse animal diet group. This study was conducted to investigate the effect of soybean-yogurt supplementation on the high fat diet (HFD), high fat diet (CLA) and high fat diet (25% Kcal, normal diet, ND) (SPY).

Each of the four groups of mice received a total of 3 g of dietary food during 12 weeks of light and dark conditions for 12 weeks of feeding, and dietary supplementation was performed every 2 days at 8:30 in the morning, And the results are shown in Fig.

As shown in FIG. 9, in the SPY group of the soybean-yogurt diet according to the present invention, CLA group (37.79 g) fed with conjugated linoleic acid (CLA) Almost the same effect was obtained.

<Functional test for improving blood sugar>

Blood samples were withdrawn from the heart using an injection syringe, and blood glucose and c-peptide content were measured by centrifugation for 10 minutes to collect the serum. 10.

In the SPY group according to the present invention, the blood glucose level was 232.83 mg / dL, which was lower than that of the HFD group (253.60 mg / dL) and was close to the blood glucose level (206.14 mg / dL) (Fig. 10A).

The c-peptide content of the HFD group was the highest at 2299.47 pg / dL and the insulin resistance was the lowest at the ND group (1563.81 pg / dL). In the SPY group, 1656.33 pg / dL, (Fig. 10 (b)).

<Functional test for improving liver function>

Mice of each dietary group raised for 12 weeks were subjected to ether anesthesia to extract liver tissues. First, liver shape was observed and weight was measured. The results are shown in FIG. 11 (A).

In the liver type and color of each dietary group, the HFD group had more fat accumulation than ND group, CLA group and SPY group, and the liver was light brown and the color was also light brown. In the ND group, the liver weighed 1.58 g and increased to 2.20 g in the HFD group. The SPY group of the present invention had a weight of 1.54 g which was almost the same as that of the ND group, and the liver weight was remarkably low when compared with the HFD group (FIG. 11A).

Liver histology was observed with haematoxylin and eosin (H & E) staining and oil red O staining. H & E staining was performed by immersing the extracted liver tissues in 4% neutralized paraformaldehyde solution and then washing them with ethanol (70, 80, 90, 95, and 100%), respectively. Then, dehydrated in xylene solution for 3 minutes, washed in ethanol, and immersed in hematoxylin solution for 3 minutes. Thereafter, the cells were washed again with ethanol, finally stained with eosin solution, and the tissues were observed with a microscope. The results are shown in Fig. 11 (B). For the oil red O staining, the extracted liver tissue was fixed with 3.7% formalin for 1 hour, washed with 60% isopropanol, and stained with oil red O dye for 20 minutes. After staining, the cells were washed with distilled water three times or more, and the stained cells were observed through a microscope. The results are shown in Fig. 11 (B).

In the HFD group, a lot of adipose tissue stained with red color was observed in the HFD group and the SPY group in the present invention and CLA group in the reference group, - Yogurt was found to be effective in improving fatty liver (Fig. 11B).

From the functional test results, the soybean fermentation composition according to the present invention not only promotes the content of GABA, conjugated linoleic acid, and unglycosylated isoflavones (daidzein and genistein) at the same time, but also promotes antioxidant activity and decreases fat, , And it is useful as a material for anti-diabetic and anti-obesity functional foods due to the effect of improving the liver.

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

Claims (9)

A soybean fermentation composition containing a lactobacillus brevis WCP02 strain deposited with accession number KACC92159P and a lactobacillus plantarum strain P1201 deposited with accession number KACC91848P, which contains the promoter of the soybean fermented with soybean, the conjugated linoleic acid and the unglycosylated isoflavone &Lt; / RTI &gt;
The method of claim 1, wherein the soybean is a germination-enhancing soybean. The method of producing a soybean fermentation composition containing enhanced gabas, conjugated linoleic acid, and non-glycoside isoflavones.
The method according to claim 1, wherein, before fermentation, soybean is processed into juice to make soybean hydrolyzate,
Wherein the juice is any one selected from the group consisting of kiwifruit juice, fruit juice, pineapple juice, and papain juice.
A soybean fermentation composition comprising a promoter produced by the process according to any one of claims 1 to 3 and promoted gabas, conjugated linoleic acid and non-glycoside isoflavones.
5. The fermented soybean composition according to claim 4, wherein the soybean fermentation composition has enhanced total phenolic content, antioxidant activity, alpha-glucosidase inhibitory activity, and pancreatic-lipase inhibitory activity.
An antidiabetic and anti-obesity functional food comprising the fermented soybean composition according to claim 4 as an active ingredient.
[Claim 7] The anti-diabetic and anti-obesity functional food according to claim 6, wherein the anti-diabetic is caused by a decrease in blood glucose and c-peptide content, and the anti-obesity is caused by neutral lipid reduction, weight control and fatty liver improvement.
8. The food according to claim 7, wherein the functional food is any one selected from the group consisting of soybean milk, tofu, soybean meat, soybean snack, soybean cake, liquid yogurt, yogurt, soy sauce, Obesity functional food.
The method according to claim 1, wherein the Lactobacillus brevis WCP02 strain deposited with Accession No. KACC92159P and the Lactobacillus plantarum P1201 deposited with Accession No. KACC91848P are provided.

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