KR102044602B1 - 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

In the present invention, there is provided a method for producing a soybean fermentation composition enhanced by Gaba, conjugated linoleic acid and non-glycoside isoflavones, a complex probiotic agent used therein, and an anti-diabetic and anti-obesity functional food comprising the soybean fermentation composition as an active ingredient.
Soybean fermentation composition according to the present invention is significantly enhanced in the content of Gaba, conjugated linoleic acid and non-glycoside isoflavones (Dadezein and Genistein), and also excellent total phenolic content, antioxidant activity, digestive enzymes (carbohydrates and fat hydrolysis) Enzyme) inhibitory activity, excellent antidiabetic and anti-obesity effect, and can be used as a material for functional foods and pharmaceuticals.

Description

Preparation method of soybean fermented composition enhanced with Gaba, conjugated linoleic acid and non-glycoside isoflavone, complex probiotic used for this, and anti-diabetic and anti-obesity functional food containing the soybean fermented composition as an active ingredient {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}

The present invention relates to a method for producing a soybean fermentation composition enhanced with Gaba, conjugated linoleic acid and a non-glycoside isoflavone, a complex probiotic agent used therein, and an anti-diabetic and anti-obesity functional food comprising the soybean fermentation composition as an active ingredient. More specifically, a method for preparing soybean fermentation compositions in which GABA, conjugated linoleic acid and nonglycoside isoflavones are enhanced by using a combination probiotic of Lactobacillus brevis WCP02 and Lactobacillus plantarium P1201 as a seed, and the Lactobacillus brevis WCP02 and lactose The present invention relates to an excellent anti-diabetic and anti-obesity functional food comprising a complex probiotic of Bacillus plantarium P1201, and a soybean fermentation composition containing enhanced Gaba, conjugated linoleic acid, and a non-glycoside isoflavone.

As the diet becomes westernized, the calorie intake of adults has continuously increased, and as a result, metabolic diseases such as stroke, arteriosclerosis, hypertension, diabetes, and obesity have increased along with the excess calories. In general, obesity is due to the accumulation of fat tissue by the formation of fat cells such as differentiation. As a result, it is considered as a cause of type 2 diabetes or cardiovascular disease by causing various hormone resistance such as insulin.

Probiotics (probiotics) means 'active probiotic culture' means 'live bacteria', or living bacteria, which keep the gastrointestinal tract of a host such as humans and animals healthy. It also means microorganisms that compete competitively from the stomach to the intestine, the most difficult environment of the human body. These probiotic agents are commonly known as lactic acid bacteria and have been used in various food fermentations. It is also a microorganism generally recognized as safe through long history of ingestion. By ingesting it, it is known that the antibacterial substance secreted by probiotics has a great effect on improving colon diseases such as inhibiting harmful bacteria, stabilizing intestinal flora, lactose intolerance and irritable bowel syndrome.

Γ-Aminobutyric acid or GABA is a non-protein amino acid consisting of four carbons, which is characterized by decarboxylation reaction from glutamic acid (GA) by glutamate decarboxylase (GADase). Together, carbon dioxide is released and converted to Gabba. Gava has a wide variety of physiological functions including not only neurotransmitters in the brain but also brain function, mental stability, blood pressure lowering, diuresis, liver function improvement, obesity prevention, alcohol metabolism and deodorization. In addition, it is registered as a drug and is used for the treatment of headache, tinnitus and decreased motivation caused by stroke or cerebral artery sequelae. Gaba is a first-class dietary supplement for physiological activity of the Ministry of Food and Drug Safety, which helps to improve blood pressure, and its daily intake is set at 20 mg. Normally, the body produces gabba, but since aging is progressed due to excessive ingestion of inadequate foods or additives and insufficient intake of vitamins or minerals, there is a limit in producing gabba in the body.

Conjugated Linoleic Acid (CLA) is an isomer of linoleic acid, which has a conjugated double bond structure, and has anticancer action, body fat reduction effect, atherosclerosis prevention and alleviation effect, total- and LDL-cholesterol and TG (neutral fat; triglycerides (PARK YK et al., 1997 Lipids 32: 853-858; McCarty MF 2000. Medical hypotheses 55: 187-188). Conjugated linoleic acid (CLA) is a physiologically active grade 1 health functional food ingredient of the Ministry of Food and Drug Safety, "Helping to Reduce Body Fat", and its daily intake is set at 1.5 to 3 g. Conjugated linoleic acid is chemically synthesized or produced by microorganisms. In the case of chemical synthesis, there are many problems such as expensive equipment, too much time required for the process, and all of the isomers of conjugated linoleic acid (CLA) may be produced. Therefore, there has been an attempt to produce foods with increased conjugated linoleic acid through the process of converting linoleic acid by conjugate microorganisms to conjugated linoleic acid (registered patent 10-1201184).

Most isoflavones exist in the form of glycoside isoflavone glycosides in nature. Glycoside isoflavones are not degraded by gastric acid and are not absorbed in the body. Hydrolysates by enzymes secreted by microorganisms present in the large intestine Absorption rate is low because it is absorbed after being made. On the other hand, isoflavones in the form of non-glycosides from which sugars have been removed by microbial fermentation or enzymatic reaction are not only directly absorbed in the stomach and small intestine, but also rapidly absorbed (Patent 10-1498919). The Ministry of Food and Drug Safety is a first-class food supplement for physiological activity of Grade 1 physiological activity for 'body bone health'. The daily intake is set at 24 to 27 mg of nonglycoside isoflavones (total amount of Dyne, Glycysteine and Genistein). .

Therefore, the necessity and usefulness of the development of probiotics and fermented foods prepared using the same, which can simultaneously enhance the foods of health functional foods, Gabba, conjugated linoleic acid and non-glycoside isoflavones, are emerging.

The present inventors have studied to meet the needs of the prior art, soybean is at least 15% protein is glutamic acid (GA), at least 50% fat is linoleic acid (LA) and at least 95% isoflavone glycoside isoflavone Develop a probiotic that can efficiently convert soy glutamic acid, linoleic acid, and glycoside isoflavones into gabba, conjugated linoleic acid, and nonglycoside isoflavones simultaneously, thereby using enhanced gabba, conjugated linoleic acid, and nonglycoside isoflavones. Soybean fermented composition containing the prepared was prepared to complete the present invention.

Accordingly, it is an object of the present invention to provide a method for preparing a soybean fermentation composition in which both gaba, conjugated linoleic acid and nonglycoside isoflavones are enhanced.

Still another object of the present invention is to provide a soybean fermentation composition having an increased content of Gaba, conjugated linoleic acid and non-glycoside isoflavones prepared by the above production method.

Still another object of the present invention is to provide a combined probiotic of Lactobacillus brevis WCP02 and Lactobacillus plantarium P1201 which can be used as a fermentation seed of the above production method.

Still another object of the present invention is to provide an anti-diabetic and anti-obesity functional food comprising a soybean fermentation composition containing enhanced Gaba, conjugated linoleic acid and non-glycoside isoflavones as an active ingredient.

In order to achieve the above object, the present invention is fermented soybean fermented composition containing enhanced Gaba, conjugated linoleic acid and non-glycoside isoflavones by fermenting soybean with a complex probiotic seed of Lactobacillus brevis WCP02 strain and Lactobacillus plantarium P1201 strain Provide a way to.

콩) beans

Raw beans, germinated beans, steamed beans and germinated-seed beans may be used as the soybean in the production method of the present invention, but germination-seed beans which have been increased at 100 to 120 ° C. for 30 to 60 minutes are most preferred.

In addition, before fermentation, soybeans may be processed into a soybean hydrolyzate after treating with fruit juice and then fermented. The throughput of the juice is preferably 1% to 10% (v / v).

The juice is preferably a juice of kiwi, radish, pineapple or papain, which has excellent proteolytic ability. Such juice treatment can further increase the productivity of the bioactive substances (Gaba, CLA, and nonglycoside isoflavones).

Ii) spawn

In the preparation method of the present invention, a complex probiotic agent of Lactobacillus brevis WCP02 strain and Lactobacillus plantarium P1201 strain is used.

The Lactobacillus brevis WCP02 strain is a novel strain isolated / identified from the kimchi by the present inventors, and has excellent probiotic properties (gastric acid resistance, bile acid resistance) and excellent GABA production ability, and is the National Institute of Agricultural Science (KACC). Was deposited on December 12, 2016, and was granted accession number KACC92159P (patent application 2017-0102863).

Lactobacillus brevis WCP02 strain is white oval, coarse, grows from pH 3 to 11, up to 6% salt concentration and from 10 to 50 ° C, and 12 species including L-arabinose are available. As the saturated fatty acid, palmitic acid (C16: 0) is 32.37% and has a fatty acid composition of the genus Lactobacillus (FIGS. 1 to 3).

Lactobacillus brevis WCP02 strain showed a survival rate of 39% or more after 4 hours in gastric acid solution at pH 2, 86% or more after 48 hours in bile acid at pH 3.0, and 0.1% (w / v) of monosodium glutamic acid (monosodium glutatmate, MSG) has a Gaba conversion ability of more than 95% (Table 1 and Table 2).

The Lactobacillus plantarium P1201 strain is a novel strain obtained by the present inventors by separating / identifying strains having excellent probiotic properties such as acid resistance and bile acid resistance and excellent conjugated linoleic acid productivity from fermented foods. (KACC) was deposited on July 19, 2013 and was given accession number KACC91848P (registered patent 10-154418).

 Lactobacillus plantarium P1201 strains are yellow, round and smooth in shape, capable of growing from pH 4 to 11, salt concentration of 4% and temperature from 10 ° C to 45 ° C, and 24 species including lactose are available. In the saturated fatty acid, palmitic acid (C16: 0) is 37.81% and has a fatty acid composition of the genus Lactobacillus (FIGS. 1 to 3).

Lactobacillus plantarium P1201 strain showed a survival rate of 62.22% or more after 4 hours in gastric acid solution at pH 2.5, 89.62% or more after 48 hours in bile acid at pH 3.0, and excellent (250 ~ 1000 μg / ml 44.97-93.66 μg / ml) at linoleic acid concentrations have the productivity of conjugated linoleic acid (Table 1, Table 2, FIG. 4).

In the present invention, the complex probiotic agent may be added as a separate culture medium for each of the Lactobacillus brevis WCP02 strain and the Lactobacillus plantarium P1201 strain, or may be added as a mixed culture mixed culture.

The mixing ratio of the two strains in the complex probiotic can be mixed in a 3: 1 to 1: 3.

Iii) fermentation

Fermentation may be carried out by inoculating the seed culture with soybeans and fermenting at 20 to 40 ° C. for at least 12 hours.

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

In fermentation, the fermentation properties of the two strains are complemented and synergistically enhanced, thereby enhancing the contents of conjugated linoleic acid, gabba and nonglycoside isoflavone compounds, Dyzein and Genistein, as well as effective physiological activities such as total phenolics. The content of the substance is enhanced, and the antioxidant activity and the digestive enzyme (carbohydrate and fat hydrolase) inhibitory activity are all enhanced (Example 3).

According to another object of the present invention, the present invention provides a soybean fermentation composition prepared by the above production method, the content of Gaba, conjugated linoleic acid and non-glycoside isoflavones is enhanced.

The soybean fermentation composition according to the present invention has an enhanced content of Gaba, conjugated linoleic acid and non-glycoside isoflavones (particularly, Dydazein and Genistein), and also has an excellent total phenolic content, antioxidant activity, digestive enzymes (carbohydrates and fat hydrolysates). Degrading enzyme) also has an inhibitory activity and is excellent in antidiabetic and anti-obesity effects (Example 4).

According to another object of the present invention, there is provided a complex probiotic formulation of Lactobacillus brevis WCP02 strain and Lactobacillus plantarium P1201 strain that can be used as the fermentation seed of the production method.

The complex probiotic according to the present invention can also be used for the edible of animals including humans, specifically, it can also be used as food or pharmaceutical material. Probiotic can be supplied in various forms such as dried cell form or fermented product.

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

Functional food in the present invention may be in the form of soy milk, tofu, soybeans, soy snacks, legumes, liquid yogurt, staple yogurt, jang, seasonings, sauces, beverages, but is not limited thereto.

The production method of the present invention, compared to the conventional method, produces soybean fermentation compositions with significantly enhanced gabba, conjugated linoleic acid and nonglycoside isoflavones.

Soybean fermentation composition according to the present invention is significantly enhanced in the content of Gaba, conjugated linoleic acid and non-glycoside isoflavones (Dadezein and Genistein), and also excellent total phenolic content, antioxidant activity, digestive enzymes (carbohydrates and fat hydrolysis) Enzyme) inhibitory activity, excellent antidiabetic and anti-obesity effect, and can be used as a material for functional foods and pharmaceuticals.

Functional food according to the present invention has excellent anti-diabetic and anti-obesity functionality, fat suppression effect, weight control, cholesterol lowering, hyperlipidemia improvement, arteriosclerosis relief, diabetes relief, blood circulation improvement, immunity improvement, hot flashes improvement and osteoporosis It is useful for improving menopausal and menopausal symptoms caused by female hormone imbalance.

The composite probiotic of the present invention is a seed which greatly increases the productivity of both gaba, conjugated linoleic acid, and nonglycoside isoflavones, and also has excellent resistance to gastric acid and bile acids, and thus has great utility as a probiotic, compared to ordinary seed.

1 is a phylogenetic tree of Lactobacillus brevis WCP02 strain and Lactobacillus plantarium P1201 strain with other lactic acid strains.
2 shows a comparison of morphological, biologic and biochemical properties of Lactobacillus brevis WCP02 strain and Lactobacillus plantarium P1201 strain.
Figure 3 shows the results of analyzing the cell fatty acid composition of Lactobacillus brevis WCP02 strain and Lactobacillus plantarium P1201 strain.
4 is A graph showing the CLA productivity of the Lactobacillus plantarium P1201 strain.
FIG. 5 is a Gaba, conjugated linoleic acid and isoflavone analysis chromatogram of soy-yogurt according to the present invention. FIG. FIG. 5A is a Gaba Analytical Amic Acid Analyzer chromatogram, FIG. 5B is a conjugated linoleic acid analytical gas (GC) chromatogram, and FIG. 5C is an isoflavone analytical high pressure liquid (HPLC) chromatogram.
Figure 6 shows the antioxidant 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.
Figure 7 shows the digestive enzyme inhibitory activity of soybean-yogurt according to the present invention. a shows alpha-glucosidase inhibitory activity and b shows pancreatic-lipase inhibitory activity.
Figure 8 shows the fat reduction effect in 3T3-L1 adipocytes of soybean-yogurt according to the present invention. Figure 8a is the result of oil red O staining, Figure 8b shows the neutral lipid content.
Figure 9 shows the weight loss effect according to the diet for 12 weeks in the mouse animal model of soybean-yogurt according to the present invention.
10 shows a blood glucose improvement effect after 12 weeks diet in a mouse animal model of soybean-yogurt according to the present invention. Figure 10a shows the blood sugar content, Figure 10b shows the c-peptide content.
Figure 11 shows a fatty liver improvement effect as a result of liver tissue analysis after 12 weeks diet in a mouse animal model of soybean-yogurt according to the present invention. (A) and (B) show the shape and weight of the liver, and (C) shows the HN and oil red O staining results of liver tissue.

The invention is explained in more detail by the following examples. These examples are intended to illustrate the invention and the scope of the invention should not be limited by them.

Example

Example 1 Activity Analysis of Lactobacillus Brevis WCP02 and Lactobacillus Plantarium P1201 Strains

The activities (characteristics) of the Lactobacillus brevis WCP02 strain and the Lactobacillus plantarium P1201 strain were analyzed in comparison with the respective strains.

Comparative analysis of gastric acid resistance

After the artificial gastric acid was prepared by adding 1% of pepsin (pensin) to the MRS liquid medium having adjusted pH (2.0, 2.5, 3.0), the strains ( L. brevis) were prepared. KCTC 3320, L. plantarum KCTC 3012), WCP02 strain and P2101 strain cultures were inoculated at 5% and incubated at 37 ° C for 4 hours. Each of the lactic acid bacteria was identified by virtue of stepwise dilution in MRS solid medium immediately after culturing and at 2 and 4 hours of cultivation. The survival rate was determined by the number of viable cells at the initial stage of culture and the number of colonies formed on MRS solid medium at 2 and 4 hours of cultivation. In comparison, the survival rate (%) was calculated and shown in Table 1.

 % Survival = (initial viable count ÷ viable count after culture) × 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, gastric acid resistance was better in the WCP02 strain and the P1201 strain than the test strain. At pH 2.0, the WCP02 strain showed 39.64% survival at 4 hours of culture, and at pH 2.5, the P1201 strain showed 62.22% at 4 hours of culture.

Comparative Analysis of Bile Acid Resistance

The bile acid was prepared by adding 1% pancreatin and bile salt to the MRS liquid medium adjusted to pH (2.0, 2.5, 3.0), and then the strains ( L. brevis). KCTC 3320, L. plantarum KCTC 3012), WCP02 strain and P2101 strain were inoculated with 2.5% of the culture medium, and cultured for 48 hours at 37 ° C. The calculation is 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 of Table 2, bile acid resistance was higher in the WCP02 strain and the P2101 strain than in the test strains at both pH 2.5 and pH 3.0.

Example  2. Composite Probiotic  Spawn Productivity  analysis

The production capacity of the lactic acid bacteria strain fermenting the combined probiotic alone and the fermentation of the mixed probiotic species (mixed species) were compared and analyzed for changes in the production capacity according to the juice treatment.

A. Comparison of production of single and mixed seed

<Production of Fermentation>

Lactobacillus brevis WCP02 strain, Lactobacillus plantarium P1201 strain WCP02 strain and test strains ( L. brevis) KCTC 3320, L. plantarum KCTC 3012) was inoculated in MRS liquid medium and incubated at 30 ° C. for 2 days to prepare a culture medium as a seed.

After tapping the tap water to double the weight of the raw soybeans, and immersing it for 12 hours at room temperature, removing water by applying it to the rice tray and germinating so that it doubles as the raw grains in the bean sprouts growing machine. The germination-powdered soybean powder prepared by steaming for 30 minutes at ℃, hot air drying and grinding at 55 ℃ for 2-3 days was prepared.

After adding and mixing 100 ml of distilled water to 10 g of germinated-seed bean powder and sterilizing at 121 ° C. for 15 minutes and cooling at room temperature (25 ° C.), each seed prepared above was 5% (v for inoculation alone). / v), in the case of mixed seed inoculation, fermented product was prepared by fermenting two seed species by 2.5% (v / v), respectively, and fermenting at 30 ° C. for 72 hours.

<Gaba production capacity comparison>

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

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, even when the inoculation amount of the Lactobacillus brevis WCP02 strain (which is a Gabba-producing strain) at the time of the mixed seed fermentation showed the same Gaba generating ability, even when it was only 1/2 as compared to that at the single seed fermentation.

<Comparison of Conjugated Linoleic Acid Production Capacity>

Conjugated linoleic acid (CLA) production capacity analysis was performed by taking 2 ml of each fermentation product before the fermentation (0 hour) and after the fermentation (72 hours), adding 3 ml of methanolic 0.5 N NaOH, heat-processing at 100 ° C for 10 minutes, and then BF ₃ Fatty acid was extracted by adding 2ml, and the extracted fatty acid was subjected to saponification and methylation, followed by filtration, and analyzed using GC chromatogram according to the fatty acid analysis method in the general ingredient analysis method of the Food Code. 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 0.04 0.51 nd: not detected

As shown in Table 4, the amount of inoculation of the Lactobacillus plantarium P1201 strain (conjugated linoleic acid-producing strain) at the time of mixed seed fermentation showed almost the same conjugated linoleic acid production ability even if it was only 1/2 compared to that at the time of single seed fermentation. .

<Comparison of Nonglycoside Isoflavones Production Capacity>

After freeze-drying the fermented product for isoflavone content analysis, 50% methanol was added 10 times to 1 g of the ground powder, and the extract extracted at room temperature for 12 hours was centrifuged for 30 minutes, and the supernatant was filtered through a 0.45 μm filtration filter (Dismic-25CS). Was used as a sample.

Isoflavones were analyzed by HPLC. The analytical column used a Lichrophore 100 RP C18 column (4.6 × 250 mm, 5 μm, Merck, Germany) and the mobile phase solvents were 0.2% glacial acetic acid (solution A) and 0.2% acetonitrile (in glacial acetic acid). It was analyzed by (solution B). Mobile phase conditions were maintained at 0 minutes-100%, 15 minutes-90%, 25 minutes-80%, 35 minutes-75%, 45 minutes-65% and 50 minutes-65% based on solvent A. 20 μl of the sample prepared above was injected and the mobile phase speed was maintained at 1 ° C./min at 30 ° C. 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 inoculation amount of the Lactobacillus plantarium P1201 strain at the time of mixed seed fermentation is only 1/2 compared to that at the time of single seed fermentation, it is almost equivalent to the production of non-glycoside isoflavones (especially Dyzedine, Genistein). Indicated.

Taken together, the combined probiotic spawn of the present invention synergistically complements the fermentation properties of the two strains during fermentation, Dadezein and the conjugated linoleic acid, Gaba and non-glycoside isoflavone compounds It can be seen that the content of genistein is significantly enhanced (relative to the inoculation amount).

B. Analysis of Production Capacity Changes According to Juice Processing

Kiwi juice was prepared with a Hurom mixer (HUROM HU-400 / HU-400G) to prepare the kiwi juice.

After adding and mixing 100 ml of distilled water to 10 g of germinated-seed beans powder prepared as in A., the kiwi juice was added at 2.5, 5.0, and 10.0% (v / v), respectively. After shaking for 1 hour and shaking for 1 hour, sterilizing for 15 minutes at 121 ℃, and inoculated with 2.5% each of the culture medium of Lactobacillus plantarium P1201 strain and Lactobacillus brevis WCP02 strain prepared as in A. 72 hours culture To prepare a fermented product.

<Cava and Conjugated Linoleic Acid Production Capacity Change>

Gaba and conjugated linoleic acid production capacity was performed in the same manner as described in A. above, and the results are shown in Table 6.

Kiwi juice 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 Kiwi juice 0%  Before fermentation (0 hr) nd nd nd 1.07 0.60  After fermentation (72 hr) 0.61 0.06 0.67 0.32 0.88 Kiwi juice 2.5%  Before fermentation (0 hr) nd nd nd 1.02 0.61  After fermentation (72 hr) 0.63 0.06 0.69 1.34 0.97 Kiwi juice 5.0%  Before fermentation (0 hr) nd nd nd 1.06 0.65  After fermentation (72 hr) 0.72 0.07 0.79 2.77 1.25 Kiwi juice 10%  Before fermentation (0 hr) nd nd nd 1.04 0.68  After fermentation (72 hr) 0.84 0.8 0.92 5.25 1.60 nd: not detected

As shown in Table 6, the conjugated linoleic acid (CLA) content and the Gaba content of the fermented product increased further when the kiwi juice was added, especially the conjugated linoleic acid content and the Gaba content when the kiwi juice was added at 5 to 10.0% (v / v). We can see that the increase is bigger.

Example 3. Preparation and Activity Analysis of Soybean Fermentation Composition

Soy-Yogurt Manufacturing

After dissolving 10 L of distilled water in 1,000 g of germinated-seed bean powder and dissolving it for 15 minutes at 121 ° C., after cooling to room temperature, the culture solution of Lactobacillus plantarium P1201 strain and Lactobacillus brevis WCP02 strain were respectively 2.5% ( v / w) and fermented at 30 ° C. for 60 hours to prepare soybean-yogurt, a soybean fermentation composition according to the present invention.

<Analysis sample preparation>

Soy-yogurt was frozen at 70 ° C. for 2 days, lyophilized and ground to powder. 50% methanol was added 10 times to 1 g of lyophilized powder, extracted for 12 hours at room temperature, and the extract was centrifuged for 30 minutes, and only the supernatant was filtered through a 0.45 μm filtration filter (Dismic-25CS). Analytical samples were used, and the remaining filtrate was concentrated under reduced pressure, lyophilized powder was prepared, and then 0.1-1 mg / ml was used as a physiological activity (gava, conjugated linoleic acid, antioxidant activity, functional) measurement sample.

<Determination of Gaba, Conjugated Linoleic Acid and Isoflavone Contents>

Gaba, conjugated linoleic acid and nonglycoside isoflavone contents of soy-yogurt were measured in the same manner as described in Example 2 using the assay samples prepared above, and the results are shown in Table 7 and FIG. 5.

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, the soy-yogurt according to the present invention was produced in about 2.9-fold enhanced amount compared to before fermentation (GABA) (FIG. 5A),

Conjugated linoleic acid (CLA) was not even detected prior to fermentation, but after fermentation, cis 9, trans 11 CLA 0.94 mg / g, trans 10, cis 12 CLA 0.09 mg / g resulted in a significant increase in total conjugated linoleic acid to 1.03 mg / g. Was produced in sheep (Figure 5b),

The conversion from glycoside isoflavones to nonglycoside isoflavones was very good at 96%, and nonglycoside isoflavones were produced in an amount of 10-fold enhancement in all of the didzein, glycidine, and genistein levels prior to fermentation (FIG. 5C). .

<Measurement of Total Phenolic Content>

The total phenolic content was measured by the Folin Denis method (1952) the samples prepared above. Specifically, 0.5 ml of sample was dispensed into the test tube and 25% Na ₂ CO ₃ 0.5 ml of solution was added and allowed to stand for 3 minutes. Then, 0.25 ml of 2 N Folin-Ciocalteu phenol reagent was added and mixed, and the mixture was left to stand at 30 ° C. for 1 hour for color development. The developed blue color was measured at 750 nm using a spectrophotometer (Spectronic 2D). At this time, the total phenolic content was calculated from the 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 the soy-yogurt according to the present invention was produced in about 50% (0.63 mg / g) enhanced amount compared to before fermentation (0.41 mg / g).

Antioxidant activity

DPPH radical scavenging activity was 0.8 ml of DPPH solution (1.5 × 10 ⁻⁴ M) in 0.2 ml of the samples prepared above (0.25 mg / ml, 0.5 mg / ml, 0.75 mg / ml, 1 mg / ml, respectively). After the mixture was added and left for 30 minutes to mix uniformly, absorbance was measured at 525 nm. Negative control of DPPH radical scavenging activity proceeded in the same manner using distilled water instead of the sample to calculate the difference in absorbance as a percentage (%) by the following equation, the results are shown in Figure 6a.

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

For ABTS radical scavenging activity, 5 ml of 7 mM ABTS reagent and 5 ml of 140 mM K ₂ S ₂ O ₈ (FW 270.3, Sigma 9392) were mixed and left for 14 to 16 hours in the dark to generate cationic radicals, which were then mixed with methanol. The ABTS solution adjusted to the absorbance value of the control at 732 nm to 0.7 ± 0.02 was used. 0.1 ml of the sample (0.25 mg / ml, 0.5 mg / ml, 0.75 mg / ml, 1 mg / ml each) and 0.9 ml of ABTS solution were mixed for 3 minutes and absorbance was measured at 732 nm. The negative control of the ABTS radical inhibitor also proceeded in the same manner using distilled water instead of the sample to calculate the difference in absorbance as a percentage (%) by the above equation, and the results are shown in b of FIG. 6.

The hydroxyl radical scavenging activity was measured in 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 (0.25 mg / ml, 0.5 mg / ml, 0.75 mg / ml, 1 mg / ml concentration, respectively) 4) After mixing 1.4 ml and reacting at 37 ° C. for 4 hours to form a mixed solution, 1 ml of 1% thiobarbital acid (in DW) and 2.8% trichloroacetic acid (in DW) was added to the mixture and 100 ° C. After cooling for 20 minutes, the absorbance was measured at 520 nm. PBS (8.76 g NaCl, 0.11 g NaH ₂ PO ₄ , 0.596 g Na ₂ HPO ₄ ) was used instead of the sample as a negative control. As for the hydroxyl radical scavenging activity, the difference in absorbance between the added and non-added spheres of the sample solution was calculated as a% value by the above equation, and the results are shown in FIG. 6C.

As shown in Figure 6, the antioxidant activity of the soybean-yogurt according to the present invention was enhanced compared to before the fermentation, and it can be seen that the enhancement width is 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 to evaluate anti-obesity effect was measured.

Alpha-glucosidase inhibitory activity was measured by 50 μl of 0.5 U / ml alpha-glucosidase enzyme solution in 50 μl of the sample (four concentrations of 0.25 mg / ml, 0.5 mg / ml, 0.75 mg / ml and 1 mg / ml, respectively). 50 μl of 200 mM sodium phosphate buffer (pH 6.8) was added thereto, pre-incubated at 37 ° C. for 10 minutes, and then 100 μl of sodium phosphate buffer (pH 6.8) was added and reacted at 37 ° C. for 10 minutes. 0.75 ml of 100 mM Na ₂ CO ₃ was added to the reaction solution to stop the reaction, and the absorbance was measured at 420 nm. The negative control proceeded in the same manner using distilled water instead of the sample. It was calculated as, and the result is shown in FIG.

Alpha-glucosidase inhibitory activity (%) =

 [1- (negative control absorbance ÷ experiment control absorbance)] × 100

The carbohydrate enzyme alpha-glucosidase inhibitory activity of the present invention was enhanced compared to before fermentation, and it can be seen that the enhancement range was increased in a concentration dependent manner (FIG. 7 a).

Pancreatic-lipase inhibitory activity was measured in 50 μl samples (0.25 mg / ml, 0.5 mg / ml, 0.75 mg / ml, 1 mg / ml, respectively), 50 μl of 1.0 U / ml pancreatic lipase enzyme solution, and 200 mM sodium phosphate buffer. (pH 6.8) 50 μl was mixed and pre-incubated at 37 ° C. for 10 minutes, and then 100 μl of sodium phosphate buffer (pH 6.8) was added and reacted at 37 ° C. for 10 minutes. 0.75 ml of 100 mM Na ₂ CO ₃ was added to the reaction solution to stop the reaction, and the absorbance was measured at 420 nm. The negative control proceeded in the same manner using distilled water, and the difference in absorbance was expressed as a percentage by the following equation. It was calculated and the result is shown in FIG.

Pancreatic-lipase inhibitory activity (%) =

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

Pancreatic-lipase inhibitory activity, which is a lipolytic enzyme of soybean-yogurt according to the present invention, was also enhanced compared to before fermentation, and the extent of enhancement was increased in a concentration-dependent manner (FIG. 7). B).

Example 4 Functional Assay of Soybean Fermentation Composition

<Fat reduction functional test>

The effect of fat reduction of the soy-yogurt according to the present invention was measured using 3T3-L1 adipose precursor cells (ATCC).

3T3-L1 adipocytes were cultured at 37 ° C. and 5% CO ₂ using DMEM medium (Dulbecco's modified Eagle's medium) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. To induce the differentiation of 3T3-L1 adipocytes into adipocytes, the cells were dispensed at 5 × 10 ⁵ cells / μl in 60 cm plates and the first day was 1.5 μg / ml insulin, 1 μM dexamethasone, 500 μM IBMX and 1 μM rosiglitazone (3T3-L1 adipocytes). DMEM medium to which L1 differentiation kit, Biovision) was added was treated.

Three days after the induction of adipocyte differentiation, the soy-yoghurt extract sample was incubated for 14 days with DMEM containing 1.5 μg / ml insulin once every two days. The medium was removed from cultured 3T3-L1 adipocytes, washed three times with PBS, stained with oil red O, and measured for the amount of triglyceride (TG). After removing the medium from the L1 progenitor cells and washing three times with PBS, the cells were collected using a rubber policeman and observed under a microscope. The results are shown in FIG. 8A.

Suspended by adding an appropriate amount of TG standard diluent (Triglyceride colorimetric assay kit, Cayman chemicals) to the collected cells, and then treated with ultrasound (1 second, 20 ×) and centrifuged for 15 minutes at 12000rpm. After centrifugation, TG in the supernatant was measured by Triglyceride colorimetric assay kit. The results were shown in FIG. 8B with the control adjusted to 100. FIG.

As shown in FIG. 8A, as a result of oil red O staining, 3T3-L1 adipocytes were not treated with differentiation inducers, but neutral lipids were not generated (untreated), and when treated with differentiation inducers. Neutral lipids of the red line were produced (treated). When the soy-yogurt sample was added to the cells that produced the neutral lipid by adding the differentiation inducing substance to the fat precursor cells, the staining was similar to the case of treatment of the differentiation inducing substance because the inhibitory effect of neutral lipid production was not large. When fermented soy-yogurt samples were processed after fermentation, it was confirmed that the neutral lipids were greatly reduced and the neutral lipid formation inhibitory effect was observed.

As shown in FIG. 8B, the neutral lipid content was also significantly reduced when the soy-yogurt sample was added after fermentation.

<Weight loss functional test>

The mouse animal diet group was used to assay the weight loss effect of the soy-yogurt of the present invention. Normal diet group (25% Kcal, normal diet, ND), high fat diet group (60% Kcal, high-fat diet HFD), high-fat conjugated linoleic acid group (CLA) and high-fat diet soy- yogurt group 4 groups of (SPY) were set.

Each of the four groups of mice consumed a total of 3 grams of diet in 12 hours of light and dark conditions for 12 weeks of breeding, and was fed at 8:30 am once every two days and weighed weekly. And recorded, the results are shown in FIG. 9.

As shown in Figure 9, the soy-yogurt diet SPY group according to the present invention showed a weight of 38.73 g at 12 weeks of breeding, CLA group (37.79 g) fed conjugated linoleic acid (CLA) known to have a weight loss effect and Almost the same effect.

<Glucose improvement functional assay>

The mice of each diet group bred for 12 weeks were subjected to ether anesthesia, blood samples were drawn from the heart using a syringe, and centrifuged for 10 minutes to recover serum to measure blood glucose and c-peptide content. 10 is shown.

SPY group according to the present invention showed a tendency to decrease the blood sugar level to 232.83 mg / dL (253.60 mg / dL) when compared to the HFD group, close to the blood sugar content (206.14 mg / dL) of the general diet group (ND group) (FIG. 10A).

The c-peptide content of the HFD group was 2299.47 pg / dL, which was the highest insulin resistance, and the ND group was the lowest, 1563.81 pg / dL, and the SPY diet group according to the present invention was 1656.33 pg / dL. Insulin resistance similar to that was shown to be effective in reducing blood glucose (Fig. 10b).

Fatty liver improvement functional test

The mice of each diet group bred for 12 weeks were subjected to ether anesthesia, and liver tissues were extracted. First, the liver morphology was observed and weighed, and the results are shown in FIG. 11A.

The liver shape and color of each diet group showed that the HFD group had more fat than the ND group, CLA group, and SPY group, resulting in hypertrophy of the liver, and the color was light brown. In the ND group, liver weight was measured to be 1.58 g and in HFD group, it increased to 2.20 g. In the SPY group of the present invention, 1.54 g showed almost the same weight as the ND group, and the liver weight was significantly lower when compared with the HFD group (FIG. 11A).

Liver histological observations were made by Haematoxylin and eosin (H & E) staining and oil red O staining. In H & E staining, the extracted liver tissue was fixed in 4% neutralized paraformaldehyde solution, and then washed in ethanol (70, 80, 90, 95 and 100%), respectively. After dehydration in xylene solution for 3 minutes, washed in ethanol and soaked in hematoxylin solution for 3 minutes. After washing again in ethanol and finally stained in the eosin solution to observe each tissue under a microscope, the results are shown in Figure 11 (B). Oil red O staining was performed by fixing the extracted liver tissue with 3.7% formalin for 1 hour, washing with 60% isopropanol, and staining with oil red O dye for 20 minutes. After staining, the cells were washed three times or more with distilled water, and the stained cells were observed through a microscope, and the results are shown in FIG. 11B.

HN and oil red O staining results were also observed a lot of adipose tissue stained in red in the HFD group, a lot of adipose tissue in the SPY group and the reference group CLA group of the present invention, soybean according to the present invention -Yogurt was found to be effective in improving fatty liver (FIG. 11B).

From the results of the above functional assay, the soybean fermentation composition according to the present invention not only simultaneously increases the content of Gaba, conjugated linoleic acid and nonglycoside isoflavones (Daidzein and Genistein), but also enhances antioxidant activity, decreases fat, reduces weight, and improves blood sugar. In addition, it can be seen that it is useful as an anti-diabetic and anti-obesity functional food because it has an effect of improving fatty liver.

Institute of Agricultural Biotechnology KACC92159P 20161212 Institute of Agricultural Biotechnology KACC91848P 20130719

Claims (9)

A method of preparing a soybean fermentation composition containing enhanced Gabba and conjugated linoleic acid by fermenting soybeans with the seed of Lactobacillus brevis WCP02 strain deposited with accession no. to,
The soybean is germinated-steamed soybeans sprouted 30 to 60 minutes at 100 ~ 120 ℃,
The complex probiotic spawn is a method in which the Lactobacillus brevis WCP02 strain and the Lactobacillus plantarium P1201 strain are mixed at a mixing ratio (v / v) of 1: 1.
delete The process for preparing a soybean fermentation composition containing enhanced Gaba and conjugated linoleic acid according to claim 1, wherein before fermentation, the beans are processed into kiwi juice to make soybean hydrolysates.
A soybean fermentation composition prepared by the process according to claim 1 or 3, which contains enhanced Gaba and conjugated linoleic acid.
delete Functional food for diabetic improvement comprising soybean fermentation composition according to claim 4.
Functional food for improving obesity comprising soybean fermentation composition according to claim 4. delete delete

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