KR100370796B1 - Bacillus subtilis CG-1 and Lactobacillus delbrueckii CG-2, and the preparation of fermented soybean by mixed culture of them - Google Patents

Abstract

The present invention relates to novel microbial Bacillus subtilis CG-1 and Lactobacillus delbrueckii CG-2 microorganisms, and soybean fermented products by mixed culture of these microorganisms. It relates to a manufacturing method thereof.

The soybean fermented product prepared by inoculating and culturing the novel microorganisms of the present invention has high enzymatic activity, increased effective physiologically active ingredients, stable preservation and high intestinal survival rate.

The soybean fermented product prepared above may be dried and powdered, and then formulated into granules, capsules, pastes or tablets to simplify taking.

Description

Bacillus subtilis CG-1 and Lactobacillus delbrueckii CG-2, and the preparation of fermented soybean by mixed culture of them}

The present invention is a new microorganism Bacillus subtilis CG-1 and Lactobacillus delbrueckii CG-2, and soybean fermented products by mixed culture of these microorganisms and their It relates to a manufacturing method.

More specifically, the present invention provides Bacillus subtilis CG-1, a new microorganism that produces proteolytic and thrombolytic enzymes, and Lactobacillus delbruecchi, which is a microorganism having excellent acid and bile resistance. -2 (Lactobacillus delbrueckii CG-2), and soybean fermentation by the mixed culture of these microorganisms and a method for producing the same, soybean fermentation produced by the mixed culture of the microorganisms is improved antioxidant, carcinogen removal, thrombus It exhibits a wide range of physiological activities, such as dissolution and immune enhancement, as well as high amounts of active isoflavones.

Soybean fermentation products include soybean nutrients as well as enzymes such as amylase, protease, lipase and thrombolytic enzymes produced by microorganisms, and peptides, amino acids, oligosaccharides, fatty acids, and active isoflavones produced by decomposition of these enzymes. In addition, it contains a lot of bioactive substances such as phytosterol, lecithin, saponin.

Foods using soybean fermented products have been converted to more desirable foods than raw materials by enzymatic activity of microorganisms, and have improved nutritional value and shelf life. Major soybean fermented foods include doenjang, soy sauce, red pepper paste, and cheonggukjang. Natto and tempeh in Southeast Asia. Such fermented foods vary depending on the microorganism used. The microorganisms used for naphtha and cheonggukjang are bacteria, but other soybean fermented foods are fermented by fungi.

Patents related to soybean fermentation using Bacillus are mainly related to the production of functional Cheonggukjang or odorless Cheonggukjang (Application No. 2001-72808, 2000-17092, 1999-33912), and patents related to enzymes or viscous substances (Application No. 2000-15153, 2000-9885).

Related patents using lactic acid bacteria include odorless Cheonggukjang functional drink (Application No. 2000-72360), Soybean porridge containing Lactobacillus as its main ingredient, and its manufacturing method (Application No. 2000-2307), Lactobacillus fermented miso and Cheonggukjang (Application No. 1998-1090), a method for preparing fermented soy milk (Application No. 1998-1570), and a method for preparing a separated soy protein lactobacillus fermentation product containing an fruit vegetable fermentation extract (Application No. 1997-924).

The above inventions ferment the Bacillus or lactic acid bacteria alone or in a stepwise fermentation, using fermented products such as functional Cheonggukjang or odorless Cheonggukjang, enzymes or viscous substances, functional drinks, lactic acid bacteria fermentation products.

On the other hand, when mixed and cultured with different microorganisms, the characteristics of each microorganism can be exerted to produce a material having a complex function, but the environment or conditions must be adjusted to an optimal state for culturing mixed microorganisms. There are limitations. Therefore, in order to obtain an effect over the single culture by mixing culture, mixed culture is possible first, and it is necessary to select microorganisms with synergistic effects and optimize the culture conditions of these microorganisms.

The inventors of the present invention showed that the microorganism Bacillus subtilis CG-1 and Lactobacillus delbrueckii CG-2 of the present invention were mixed with each other in culture. Containing a large amount of useful ingredients, confirming that the physiological function is also enhanced to complete the present invention.

The present invention provides a novel Bacillus subtilis CG-1 microorganism (KCCM 10337) for producing proteolytic and thrombolytic enzymes and a novel Lactobacillus delbru with acid and bile resistance. Provides Lactobacillus delbrueckii CG-2 microorganism (KCCM 10359).

In addition, the present invention provides a soybean fermented product and a preparation method thereof and a formulation of the soybean fermented soybean prepared by mixing and culturing the microorganism to increase the content of useful components.

The microorganisms of the present invention are isolated from the soybean and kimchi distributed in the soil and market of Korea, Bacillus subtilis Cage-1 microorganisms secrete a large amount of protease and thrombolytic enzymes, Lactobacillus delbrueki seed-2 microorganisms are acid resistant , Has bile resistance. These microorganisms were identified as a new microorganism belonging to Bacillus subtilis and Lactobacillus delbrueckii, respectively. As of March 7, 2002, it was deposited with the Korean Culture Center of Microorganisms and received KCCM 10337 and KCCM 10359, respectively.

When the soybean fermented product was prepared by mixing the microorganisms, enzymes such as amylase, protease, lipase, and thrombolytic enzyme, and peptides, amino acids, oligosaccharides, fatty acids, and active isoflavones produced by decomposition of these enzymes were produced. Antioxidant, carcinogen removal, thrombolysis, immune enhancement and intestinal survival rate were shown to show superior efficacy compared to conventional soybean fermented food or lactic acid bacteria food.

Screening and Identification of Bacillus Subtilis Cage-1

Microorganisms with excellent proteolytic and thrombolytic enzyme-producing ability were selected from soils collected in Icheon and commercially purchased intestines.

Samples were diluted in sterile sterile water and incubated for 48 hours in pre-prepared NA + Skim milk medium, and then separated according to colony size and shape and the presence / absence of circulation. Each microorganism isolated was inoculated in NA + Fibrin (2%) medium and incubated at 37 ° C. for 48 hours to measure the size of the clear zone. Among the first screened microorganisms, microorganisms having high protease activity were selected, and each microorganism was further cultured in liquid to further raise fibrin-decomposing microorganism Bacillus subtilis (KCCM 10337; Dec. 5, 2001).

This microorganism was a genus of Bacillus, Gram positive, rod-shaped with spores, and catalase positive. The microorganism was able to grow up to 20 ~ 55 ℃ and was flameproof because it could be grown at 10% NaCl. It also secreted proteases and amylases.

Automated strain identification VITEK system (bioMereux co.) Was used to investigate the sugar utilization of this microorganism.

The properties of the novel microbial Bacillus subtilis CG-1 according to the present invention are described in Tables 1, 2 and 3.

Table 1. Morphological and Cultural Characteristics of Bacillus subtilis CG-1

Item characteristic Gram dye reaction + shape Bacillus motility + Sporulation +

Table 2. Physiological and Biochemical Properties of Bacillus subtilis CG-1

Item characteristic Hemolytic - Oxidase - Catalase + Oxygen composition Conditional anaerobic Indole formation - Reducing ability of nitric acid + Urease formation + Methyl red test - Starch hydrolysis + Bogas-Proscauer test +

Table 3. Organic Utilization of Bacillus subtilis CG-1

Item characteristic Glucose + Sucrose + Rhamnose _ Maltose + Inositol _ Sorbitol + Maltose + Arabinose + Gluconate + Malonate +

Screening and Identification of Lactobacillus Debruecki Seed-2

Lactic acid bacteria with acid resistance and bile resistance were purely isolated from fermented foods collected in the market, and microorganisms having excellent flavor and color were selected during substrate fermentation.

The screening method of the excellent acid resistance microorganism is to adjust the initial pH of MRS broth to 5.0, 4.5, 4.0, 3.5 and 3 respectively, and look at the growth state and dilute it to 10 ^-5 ~ 10 ^-7 in MRS agar at 30 ℃ 48 The colony number was measured after time incubation. In addition, to select bile-resistant microorganisms, the pH of the MRS broth was adjusted to 7.0, and the concentration of bile acids (Oxgall, Difco) was adjusted to 0, 0.5, 1.0, 1.5, 3, and 5%, respectively. Measured.

Lactobacillus delbrueckii (KCCM 10359; Mar. 7, 2002), which has been found to be excellent in acid resistance, bile resistance and cholesterol lowering ability by testing cholesterol lowering function among microorganisms found to be excellent in the above process. Deposited).

This microorganism was of the genus Lactobacilli, Gram positive, rod-shaped, and catalase negative. The microorganism was able to grow up to 20 ~ 45 ℃ and the sugar utilization of this strain was investigated using the automated strain identification VITEK system (bioMereux co.).

The properties of the novel microbial Lactobacillus delbrueckii CG-2 according to the present invention are described in Tables 4, 5 and 6.

Table 4. Morphological and Cultural Characteristics of Lactobacillus delbrueckii CG-2

Item characteristic Gram dye reaction + shape Bacillus motility - Sporulation -

Table 5. Physiological and Biochemical Properties of Lactobacillus delbrueckii CG-2

Item characteristic Hemolytic - Oxidase - Catalase - Oxygen composition Aerobic Indole formation - Reducing ability of nitric acid - Urease formation - Methyl red test - Starch hydrolysis - Gelatin hydrolysis - O / F test O + F Sight rate test +

Table 6. Sugar Utilization of Lactobacillus delbrueckii CG-2

Item characteristic Glucose + Fructose + Mannose + Sobos - Rhamnose - Mannitol - Sorbitol - Cellobiose + Maltose - Sucrose - Ribose - Arabinose - Gluconate - Glycerol - Zyritol -

Mixed Culture of Bacillus Subtilis Seed-1 and Lactobacillus delbruequie Seed-2

Bacillus subtilis seed-1 is dissolved in 10 g of soybean powder, 3 g of juice, 5 g of peptone in 1 L of water and cultured in a shaker at 37 ° C. for 2 days to use as a spawn seed. 10 g of peptone, 10 g of broth, 5 g of yeast extract, ₂ g of K ₂ HPO _4, 2 g of Ammoniumcitrate, 2 g of glucose, 1 g of Tween80, 5 g of sodium acetate, 5 g of NaCl, 5 g of MgSO ₄ · 7H ₂ O, 0.5 g of MnSO ₄ · 2H ₂ O Dissolve in 1 L of water and incubate at 37 ° C. for 2 days in a medium prepared as a spawn seed. Then, inoculate 1 to 5% of each spawn seed with soybeans, incubate at 30 to 45 ° C, incubate, and proceed with mixed fermentation until a sticky viscous substance and a sour aroma are produced. Preferably fermentation is carried out for 2 to 3 days. Most preferably, 1 kg of selected soybeans are washed with water, immersed in water for 12 hours, cut for about 1 hour, and then steamed, and then inoculated in the previously cultured seedlings in a fermentation vessel at 37 ° C. (humidity 80 to 90%). Incubate for 48 hours. After the fermented soybeans prepared in the mixed fermentation step were put in a aging vessel at 2 to 10 days at 20 ° C., the fermented products were lyophilized at −50 ° C. for 2 days or dried at 80 ° C. for 2 days. Refrigerate (or freeze) at 4 ° C.

Preparation and Activity Measurement of Soybean Fermented Extract

In order to prepare a soybean fermentation extract dried high solubility enzyme from the soybean fermentation prepared above, butanol was added, mixed well, and then concentrated under reduced pressure concentrator. The concentrated coenzyme solution was dialyzed with 20 mM Tris-HCl buffer (pH 7.5), fractionated and precipitated with EtOH 60%, centrifuged (12,000 rpm, 20 minutes), and the precipitate was collected in 20 mM Tris-HCl buffer (pH 7.5). After dissolving, dialysis, and assaying for activity, the enzyme was purified using DEAE-sephadex A-50 Column to obtain a highly inactive enzyme solution. The fractionated protein was lyophilized at -50 ° C at 0.5 atmosphere to obtain a crude enzyme product in powder form.

The lyophilized soybean fermented product was extracted for 24 hours in 70% ethanol containing 0.1% acetic acid and then centrifuged at 12,500 rpm for 5 minutes and the supernatant was taken. The isoflavone was adsorbed while passing the supernatant at a constant rate to the column containing the activated adsorptive resin, washed with about two volumes of purified water, and recovered by desorbing the isoflavone with 95% ethanol. The recovered isoflavones were lyophilized at -50 ° C 0.5 atm to form a powder.

The functional activity of the prepared soybean fermentation was measured for thrombolytic activity, antioxidant activity, nitrite scavenging activity, isoflavone extraction and content measurement of phenolic compounds, anticomplementary action (immune function), and gastrointestinal stability test. Antioxidant activity was measured by electron donating ability by DPPH (α, α-Diphenyl β-picryl-hydrazyl), peroxide value and peroxidation inhibitory effect on hepatic lipid. Stability test was carried out for enzyme activity and lactic acid bacteria of soybean fermentation after drying. Acid resistance change was measured.

Meanwhile, the soybean fermented product prepared according to the present invention is dry powdered in a conventional manner to prepare granules, capsules, pastes, or tablets.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.

Example 1

In order to prepare a soybean fermented product by co-culture with the new microorganisms described in the present invention, Bacillus subtilis seed-1 and Lactobacillus del brewier seed-2 were each seed cultured. 1 kg of the selected soybeans were washed with water, immersed in water for 12 hours, cut for about 1 hour, and then steamed at 1.5 kg / cm 2 for 50 minutes in an autoclave. The fermented container was cooled to 50 ° C., and the seed beans were uniformly inoculated at 2% level and cultured at 37 ° C. (80% humidity) for 48 hours. After homogeneous mixing, the mixture was placed in a aging vessel and aged at 20 ° C. for 2 days to 10 days, and the soybean fermented product was lyophilized after rapid freezing treatment.

Comparative Example 1-1

Soybean fermented product was prepared by incubating only the novel microbial Bacillus subtilis seed-1 in Example 1 for 48 hours.

Comparative Example 1-2

Soybean fermented product was prepared by incubating only the novel microorganism Lactobacillus delbrueckee seed-2 in Example 1 for 48 hours.

Comparative Example 1-3

Soybean fermented product was prepared by incubating the novel microbial Bacillus subtilis seed-1 for 48 hours and then inoculating Lactobacillus delbruecki seed-2 for 48 hours.

Example 2

An enzyme solution was prepared to measure the activities of amylase, protease and thrombolytic enzymes from the soybean fermentation products prepared in Example 1 and Comparative Examples 1-1 to 1-3. Enzyme liquid was added to distilled water to 12 times the weight of the sample, shaken and extracted for 3 hours at room temperature, filtered (Toyo filter paper No. 2) and the filtrate was used as a crude enzyme solution. Amylase activity was measured according to the DUN (Dextrinogenic Unit of Nagase) method. 1 ml of enzyme solution was added to 3 ml of 1% starch liquor (pH 4.8) and reacted in a constant temperature water bath at 40 ° C. for 10 minutes, and then 10 ml of 0.1 M-HCl was added to 1 ml of the reaction solution. 10 ml of 0.005% I ₂ -0.05% KI solution was added to 1 ml of the reaction stopping solution, and the absorbance was measured at 660 nm. Protease activity was measured according to the method of Anson (ML) and Chuwonmun (이 原文 二). 5 ml of 0.6% milk casein solution dissolved in sodium phosphate buffer (pH 7.0) was added to 1 ml of the crude enzyme solution. Then, 2 ml of the filtrate was added with 5 ml of 0.5 M Na ₂ CO ₃ and ₁ ml of a 3-fold dilution of the Pauline-Kiocalte oil reagent, followed by color development in a 30 ° C. water bath for 30 minutes. The absorbance was measured at 660 nm using 8452A) and expressed in terms of tyrosine content from a separately prepared standard curve. For thrombolytic activity, 1 ml of coenzyme solution was added to 5 ml of substrate dissolved in 0.65% fibrin in 1 / 15M phosphate buffer, reacted at 40 ° C for 10 minutes, and 5 ml of 0.44M TCA solution was added to stop the reaction. (Whatman No. 2) was added to 5 ml of 0.55M Na ₂ CO ₃ solution to ₂ ml of the filtrate, 1 ml of Folin reagent was added and the absorbance was measured at 660 nm with a UV-vis spectrophotometer. The control was used as a control after deactivating the enzyme activity. The enzyme activity unit was 1 unit of the amount of the enzyme which releases 1mM / ml of tyrosine for 1 minute at 40 ° C. The measurement results are shown in Tables 7, 8 and 9.

Table 7. Comparison of Amylase Production from Soybean Fermentation

division Amylase Enzyme Activity (D.U.N. unit / g) Example 1 Comparative Example 1-1 Comparative Example 1-2 Comparative Example 1-3 After fermentation 268.5 260.7 161.3 254.3 Aging 1 day 283.3 272.9 170.9 250.3 2 days of aging 259.5 244.1 124.3 230.3 Aging 3 days 232.1 214.9 114.4 211.7

Table 8. Comparison of Protease Production from Soybean Fermentation

division Protease Enzyme Activity (unit / g) Example 1 Comparative Example 1-1 Comparative Example 1-2 Comparative Example 1-3 After fermentation 143.7 141.8 123.4 131.1 Aging 1 day 166.3 147.1 122.3 134.7 2 days of aging 179.5 152.2 134.2 153.4 Aging 3 days 186.4 143.8 135.6 154.9

Table 9. Comparison of Thrombolytic Enzyme Activity of Soybean Fermentation

division Thrombolytic enzyme activity (unit / g) Example 1 Comparative Example 1-1 Comparative Example 1-2 Comparative Example 1-3 After fermentation 23.5 20.2 5.2 18.5 Aging 1 day 32.7 19.4 16.5 19.4 2 days of aging 35.8 23.1 10.2 20.5 Aging 3 days 38.5 24.5 8.5 18.4

In the above results, the activity of amylase, protease and thrombolytic enzyme was confirmed to be the highest in Example 1, which was a mixed culture of the novel microbial Bacillus subtilis Cage-1 and Lactobacillus delbruecchi Sig-2 in the present invention. It is shown that it can produce a lot of amylase, protease and thrombolytic enzyme.

Example 3

From the soybean fermentation product prepared in Example 1, a soybean fermentation extract dried with high inactivation of enzyme can be prepared. To prepare this, butanol was added, mixed well, extracted, and concentrated under reduced pressure. The concentrated coenzyme solution was dialyzed with 20 mM Tris-HCl buffer (pH 7.5), fractionated and precipitated with 60% EtOH, centrifuged (12,000 rpm, 20 minutes), and the precipitate was collected in 20 mM Tris-HCl buffer (pH 7.5). After dissolving, dialysis, and assaying for activity, the enzyme was purified using DEAE-sephadex A-50 Column to obtain a highly inactive enzyme solution. The fractionated protein was lyophilized at -50 ° C and 0.5 atm to obtain powder coenzyme.

10 g of lyophilized soybean fermented product was added with 100 ml of distilled water, extracted by stirring at 37 ° C. for 24 hours, and centrifuged and filtered to prepare a water extract. 100 ml of ethanol was added back to the filtered residue, followed by extracting with stirring at 37 ° C. for 24 hours to prepare an ethanol extract by centrifugation and filtration. Antioxidative activity, nitrite scavenging activity, and anti-complement activity were measured for the soybean fermented extract.

The lyophilized soybean fermented product was extracted for 24 hours in 70% ethanol containing 0.1% acetic acid, centrifuged at 12,500 rpm for 5 minutes, the supernatant was taken, concentrated and dried. Meanwhile, the isoflavone was adsorbed while passing the supernatant at a constant rate to the column containing the adsorption resin activating the ethanol extract, and then washed with purified water having a volume of about 2 times and recovered by desorbing the isoflavone with 95% ethanol. The recovered isoflavones were lyophilized at -50 ° C 0.5 atm to form a powder. Isoflavone content analysis was performed using this.

end. Antioxidant activity measurement

Antioxidant activity was measured by electron donating ability by DPPH (α, α-Diphenyl β-picryl-hydrazyl), peroxide value and peroxidation inhibitory effect on hepatic lipid.

The electron donating ability by DPPH is to measure the reducing power of the compound as each extract measures the electron donating effect on DPPH. 2.4 ml of 4 * 10 ^-4 M DPPH solution (dissolved in 99% ethanol) was added to 0.6 ml of the extract, shaken with vortex for 10 minutes, and reacted at room temperature for 10 minutes to measure absorbance at 526 nm. The electron donating effect is expressed as a percentage by using the absorbance of the sample addition group and no addition group as follows.

A: absorbance at sample addition port at 526 nm

B: Absorbance of the sample to which distilled water was added at 526 nm

Peroxide value was measured by antioxidant activity of linoleic acid. After dissolving 1 g of linoleic acid in 20 ml of ethanol, 25 ml of 0.2M phosphate buffer solution (pH 7.0) was added, and water extract and 5 ml of ethanol extract were mixed with linoleic acid-Ethanol solution. The solution was stored in an incubator maintained at 45 ° C., followed by shaking with the addition of 25 ml of chloroform to the reaction solution. The lower layer was taken and 25 ml of acetic acid and 1 ml of KI saturation were added. 50 ml of distilled water is added and titrated with 0.01 N sodium thiosulfate solution to calculate the peroxide value.

S: Proper Consumption of 0.01N Sodium Sulfate Solution (ml)

f: 0.01N sodium thiosulfate solution factor

Peroxidation inhibitory effect on hepatic lipids was investigated in vitro using liver homogenates of rats. In other words, the livers of the rats were extracted and homogenized with a phosphate buffer solution (pH 7.4), and then, H ₂ O ₂ (1M), FeSO ₄ (50 mM) and 0.05 ml of each extract were added thereto, followed by incubation for 40 minutes at 37 ° C. Thiobarbituric acid reactivesubstance (TBARS) content was measured.

Table 10. Antioxidant Activity of Soybean Fermentation According to the Present Invention

division Electron donating ability Peroxide value Inhibition of hepatic lipid peroxidation % meq / kg Mol MDA / g liver Soybean fermentation product of the present invention 37.6 21.7 1018 Raw soybeans 19.1 31.8 ND Bacillus sp. 27.2 25.8 1671 Lactobacillus sp. 22.1 34.5 1689 Asp. oryzae 27.4 35.9 ND Streptococcus sp. 31.0 ND ND Lacto drink ND 32.3 ND

Comparing the above results with other microorganisms, the soybean fermentation product of the mixed culture of Bacillus subtilis seed-1 and Lactobacillus delbrueckee seed-2 in the present invention has a large electron donating ability by DPPH and lipid The antioxidant activity was also great, and the antioxidant activity was judged to be excellent.

I. Nitrite Scavenging Activity

Nitrite is a carcinogenic precursor and is associated with a cancer-causing substance and usually reacts strongly in strong acidity. High nitrite scavenging ability may have anticancer effect due to high scavenging ability of carcinogens in the stomach. To measure the nitrite scavenging ability, add 1 ml of the sample extract to ₂ ml of 1 mM NaNO ₂ and calibrate the volume to 10 ml using 0.1 N HCl (pH 1.2) or 0.2 M citric acid buffer (pH 3.0). After 1 hour of reaction at 1 ml each of the reaction solution was taken. 5 ml of 2% acetid acid and 0.4 ml of Griess Reagent (mixed 1% sulfanylic acid and 1% naphtylamine each prepared with 30% acetic acid in a 1: 1 ratio, prepared immediately before use) were mixed well and reacted at room temperature for 15 minutes. After absorbance was measured at 520 nm, the value was calculated by the following equation.

N: Nitrite Scavenging

A: Absorbance of nitrite solution + sample

B: Absorbance of NaNO ₂ solution

C: Absorbance of sample

Table 11. Comparison of Nitrite Scavenging Ability over Time with Soybean Fermentation According to the Present Invention

division Nitrite Scavenging Capacity by Period (%) At the time of manufacture 1 month 2 months 3 months Soybean fermentation product of the present invention 72.6 79.4 79.4 79.6 Raw soybeans 44.9 42.8 38.7 34.6 Bacillus sp. 45.2 45.7 37.3 36.2 Lactobacillus sp. 51.2 61.9 61.7 60.3 Asp. oryzae 48.5 55.5 54.5 49.7 Lacto drink 52.2 54.1 53.3 53.3

Comparing the results with those using other microorganisms, the soybean fermentation by the mixed culture of Bacillus subtilis Seed-1 and Lactobacillus delbrueckee Seed-2 according to the present invention increases or decreases the nitrite scavenging ability according to the storage period. The scavenging ability of nitrite, a carcinogen, was excellent.

All. Thrombolytic activity

Thrombolytic activity was measured according to the method described in Example 2. The thrombolytic ability was measured by comparing soybean fermented product and fermented product with several other microorganisms in the present invention and the results are shown in Table 12.

Table 12. Thrombolytic Activity of Soybean Fermentation According to the Present Invention

division Soybean fermentation product of the present invention Bacillus sp. Soybean fermentation Lactobacillus sp. Bacillus sp. Thrombolytic ability (unit / g) 37.6 23.1 10.2 24.3

D.measurement of anticomplement action

In general, anti-tumor substances are known to have anticancer activity by non-specifically activating the immune system, and by measuring the anti-complementary action of soybean fermentation according to the present invention, it was confirmed whether there was a non-specific immunoactivating action. Soybean fermented extract prepared in Example was prepared at various concentrations and mixed with complement (Gibco, guinea pig complement) and reacted for 30 minutes at 37 ℃ (Total Hemolytic Complement, TCH ₅₀ ) Was measured using an amount of erythrocyte sensitized with hemolysin (Antisheep-hemolysin) (1 × 10 ⁹ cells / ml). The control group was used without fermented extract and the anti-complement activity was calculated as follows and the results are shown in Table 13.

Table 13. Comparison of anticomplement effect of soybean fermentation according to the present invention

division Anti-complement effect by dose (%) 0.5 g / L 1.0 g / L 2.5 g / L 5.0 g / L Soybean fermentation product of the present invention 38.0 60.9 75.3 81.5 β-glucan 55.7 72.9 91.3 89.9 Lentinan 60 88.7 95.7 ND

Like betaglucan and lentinan, which are already known to exhibit anticancer activity due to activation of nonspecific immune actions such as anticompletion, soybean fermentation products in the present invention have been confirmed to have anticomplementary action, a nonspecific immunopotentiating action. .

hemp. Isoflavone Content Analysis in Polyphenol Compounds

The quantification of the isoflavone component was performed by HPLC method which was partially modified by Wang et al. 2 g of fully dried soybean fermented extract was filtered by mixing 2 ml 0.1N HCl and 10 ml acetonitrile, dissolved in 10-fold 80% methanol, and filtered and used as an analytical sample. For the column, ODS-based YMC AM303 was used, and acetonitrile containing 0.1% acetic acid and water containing 0.1% acetic acid were used as mobile phase solvents. Acetonitrile was adjusted to a concentration gradient of 35% for 15 minutes at initial 15%, flow rate was 1.0 ml / min, and measured by UV detector at 254 nm. The experimental results were performed three times and calculated as average values.

Table 14. Active Isoflavone Content of Soybean Fermentation According to the Present Invention

division Active Isoflavone Content (Unit: ㎍ / g) Total isoflavone content Genistein Glystein Dyed Jane Soybean fermentation product of the present invention 1356.7 518 911.2 2785.9 Raw soybeans 578.6 536.2 362.9 1477.7 Bacillus sp. 912.9 215.9 816.2 1954

Among the polyphenol compounds isoflavones, Genistein, which acts as a female hormone estrogen-like substance, is excellent in preventing menopause in women, and dyzein is well known to help absorb calcium and strengthen bones. According to Table 14, the content of the zenithine and dyed zein contained in the soybean fermentation product by the mixed culture of Bacillus subtilis seed-1 and Lactobacillus delbrueckee seed-2 in the present invention is about 2 than soybean itself. It was more than doubled and produced more than in similar soybean fermented products.

Example 4

When the fermented foods containing microorganisms are ingested, the stability of the microorganisms is reduced by gastric acid, and thus the destruction or death or intestinal fixation rate is lowered, thus preventing the function of the microorganisms from retaining. Therefore, in order to show the effects of fermentation microorganisms normally, acid resistance and bile resistance are required. In order to measure this, samples incubated for a certain period of time on artificial media prepared with a composition similar to gastric acid and bile acids in the human body using the soybean fermented product and other fermented foods prepared in Example 1 were inoculated into flat medium and subjected to acid and bile resistance. The number of microorganisms (excluding lactic acid bacteria) was measured, and the lactic acid bacteria were measured under the same conditions to determine the stability of the lactic acid bacteria in the intestine.

Table 15. Microbial Stability Measurement of Soybean Fermentation According to the Present Invention

division Survival Microorganisms in Gastric / Bile Acid Environments (CFU / g,% Survival) Survival Lactobacillus in Gastric / Bile Acid Environments (CFU / g,% Survival) No treatment 1 hour treatment 2 hours treatment 3 hours treatment No treatment 2 hours 4 hours 6 hours Soybean fermentation product of the present invention 1.0 * 10 ⁹ 100% 8.0 * 10 ⁸ 80% 2.0 * 10 ⁸ 20% 9.0 * 10 ⁷ 9% 2.0 * 10 ⁹ 100% 1.1 * 10 ⁹ 55% 3.0 * 10 ⁸ 15% 1.3 * 10 ⁸ 6.5% Mixed food 1.0 * 10 ⁹ 100% 4.6 * 10 ⁷ 4.6% 2.3 * 10 ⁶ 0.23% 7.0 * 10 ⁴ 0.007% 1.0 * 10 ⁹ 100% 8.0 * 10 ⁷ 8% 4.0 * 10 ⁷ 4% 1.2 * 10 ⁷ 1.2% Lactic acid bacteria beverage 1.0 * 10 ⁹ 100% 2.4 * 10 ⁷ 2.4% 4.0 * 10 ⁵ 0.04% 1.0 * 10 ⁴ 0.001% 1.2 * 10 ⁹ 100% 2.4 * 10 ⁷ 2% 1.0 * 10 ⁷ 1% 8.0 * 10 ⁶ 0.8%

In addition, in order to confirm the stability during storage, the soybean fermentation product of the present invention was measured at room temperature and measured for each activity change.

Table 16. Comparison of Stability with Preservation Period of Soybean Fermentation According to the Present Invention

division unit Manufacturing value Remaining amount by retention period (%) 2 months 4 months 6 months pH 6.8 - - - Water content % 4.7 - - - microbe Viable count CFU / g 5.0 * 10 ¹⁰ 84 66 50 enzyme Thrombolytic ability unit / g 37.7 96 90.2 78.2 Antioxidant activity Electron donating ability % 33.9 111 122 108 Hepatic lipid peroxidation Mol MDA / g liver 984 96.5 82.4 71.6 Anticancer Nitrite scavenging activity % 72.6 109.4 109.4 109.6

From the above results, the soybean fermented product prepared by the mixed culture using Bacillus subtilis Seed-1 and Lactobacillus delbrueckee Seed-2 according to the present invention has excellent acid resistance, bile resistance, and high intestinal viability, even when ingested. It was determined that the physiological activity possessed at the time of manufacture could be maintained in the body, and each activity was relatively stable even at room temperature storage after manufacture.

It is possible to prepare a mixed soybean fermented product with increased enzymatic activity and effective physiologically active ingredients by mixed culture of the novel microorganism according to the present invention. In addition, the prepared soybean fermented product has good preservation, good biological activity and high intestinal survival. Therefore, the soybean fermentation product of the present invention can be applied to new food and pharmaceutical materials that can increase bioactivity by reinforcing enzyme activity and effective physiologically active ingredients.

Claims (5)

Bacillus subtilis CG-1 microorganism producing proteolytic and thrombolytic enzymes (KCCM 10337). Lactobacillus delbrueckii CG-2 microorganism (KCCM 10359) with acid and bile resistance. A method for producing a soybean fermented product by mixing and culturing the microorganism of claim 1 and the microorganism of claim 2. Soybean fermented product prepared by inoculating the microorganism of claim 1 and the microorganism of claim 2 together. Granules, capsules, pastes or tablets prepared by dry powdering the soybean fermentation product of claim 4.