KR101562653B1 - Bacillus subtilis CS135 strain and preparation method of Chounggukjang containing Momordica charantia using the same - Google Patents

Abstract

The present invention relates to a novel Bacillus subtilis CS135 strain having excellent antimicrobial activity against food-borne microorganisms and having efficacy from Yeoju Cheonggukjang while reducing bitterness and odor characteristic of Cheonggukjang, antioxidative activity having excellent palatability and enhanced use thereof, and The present invention also provides a method for preparing Cheonggukjang, which has an anti-diabetic and anti-obesity improving effect, and a method for producing the same.
The fermented Cheonggukjang of the present invention has an antioxidant activity and an alpha-glucosidase activity and a pancreatic lipase inhibiting activity, which are excellent in palatability and reduced odor of Yeoju and odor unique to Cheonggukjang. In addition, the health functional food including the cheonggukjang of the present invention has an anti-diabetic effect and anti-obesity improvement effect. In addition, according to the present invention, the Cheonggukjang can be powdered and easy to ingest, thus being highly useful as a health functional food.

Description

Bacillus subtilis CS135 strain and a method for producing Cheonggukjang of Yeoju using the same,

The present invention relates to a strain of Bacillus subtilis CS135 and a method for producing the yeast chungkukjang. More particularly, the present invention relates to a method for producing a yeast strain of Chungkukjang, which has excellent antimicrobial activity against food-borne microorganisms, The present invention relates to a novel strain of Bacillus subtilis CS135, an excellent anticorrosive activity using the same, an enhanced antioxidative activity, and an antidiabetic and anti-obesity effect.

Modern people are exposed to excessive stress and negative health environment. In particular, lack of exercise, stress, and unbalanced intake of nutrients deteriorate the health status of modern people, and it is a major cause of increasing the incidence of various adult diseases and chronic diseases. As interest in health has increased, interest in health foods such as chongkukjang is increasing.

Chungkukjang is a representative of fermented soybeans in Korea. It is fermented by Bacillus strains such as rice straw, boiled soybeans, and fermented at high temperature without adding salt. Chungkukjang contains various physiologically active substances such as isoflavone, fucic acid, saponin, trypsin inhibitor, tocopherol, unsaturated fatty acid, dietary fiber and oligosaccharide derived from soybean, antioxidant substance and thrombolytic enzyme in a large amount. However, in the case of Chungkookjang, the soybean protein degradation by the enzyme produced from the bacterium during the proliferation of the Bacillus strains has caused a characteristic odor of the Chungkookjang.

A method of using green tea (Patent Document 10-2006-0103570), a method of adding dietary fiber (Patent 10-0979093), a method of adding bamboo salt (Patent 10-1162539), etc. , But the effect was not complete and therefore new methods are still required to be developed.

Yeoju is a vine plant with pak and English name is bitter melon. The official name is Momordica charantia. Origin is not known, but it is estimated to be tropical Asia, including India. Yeoju is a summer vegetable fruit bearing fruit from June to August, harvested in summer with plenty of sunshine, and harvested with less fruit. The more greenish and lumpy the more the lump, the more bitter it is, and the more effective it is. The major component of the yeast is the peptide, charantin, which is contained in the fruit and has a molecular weight of about 93.7 kDa. It is known that insulin-like substances, which are activated by beta-cells that play a crucial role in insulin secretion, contribute to blood glucose regulation ultimately. Vitamin C is especially found in 120 milligrams of 100 grams, which is much higher than strawberry (80 mg / 100 g), lemon (90 mg / 100 g) and cabbage (40 mg / 100 g). Especially, vitamin C contained in Yeoju, unlike other fruits, is stable to heat and does not destroy vitamin C more than other fruits. Yeoju also contains beta-carotene and minerals such as iron and potassium that are converted into vitamin A in the body.

In the past, there have been instances where yeast was processed by tea or ring, but it was rarely used for food because of the bitter taste of Yeoju. Therefore, it is required to develop a new functional food that combines excellent characteristics of Yeoju.

As a result of continuous research to meet the demand in the prior art, the present inventors have isolated and identified a novel strain of Bacillus subtilis CS135 in soy sauce, and found that this strain has excellent antimicrobial activity against food microorganisms, And it is confirmed that it has the efficacy of Yeoju Cheonggukjang, and the present invention is completed.

Accordingly, an object of the present invention is to provide a novel strain of Bacillus subtilis CS135.

It is a further object of the present invention to provide a method for the treatment of Bacillus subtilis CS135, which comprises the step of reducing the bitterness and odor characteristic of Cheonggukjang using the strain Bacillus subtilis CS135, which has excellent palatability and enhanced antioxidative activity and alpha-glucosidase and pancreatic lipase- And a method for producing the same.

It is still another object of the present invention to provide a health functional food for improving antidiabetic and anti-obesity comprising the chrysanthemum of the present invention.

In order to achieve the object of the present invention, the present invention provides Bacillus subtilis CS135 strain having excellent antimicrobial activity against food-borne microorganisms and having the efficacy of Yeoju Cheonggukjang while reducing the bitter taste of Yeoju and odor characteristic of Chungkukjang.

The present inventors isolated various Bacillus species from soybean paste (soybean paste) and evaluated the antimicrobial activity against food-borne microorganisms, the ability to breed according to the addition of yeast having strong antimicrobial activity, the bitter and bitter taste of Cheonggukjang, the efficacy of Yeoju Cheonggukjang, And fermented Cheonggukjang were investigated for their antioxidant activity, alpha-glucosidase inhibitory activity and pancreatic lipase inhibitory activity. These strains were identified and named as 'Bacillus subtilis CS135' (KACC) on December 12, 2012 (accession number: KACC 91771P).

Gram stain and transmission electron microscopy were used for morphological observation of Bacillus subtilis CS135, and API50CHB kit for testing biochemical biochemical characterization. Cell wall fatty acid analysis (MIDI analysis) and 16S rDNA sequence analysis. The Bacillus subtilis CS135 of the present invention based on the 16S rRNA base sequence belonged to the conventional Bacillus sp. Strains (Fig. 2).

The antimicrobial test for the strain of the present invention was tested for the inhibitory effect against the growth of food-borne microorganisms distributed in the Korean Collection for Type Cultures (KCTC), and the strain of the present invention was Bacillus cereus Bacillus cereus KCTC1012) and Listeria monocytogenes KCTC 3569 (Fig. 1).

The strain of the present invention has a high survival rate comparable to the case where no yeast is added even when the yeast having a strong antibacterial activity is added, and thus it has the characteristics as fermenting bacteria of Yeongju Cheonggukjang (Figs. 4A and 4B).

In addition, the strain of the present invention has the property of imparting excellent palatability by reducing the bitter taste of Yeoju and the odor intrinsic to Cheonggukjang during fermentation of Cheonggukjang (see Table 1).

According to another object of the present invention, there is provided a pharmaceutical composition for preventing or treating odor of chrysanthemum which has excellent odor, reduced antioxidant activity and anti-diabetic activity and anti-obesity effect by using Bacillus subtilis CS135 strain Provides a manufacturing method of Cheonggukjang of Yeoju.

The manufacturing method of the Yeoju cheonggukjang of the present invention

I) providing a mixture of 95 to 99% by weight of bean called water and 1 to 5% by weight of flax powder;

Ii) cooling the mixture to 35 to 45 DEG C by increasing the temperature at 100 to 120 DEG C for 15 to 60 minutes;

Iii) inoculating 2.5 to 5.0% (v / w) of Bacillus subtilis strain CS135 and fermenting at 35 to 45 ° C for 2 to 5 days.

The production method of the present invention may further comprise, if necessary, after step (iii), drying and pulverizing the produced cheonggukjang.

Step i): Yeoju-bean mixture

A mixture of 95 to 99% by weight of beans called water and 1 to 5% by weight of flax powder.

In the present invention, the soybean may be white rice (yellow soybean), black rice (black soybean), or black soybean (soybean), but is not limited thereto. Soak the beans 2-3 times as much as water for 6 - 24 hours, remove the water and prepare the beans called water.

Yeoju powder is washed and then cut and dried appropriately and used as a powder of 80 ~ 100 mesh size by a pulverizer.

When the yeast powder is added in an amount exceeding the above amount, the fermentation of the fermentation microorganism is insufficient due to the strong antibacterial activity of Yeoju, so that the fermentation of Cheonggukjang is not performed well.

Step ii): Addition and Cooling

Boil-loose soybean mixture from step i) is cooled to 35-45 ° C by heating at 100-120 ° C for 15-60 minutes.

It is possible to sterilize Yeoju - called soybean mixture through the increase of sugar, so that the fermentation does not propagate germs and the macromolecule protein is converted into a low - molecular protein, thereby increasing the efficiency of fermentation of Bacillus strains and fermentation of Bacillus strains.

Step iii): Inoculation and fermentation

The mixture is inoculated with 2.5 ~ 5.0% (v / w) of Bacillus subtilis CS135 and fermented at 35 ~ 45 ℃ for 3 ~ 5 days.

The Bacillus subtilis strain CS135 is used for seed culture according to a conventional method. The culture can be carried out as a culture medium in a TSB (Trytic Soy Broth) liquid medium for about 12 hours, but is not limited thereto.

It is preferable that the inoculation amount is 2.5 to 5.0% (v / w). If it is more than 5.0% (v / w) Which may affect the fermentation and quality of Chungkookjang.

Step iv): Preparation of Chungkukjang Powder

If necessary, the chungkukjang is dried and pulverized.

The drying is preferably, but not limited to, drying at a temperature of 50 to 55 占 폚 for 2 days or more.

The dried chungkukjang is pulverized with a grinder and pulverized into 80 ~ 100 mesh pulverized powder.

According to another object of the present invention, there is provided a process for producing a fermented soybean oil, which is produced according to the production method of the present invention and has reduced bitterness and odor characteristic of Chungkookjang, and has excellent palatability, antioxidant activity, and alpha-glucosidase and pancreatic lipase inhibiting activity, And Cheonggukjang powder of Yeoju.

According to still another aspect of the present invention, there is provided a health functional food for improving anti-diabetic and anti-obesity foods, including a lucrative cheonggukjang or a lucrative cheonggukjang powder of the present invention.

The Bacillus subtilis CS135 strain according to the present invention is excellent in antimicrobial activity against food microorganisms and has excellent biochemical properties by addition of yeast having high antimicrobial activity, and fermented Yejoo Cheonggukjang by decreasing the bitter taste of Yeoju and the odor characteristic of Cheonggukjang Is a novel strain having characteristics that can be achieved.

The fermented Cheonggukjang prepared according to the production method of Cheonggukjang of the present invention has an antioxidant activity and an alpha-glucosidase activity and a pancreatic lipase inhibiting activity, which are excellent in palatability and reduced odor characteristic of Yeochu and odor of Cheonggukjang. In addition, the health functional food including the cheonggukjang of the present invention has an anti-diabetic effect and anti-obesity improvement effect. In addition, according to the present invention, the Cheonggukjang can be powdered and easy to ingest, thus being highly useful as a health functional food.

1 is a photograph showing the results of an antibacterial activity test of Bacillus subtilis CS135 strain against a food-harmful microorganism. A shows antibacterial activity against Bacillus cereus, and B shows antibacterial activity against Listeria monocytogenes.
Fig. 2 is a schematic diagram showing the phylogenetic location of Bacillus subtilis CS135 strain in Bacillus.
FIG. 3 is a photograph showing the Cheonggukjang and Cheonggukjang powders prepared according to the present invention. Fig. 3 (a) is the Cheonggukjangjang of Yeoju, and Fig. 3 (b) is the powder of Cheonggukjang of Yeoju.
FIG. 4 is a graph showing the bio-cultivation performance of Bacillus subtilis strain CS135 according to the yeast content. FIG. FIG. 4A shows the growth of the strain in the cheonggukjang using the white rice, and FIG. 4B shows the growth of the strain in the cheonggukjang using the seared rice.
5 is a graph showing the total phenolic content of Cheonggukjang in Yeoju. FIG. 5A shows the total phenolic content of chungkukjang using white rice, and FIG. 5B shows the total phenolic content of chungkukjang using seasonal rice.
6 is a graph showing the antioxidative activity of Cheonggukjang in Yeoju. 6a shows the ABTS radical scavenging activity of chungkukjang using white rice, Fig. 6b shows the ABTS radical scavenging activity of chungkukjang using seaweed, Fig. 6c shows the reducing power of chungkukjang using white rice, and Fig. Quot;
FIG. 7 is a graph comparing the total phenolic contents of Yeoju chungkukjang powder and Yeoju chungkukjang powder using Yeoju chrysanthemum powder and Seoritae using white rye, white rye, and white rye.
Fig. 8 is a graph comparing antioxidative activities of Yeoju chungkukjang powder and Yeoju chungkukjang powder using white ryegrass, white rye, and white ryegrass. Figure 8a shows the ABTS radical scavenging activity and Figure 8b shows the reducing power.
Fig. 9 shows the alpha-glucosidase inhibitory activity and pancreatic lipase inhibitory activity of Yeoju chungkukjang powder using Yeoju chungkukjang powder and Seoritae using Yeoju, Baektae and Seolite, and Baektae. Figure 9a shows the alpha-glucosidase inhibitory activity and Figure 9b shows the pancreatic lipase inhibitory activity.

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 Isolation and Identification of Bacillus subtilis CS135 Strain

<Strain isolation>

10 commercial varieties of soybean paste and 8 traditional soybean paste varieties produced in Gyeongnam area were each suspended in 9 ml of sterilized physiological saline with 1 g each. After vigorous shaking, the supernatant obtained by incubation for 5 minutes was used as a sample for strain isolation Respectively.

The samples were diluted with sterilized physiological saline so that 30-300 colonies appeared on each plate, plated on a plate medium of Tryticase Soy Agar (TSA: Tryticase Soy Agar, DIFCO, USA) and incubated in a thermostat at 30 ° C for 48 After culturing for 72 hours, specific colonies of various Bacillus species were taken and purely separated using a plate medium of the same composition.

The isolates were suspended in cryoprotectant broth (TSB: Trytic Soy Broth, DIFCO, USA) containing 20% (v / v) glycerol, Respectively.

The pure isolates were inoculated into a 250-mL Erlenmeyer flask containing 50 g of boiled soybeans and fermented at 39 ° C. in a thermostat to examine the degree of viscous formation and odor generation. Was selected and named Bacillus subtilis CS135.

&Lt; Antibacterial test of strain &

The antibacterial test of the selected Bacillus subtilis strain CS135 was carried out using Escherichia coli KCTC1682, Pseudomonas aeruginosa KCTC 1750, Salmonella enteritidis (Salmonella enteritidis), which was purchased from KCTC, Korea Research Institute of Bioscience and Biotechnology ) Were performed on KCTC12400, Shigella flexineri KCTC2008, Bacillus cereus KCTC1012, Listeria monocytogenes KCTC 3569, and Stephylococcus aureus KCTC1916 .

Specifically, the food-borne microorganism was cultured in TSB liquid medium for 24 hours, the culture liquid was diluted to 10 ⁹ CFU / ml, and 100 μl was spread on TSA plate medium. Bacillus subtilis CS135 strain was cultured in TSB liquid medium for 24 hours, centrifuged, and 100 쨉 l of the supernatant was dispensed on an 8 mm paper disk. The paper disk was attached to the microbial coated TSA plate medium for food, For 24 hours, and the resulting transparent ring size was measured. The results are shown in FIG.

As shown in Fig. 1, the strain according to the present invention showed higher antibacterial activity against Bacillus subtilis strain (CSY191) against Bacillus cereus (A) and Listeria monocytogenes (B).

<Identification of Strain>

The morphological, physiological, biochemical and molecular genetic characteristics of Bacillus subtilis CS135 were investigated and the results were as follows:

The Bacillus subtilis CS135 strain was a Gram-positive bacterium, and the cell wall fatty acid analysis results of the bacterium having mobility and sporulation ability are shown in Table 1. Table 1 shows that the major fatty acids, 15: 0 ISO and 15: 0 ANTEISO, account for more than 78.87%.

fatty acid Furtherance (%) C14: 0 Iso 5.73 C14: 0 1.41 C15: 0 Iso 24.85 C15: 0 Anteiso 54.02 C16: 0 Iso 3.82 C16: 1 w11c 0.94 C16: 0 3.04 C17: 0 Iso 2.60 C17: 0 Anteiso 3.58

Biochemical characterization of the strain using the API50CHB kit (BioMerieux, France), a method for testing the sugar availability of bacteria, resulted in the magnetization of 24 carbon sources including glycerol, and was assumed to be a tuberous anaerobic Bacillus sp. Strain. The growth temperature was 15 ~ 50 ℃, the growth pH was 4.0 ~ 9.0 and the viable salt concentration was ~ 14%.

Sequence analysis of 16S rRNA as a molecular genetic method revealed that it showed 99% high homology with Bacillus subtilis strains through BLASTN (nucleotide-nucleotide BLAST) database (FIG. 2).

The isolated and identified Bacillus subtilis CS135 strain was deposited on December 12, 2012 at the Korean Agricultural Microbiology Resource Center (KACC) (accession number: KACC 91771P).

Production Example: Production of Cheonggukjang and Yeoju Cheonggukjang Powder using Bacillus subtilis CS135 strain

Using the Bacillus subtilis CS135 isolated and identified in Example 1, the Cheonggukjang of Yeoju and the Cheonggukjang powder of Yejoo were prepared as follows:

1000 g of white and yellow seaweeds were immersed in about 2 times of water for about 12 hours, and water was drained for about 2 hours, followed by mixing 1%, 2.5% and 5% (w / w) Lt; 0 &gt; C for 30 minutes.

Bacillus subtilis CS135 medium (0.5 ml, 5 ml) was inoculated in a TSB broth for 12 hours and fermented at 37 ℃ for 72 hours. (FIG. 3A). The resulting mixture was dried at 50 to 55.degree. C. for 2 days, pulverized by a pulverizer, and filtered at 80 mesh to prepare a cheonggukjang powder (FIG. 3B).

As a control, chungkukjang and chungkukjang powders were prepared in the same manner as above except that only the soybean powder was added (0%).

Each of the prepared chungkukjang prepared in Preparation Example was lyophilized and then 200 ml of 50% methanol was added to 10 g of each sample. The mixture was extracted at 160 rpm for 12 hours at room temperature, filtered through a Whatman No. 2 filter paper, Concentrated and used as an experimental sample in the following experimental examples.

Experimental Example 1: Growth and phenolics productivity of Bacillus subtilis CS135

<Fostering>

Samples were taken at 0 h, 12 h, 24 h, 48 h and 72 h fermentation during fermentation of each fermented soybean curd according to the preparation example. The samples were diluted with 90 ml sterilized distilled water, (10 ⁵ to 10 ⁹ ), diluted with TS agar medium and cultured at 30 ° C. for 48 hours. The number of viable cells was measured. The results were shown in FIG. 4A (in case of using cotton seeds) and FIG.

As shown in FIG. 4A and FIG. 4B, the strain of the present invention had a high survival rate comparable to the case of no addition of yeast (0%, control) to the addition of yeast having strong antibacterial activity during the fermentation period, . Further, it can be seen that the strain of the present invention is excellent in bio-cultivation regardless of the kind of soybean.

<Phenolic Productivity>

Total phenolic content was determined by Folin-Denis method.

Specifically, each of the experimental samples prepared in Preparation Example was diluted 100 times with 0.45 μm filter, 0.5 ml was added to a test tube, and 25% Na ₂ CO ₃ 0.5 ml was added and allowed to stand for 3 minutes. Then, 0.25 ml of 2N-Folin-Ciocalteu phenol reagent was added to the mixture, and the mixture was allowed to stand at room temperature for 1 hour to develop color. The color developed blue was measured for absorbance at 750 nm using a spectrophotometer (Spectronic 2D). The content of total phenolics was determined from the standard curves prepared using gallic acid. The absorbance was measured at 750 nm by the method described above so that the final concentration of gallic acid was 0, 25, 50, and 100 mg / l, and the results were shown in FIG. 5a In the case of using a seawater).

As shown in FIGS. 5A and 5B, the total phenolic content was increased with the addition amount of the fennel powder, and the total phenolic content of the fennel was slightly higher than that of the fennel.

Experimental Example 2: Antioxidant activity of Cheonggukjang, Yeoju

Antioxidant activity was determined by measuring the reducing power of ABTS radical scavenging activity and FRAP method.

<ABTS radical scavenging activity>

The ABTS radical scavenging activity is a method of examining the antioxidant ability according to the degree of reduction of the colored radical of 2-azino-bis. This method is a method to measure the antioxidant ability by comparing with the value of sample and reference material (Trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) (ABTS +) is discarded and decolorized to cyan. At this time, the removal degree of ABTS cation (ABTS +) can be found by measuring the absorbance. Specifically, 5 ml of a 7 mM ABTS reagent (Sigma 1888) and 5 ml of 140 mM K ₂ S ₂ O ₈ (FW 270.3, Sigma 9392) were mixed and allowed to stand in the dark for 14 to 16 hours. And the absorbance value of the control was adjusted to 0.70.02 at 732 nm.

0.1 ml of each test sample prepared in the Preparation Example and 0.9 ml of ABTS solution were mixed, shaken for 30 seconds, reacted for 3 minutes, and absorbance was measured at 732 nm. Vitamin C was used in the positive control experiment. Negative control experiments were performed with distilled water instead of samples. The ABTS radical scavenging activity was expressed as a percentage (%) by the following formula, and the results are shown in FIG. 6A (when using white rice) and FIG. 6B (when using a seared rice).

ABTS radical scavenging activity (%) =

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

<Reducing power analysis>

Reducing power was measured by using Liu et al. Based on FRAP analysis. Specifically, reducing power FRAP analysis 300mM acetate buffer in the reaction solution (pH 3.6), 10 mM 2,4,6- tree pyridyl -s- triazine (TPTZ dissolved in 40 mM _{HCl, T1253, C 18 H 12 N} 6 (MW 312.33) and 20 mM FeCl ₃ (F7134, MW 162.20) were prepared, and the acetate buffer solution, the TPTZ solution and the FeCl ₃ solution were mixed at a ratio of 10: 1: 1 (v / v / v) Preliminary reaction was allowed.

0.1 ml of each test sample prepared in the Preparation Example and 0.9 ml of the pre-reacted FRAP reagent were dispensed into a 96-well plate, reacted for 15 minutes, and the absorbance was measured at 590 nm using a microplate reader (Biorad 3055, Sweden) The results are shown in Fig. 6C (when white sheets were used) and Fig. 6D (when using a black sheet).

6A to 6D, the ABTS scavenging activity and reducing power were increased as the amount of the fumarate powder was increased.

From the above results, it can be seen that the Cheonggukjang of Yeoju according to the present invention has an excellent antioxidative activity.

Experimental Example 3. Evaluation of Palatability of Yeoju Cheonggukjang

The preference for bitterness and odor was evaluated for the cheonggukjang prepared in the production example. The palatability evaluation was conducted on 20 adult males and females using the 5-point scaling method. The results are shown in Table 2.

Chungkukjang type (Yeoju powder added amount) Bitterness of Yeoju Odor unique to Cheonggukjang Cheonggukjang (0% in Yeoju) - Control 4.2 3.0 Cheonggukjang of Bae Tai (Yeoju 1.0%) 4.2 3.4 Cheonggukjang of Bae Tai (Yeoju 2.5%) 4.3 3.7 Cheonggukjang of Bae Tai (Yeoju 5.0%) 4.3 4.2 Seolitai Chungkukjang (Yeoju 0%) - Control 4.1 3.2 Chungkookjang (1.0% of Yeoju) of Seoritae 4.2 3.5 Chungkookjang, a SaoriTae (Yeoju 2.5%) 4.2 4.0 Seolitai Chungkukjang (Yeoju 5.0%) 4.2 4.4 Note) As the bitter taste and bad smell are weaker, they are taught to give higher scores.

According to the results shown in Table 2, the bitter taste of Yeoju was similar to that of the control (no added yeast powder) in the range of 1 ~ 5% of Yeoju powder, It can be seen that the odor of Chungkookjang is also removed as the amount of added yeast powder increases.

Experimental Example 4: Comparison of total phenolic contents

The total phenolic content of each of the liquorice powder (5% of the liquor powder and the white liquor or the white liquor mixture) and the liquorice powder, white liquor and white liquor used in the preparation example of the present invention, The measurement was made in the same manner as described and the results are shown in Fig.

7, the total phenolic content was 10.84 mg / g of bitter melon, 4.72 mg / g of yellow soybean, and 10.23 mg / g of black soybean, respectively, (BM-YSC powder) was 18.24 mg / g in the case of using the cheonggukjang powder of the present invention and 25.91 mg / g in the case of using the bleaching agent (BM-BSC powder). Therefore, it can be seen that the yuju chungkukjang powder of the present invention contains a higher total phenolic content than the respective cheonggukjang materials.

Experimental Example 5. Comparison of Antioxidant Activity

The ABTS radical scavenging activity and FRAP analysis of each of the yuju chungkukjang powder (in case of using 5% yuju powder and white or seaweed mixture) according to the present invention prepared in the manufacturing example and the yuzu powder, The results are shown in FIGS. 8A and 8B.

8A and 8B, the ABTS radical scavenging activity was 88.01% for yuzu powder, 31.86% for yellow soybean and 90.81% for black soybean, respectively. (BM-YSC powder) was 120.37%, and BM-BSC powder was 166.27%.

In the case of chalkyookang powder of the present invention, the BM-YSC powder was 1.234 and the ratio of use of black soybean powder was 1.234 and that of black soybean powder was 1.34, (BM-BSC powder) was 1.784.

Therefore, it can be seen that the cheonggukjang powder of the present invention significantly enhances the antioxidative activity as compared with each of the cheonggukjang materials.

Experimental Example 6. Anti-diabetic and anti-obesity effects of Chejuookjang

The inhibitory activity of alpha - glucosidase, an indicator of antidiabetic effect, and the inhibitory activity of pancreatic lipase, which is an index of anti - obesity effect, were assayed.

&Lt; Alpha-Glucosidase Inhibitory Activity >

50 μl of each of the experimental sample solutions prepared in Preparation Example, 50 μl of 0.5 U / ml α-glucosidase enzyme solution, 50 μl of 200 mM sodium phosphate buffer solution (pH 6.8) was preliminarily cultured at 37 ° C for 10 minutes. 100 μl of sodium phosphate buffer solution (pH 6.8) in which p-NPG was dissolved to 5 mM was added, and the mixture was reacted at 37 ° C for 10 minutes . 750 ml of 100 mM Na ₂ CO ₃ was added to the reaction solution to stop the reaction, and the absorbance at 420 nm was measured. The inhibition rate was calculated using distilled water as a negative control. The results are shown in FIG. 9a. The inhibition rate was also determined in the same manner as in the case of the cheonggukjang materials, Baektae and Seolite, as a control, as shown in Fig. 9a.

According to FIG. 9A, the alpha-glucosidase inhibiting activity of the present invention of Cheonggukjang is significantly higher than that of the cheonggukjang material according to the present invention (especially when white rice is used).

From these results, it can be seen that the Cheonggukjang of Yeoju according to the present invention has an effect of improving diabetes.

<Pancreatic Lipase Inhibitory Activity>

50 μl of each test sample solution prepared in Preparation Example, 50 μl of 1.0 U / ml pancreatic-lipase enzyme solution (Sigma-Aldrich, L3126) 50 μl of a 200 mM sodium phosphate buffer solution (pH 6.8) was preliminarily cultured at 37 ° C for 10 minutes. 100 μl of a sodium phosphate buffer solution (pH 6.8) in which p-NPB was dissolved to 5 mM was added and the mixture was reacted at 37 ° C for 10 minutes . The reaction was stopped by adding 750 ml of 100 mM Na ₂ CO ₃ to the reaction solution, and the absorbance at 420 nm was measured. The inhibition rate was calculated using distilled water as a negative control. The results are shown in FIG.

9B, it can be confirmed that the pancreatic lipase inhibiting activity of Yeoju-Cheonggukjang of the present invention is remarkably higher than that of the cheonggukjang material according to the present invention (especially when white rice is used).

From these results, it can be seen that the Cheonggukjang of Yeoju according to the present invention has an effect of improving obesity.

Korea Agricultural Microbiology Resource Center KACC91771P 20121228

Claims (9)

Bacillus subtilis CS135 strain deposited with Accession No. KACC91771P.
2. The method according to claim 1, wherein the strain is bio-
Antibacterial activity against Bacillus cereus KCTC1012 and Listeria monocytogenes KCTC 3569 was strong,
A strain characterized by having the efficacy of Yeoju Cheonggukjang while reducing the bitter taste of Yeoju and the odor characteristic of Cheonggukjang.
A yeast-containing Cheonggukjang produced by using the strain according to claim 1.
As a method of manufacturing a Cheonggukjang field,
I) providing a mixture of 95 to 99% by weight of bean called water and 1 to 5% by weight of flax powder;
Ii) cooling the mixture to 35 to 45 DEG C by increasing the temperature at 100 to 120 DEG C for 15 to 60 minutes; And
Iii) fermenting the strain according to claim 1 at 2.5 to 5.0% (v / w) at 35 to 40 DEG C for 2 to 5 days.
5. The method according to claim 4, further comprising, after step (iii), drying and pulverizing the prepared cheonggukjang.
The cheonggukjang prepared by the manufacturing method according to claim 4, wherein the bitter taste of Yeoju and odor unique to Cheonggukjang are reduced and excellent palatability, and having enhanced antioxidant activity and alpha-glucosidase and pancreatic lipase inhibiting activity.
A cheonggukjang powder prepared by the method according to claim 5, which has reduced bitter taste and odor specific to Cheonggukjang and has excellent palatability, enhanced antioxidant activity and alpha-glucosidase activity and pancreatic lipase inhibiting activity.
A health functional food for improving diabetes and anti-obesity comprising the chrysanthemum according to claim 6.
A health functional food for improving diabetes and anti-obesity comprising the chrysanthemum powder according to claim 7.

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