Strain isolated from traditional meju, soybean koji preparation method using same, and soybean koji prepared by the same preparation method

Abstract

The present disclosure relates to a Bacillus amyloliquefaciens CJ14-6 strain isolated from traditional meju, a preparation method for soybean koji using the same, and a soybean koji prepared by the preparation method. The preparation method for soybean koji includes: soaking soybeans in water or adding waster to soybeans and steaming the soaked soybeans; and inoculating a Bacillus amyloliquefaciens CJ14-6 strain into the steamed soybeans, fermenting the steamed soybeans, and drying the fermented soybeans to prepare a soybean koji.

Claims

1. A method of treating obesity, comprising: administering a composition comprising a culture of Bacillus amyloliquefaciens CJ 14-6 strain, deposited at the Korean Culture Center of Microorganisms under Accession number KCCM 11718P to a subject in need thereof.

2. The method according to claim 1, wherein the Bacillus amyloliquefaciens CJ 14-6 strain is isolated from traditional meju.

3. The method according to claim 1, wherein the composition comprises a soybean koji prepared by using the Bacillus amyloliquefaciens CJ 14-6 strain as a fermentation starter.

4. The method according to claim 3, wherein the soybean koji is prepared by: soaking soybeans in water or adding water to soybeans, and steaming the soybeans; inoculating the Bacillus amyloliquefaciens CJ 14-6 strain into the steamed soybeans; and fermenting the steamed soybeans to prepare the soybean koji.

5. The method according to claim 4, wherein the preparation of the soybean koji further includes soaking selected and washed soybeans at 10 to 50° C. for 1 to 15 hours prior to the steaming step, wherein the steaming step includes forcing a saturated steam of 1.0 to 2.0 kgf/cm.sup.2 to steam the soybeans at 100 to 150° C. for 1 to 60 minutes; and cooling down the steamed soybeans to 30 to 50° C. after the steaming step.

6. The method according to claim 4, wherein the steaming step is conducted in a high-pressure steam sterilizer at 100 to 150° C. for 5 to 15 minutes.

7. The method according to claim 4, wherein the Bacillus amyloliquefaciens CJ 14-6 strain is inoculated into the steamed soybeans in an amount of 0.1 to 3.0 wt. % with respect to the total weight of raw materials.

Description

BRIEF DESCRIPTIONS OF DRAWINGS

(1) FIG. 1 is an image of adipocytes in the control group stained with anillin blue in Experimental Example 1.

(2) FIG. 2 is an image of adipocytes in the Example-2 test group stained with anillin blue in Experimental Example 2.

BEST MODES FOR CARRYING OUT THE INVENTION

(3) Hereinafter, the present disclosure will be described in detail.

(4) In accordance with a first embodiment of the present disclosure, there is provided a Bacillus amyloliquefaciens CJ14-6 strain isolated from traditional meju and having high protease activity.

(5) More specifically, a variety of strains were isolated from Korean traditional meju. As a first selection out of the various strains, Bacillus spp. strains with high protease activity were isolated from the active medium; and as a second selection from the Bacillus spp. strains, a strain with high proteolytic ability was separated from a solid culture medium using soybeans. 16s rDNA sequencing was adopted to identify the final selection of Bacillus spp. Strain.

(6) The Bacillus spp. Strain isolated as the final selection was identified as Bacillus amyloliquefaciens (Sequence No. 1). This strain with high protease activity was named “Bacillus amyloliquefaciens CJ14-6” and accepted by the Korean Culture Center of Microorganisms (KCCM) on Jul. 1, 2015 (Accession No. KCCM11718P).

(7) In accordance with a second embodiment of the present disclosure, there is provided a preparation method for soybean koji that includes a steaming step of soaking soybeans in water or adding waster to soybeans and steaming the soaked soybeans or the soybeans added water; and a making soybean koji step of inoculating a Bacillus amyloliquefaciens CJ14-6 strain into the steamed soybeans and fermenting the steamed soybeans.

(8) More specifically, the present disclosure provides a preparation method for soybean koji that includes: soaking selected and washed soybeans in water or adding water to selected and washed soybeans; steaming and cooling down the soybeans; incubating a Bacillus amyloliquefaciens CJ14-6 strain to prepare a culture medium; and inoculating the culture medium of the Bacillus amyloliquefaciens CJ14-6 strain into the cool soybeans and fermenting the soybeans to prepare a soybean koji.

(9) The preparation method may further include a step of soaking the selected and washed soybeans in a soaking water maintained at 10 to 50° C. for 1 to 15 hours prior to the step of steaming soybeans. The soybeans may be steamed with saturated steam (1.0 to 2.0 kgf/cm.sup.2) at 100 to 150° C. for 1 to 60 minutes. But, the steaming method available in the present disclosure is not limited to the specified method. The steamed soybeans may be cooled down to about 30 to 50° C., more specifically about 30 to 35° C.

(10) The soybeans may be steamed in a high-pressure steam sterilizer (Autoclave) at 100 to 150° C. for 5 to 15 minutes, more specifically at 110° C. for 10 minutes.

(11) The Bacillus amyloliquefaciens CJ14-6 strain may be used in the form of spores incubated in a culture medium. The culture medium may be inoculated uniformly into the steamed soybeans in an amount of 0.1 to 3.0 wt. % with respect to the total weight of the soybean material.

(12) The culture medium for incubation of the strain may be a soy sauce medium. The soy sauce medium as used herein may be prepared by mixing 1 to 10% of a soy sauce selected from the group consisting of a Korean-style soy sauce, a factory-made soy sauce, or a mixed soy sauce and 0.1 to 10% of a sugar selected from the group consisting of glucose, sucrose, galactose, and maltose.

(13) In another embodiment of the present disclosure, the present disclosure includes inoculating a soy sauce medium (containing a factory-made soy sauce and glucose) with a Bacillus amyloliquefaciens CJ14-6 strain, acquired by pure culture isolation and stored, and conducting an incubation in the temperature range of 30 to 42° C. for 20 to 42 hours until the production of spores to prepare a culture medium for Bacillus amyloliquefaciens CJ14-6.

(14) Following the inoculation of the Bacillus amyloliquefaciens CJ14-6, the soybeans are fermented at 30 to 45° C., more specifically at 34 to 44° C., for one to three days to prepare a soybean koji.

(15) The preparation method may further include a drying step subsequent to the fermentation step.

(16) In accordance with a third embodiment of the present disclosure, there is provided a soybean koji prepared by the preparation method for soybean koji.

(17) The soybean koji prepared by the preparation method of the present disclosure may be used in the large-quantity production of factory-made gochujang.

(18) Hereinafter, the present disclosure will be described in detail with reference to the following examples, which are given for the illustrations of the present disclosure only and not construed to limit the scope of the present disclosure.

EXAMPLES

Example 1: Isolation of Strain from Traditional Meju and Identification of Strain

(19) (1) Isolation of Strain and Identification

(20) The strain used as a fermentation starter in the present disclosure was isolated and selected from traditional meju collected from the traditional food manufacturers in the Gyeonggi-do, Gangwon-do, Chungbuk, and Chunnam Provinces.

(21) Traditional meju was diluted in sterilized water, spread on a nutrient agar (Difco), and cultured at 37° C. Five microorganisms living as dominant species in the traditional meju were selected, isolated from the traditional meju through the pure culture isolation method, and identified. The individual strains isolated were named ‘CJ 3-27’, ‘CJ 4-4’, ‘CJ 5-10’, ‘CJ 14-6’, and ‘CJ 16-57’, respectively.

(22) The isolated strains were shaking-cultured (at 200 rpm) in a nutrient broth (Difco) at 37° C. for 24 hours and then measured in regards to protease activity. The results of identification and protease activity are presented in Table 1.

(23) TABLE-US-00001 TABLE 1 Identification Result of Isolated Strains Derived from Traditional Meju Primary strain Protease activity selections (U/g) Identification results CJ 3-27 62.58 ± 0.17 Bacillus amyloliquefaciens CJ 4-4 91.44 ± 6.97 Bacillus licheniformis CJ 5-10 61.72 ± 4.13 Bacillus subtilis subsp. Subtilis CJ 14-6 140.73 ± 4.62 Bacillus amyloliquefaciens CJ 16-57 222.42 ± 0.63 Bacillus licheniformis

(24) (2) Primary Selection

(25) The primary selections from the isolated and identified strains were the strains, such as CJ 3-27, CJ-4-4, CJ 14-6, and CJ 16-57, other than CJ 5-10 that has the lowest protease activity.

(26) (3) Secondary Selection

(27) The four strains given as the primary selections were used in the preparation of soybean koji and measured in regards to protease activity. The measurement results are presented in Table 2. Through a comparison of protease activity, strains with high protease activity were designated as a secondary selection.

(28) For the preparation of soybean koji, 1 kg of soybeans were soaked in purified water for 12 hours, steamed in a high-pressure steam sterilizer (Autoclave) at 110° C. for 10 minutes, and then cooled down to 35° C. Each of the isolated strains was added to the cool soybeans in an amount of 2.0 wt. % with respect to the total weight of the material and incubated at 37° C. for 3 days. The strains thus cultured were used to prepare the individual soybean koji.

(29) To measure the protease activity, each soybean koji was subjected to extraction and filtration at 30° C. for one hour, and the filtrate was used as a coenzyme solution. An enzyme reaction solution was prepared by adding 0.5 ml of the coenzyme solution, 1.5 ml of 2% milk casein as a substrate, and 1 ml of Mcllivine buffer (pH 6.0) and causing an enzyme reaction at 38° C. for one hour. Subsequently, 3 ml of 0.4M TCA solution was added to suspend the reaction, and after a filtration, the reaction solution was sufficiently mixed with 5 ml of 0.4M Na.sub.2CO.sub.3 and 1 ml of phenol reagent. The resultant solution was color-developed at 38° C. for 30 minutes, and the absorbance at 660 nm was determined with a spectrophotometer. In the expression of the enzyme activity, the amount of an enzyme producing tyrosine corresponding to 1 μg per minute was defined as one unit. Tyrosine was used as a reference substance to form a calibration curve.

(30) TABLE-US-00002 TABLE 2 Comparison of Protease Activity for Soybean Koji Using Primary Strain Selections Primary strain selections Protease activity (U/g) CJ 3-27 258 CJ 4-4 118 CJ 14-6 225 CJ 16-57 155

(31) From the results of Table 2, CJ 3-27 and CJ 14-6 strains with high protease activity were selected.

(32) (4) Third Selection: In-Vitro 3T3-L1 Anti-Obesity Activity

(33) (A) Cell Line

(34) As for fat differentiation cells, 3T3-L1 cells as a mouse embryonic fibroblast cell line were purchased from the Korean Cell Line Bank (KCLB).

(35) (B) Cell Cultivation

(36) The cultivation of 3T3-L1 cells was performed using a DMEM culture medium containing 10% BCS and 1% penicillin-streptomycin. The cells, when proliferated to 70% of the culture dish, were centrifugally separated at 1,000 rpm and sub-cultured at a ratio of 1:5.

(37) (C) MTT Assay

(38) Based on the ability of living cellular mitochondria dehydrogenase to react with MTT and form MTT formazan crystals in a dark blue color, the MTT assay was performed according to the method disclosed by Carmichael et al. 500 μl of 3T3-L1 cells per well were plated in a 48-well plate at a concentration of 1.5×10.sup.4/ml and incubated in an incubator (37° C., 5% CO.sub.2) for 24 hours so as to immobilize the cells on the bottom of the plate. In the next day, the sample was injected into each well to a final concentration of 1000 μg/ml, 250 μg/ml, or 0 μg/ml. After 24-hour incubation, the medium containing the sample was adjusted to 5 mg/ml and a thiazolyl blue tetrazolium bromide solution was added at a rate of 50 μl per well to a final concentration of 500 μg/ml. The cells were incubated in an incubator (37° C., 5% CO.sub.2) for 4 hours. After the completion of the reaction, the medium containing the MTT reagent was removed, and 300 μl of dimethyl sulfoxide (DMSO) was added to dissolve MTT-formazan crystals, which were color-developed for 5 minutes to measure the absorbance at 540 nm using an ELISA microplate reader.

(39) (D) Induction of 3T3-L1 Cell Differentiation

(40) In order to induce cell differentiation, the cells were plated in a 6-well plate at a concentration of 2.5×10.sup.4/ml and incubated using a DMEM culture medium containing 10% FBS and 1% penicillin-streptomycin in an incubator (37° C., 5% CO.sub.2) for 4 days until they got into the post-confluent state. The positive control used in the anti-obesity test was resveratrol capable of inhibiting the differentiation of preadipocytes, accelerating adipolysis, and preventing adipogenesis by inducing the self-directed cellular ‘suicide’ of mature adipocytes.

(41) On the 0.sup.th day, the cells in the post-confluent state were treated with a differentiation-inducing medium containing 10% FBS, 1% penicillin-streptomycin, 5 μg/ml insulin, 1 μM dexamethasone (DMS), and 0.5 mM (3-isobutyl-1-methylxanthine (IBMX) for 72 hours. On the 3.sup.rd and 5.sup.th days, the cells were incubated with a DMEM differentiation-activating culture medium containing 10% FBS, 1% penicillin-streptomycin, and 10 μg/ml INS. Whenever the medium was replaced, all the samples were treated to have a concentration of 10 μg/ml, 50 μg/ml, or 0 μg/ml and adjusted to a concentration of 20 μg/ml as a positive control.

(42) (E) Oil Red O Dyeing and Quantification

(43) In order to determine the degree of differentiation of 3T3-L1 cells during incubation, the cells were stained with an Oil Red O reagent for dyeing lipid droplets according to a modification of the method disclosed by Rene et al., and Kasturi et al. on the 7th day, that is, the last day of differentiation.

(44) The cells under differentiation were immobilized in each well containing 4% para-formaldehyde (in PBS) through a reaction at room temperature for one hour or longer. The cells were removed of the immobilization solution and stained with 0.5% Oil Red O for one hour. For removal of the remaining dye, the cells were washed with flowing water twice until none of the dyeing reagent left. The stained lipid droplets were dissolved in isopropanol and measured in regards to the absorbance at 510 nm.

(45) The in-vitro anti-obesity activity was determined by comparison of the adipocyte differentiation inhibitory activity between the two stains of Bacillus amyloliquefaciens, CJ 3-27 and CJ 14-6, which proved to have high protease activity.

(46) TABLE-US-00003 TABLE 3 Adipocyte differentiation inhibition rate of secondary strain selections Concentration of culture medium Differentiation Strains (μg/ml) inhibition rate (%) Bacillus amyloliquefaciens 25 10.10 CJ 3-27 50 6.98 100 3.37 Bacillus amyloliquefaciens 25 20.57* CJ 14-6 50 26.41* 100 36.61* Statistic calibration: student-T test *P < 0.05

(47) As can be seen from the results of Table 3, the CJ 14-6 strain culture medium showed a significant level of adipocyte differentiation inhibition rate, while the CJ 3-27 strain culture medium had no significant change in the adipocyte differentiation inhibition rate.

Example 2: Preparation of Soybean Koji

(48) The final selection of novel strain in Example, Bacillus amyloliquefaciens CJ 14-6, was used as a fermentation starter to prepare a soybean koji according to the preparation method as stated in Example 1-(1).

(49) For a comparison with a conventional soybean koji using Aspergillus oryzae, a soybean koji was prepared using Aspergillus oryzae commercially available from Chungmoo Fermentation Co. as a fermentation starter according to the preparation method as stated in Example 1-(1).

(50) (1) Measurement of Protease Activity

(51) Each soybean koji was measured in regards to protease activity. The measurement results are presented in Table 4.

(52) TABLE-US-00004 TABLE 4 Enzyme activity of soybean koji using novel Bacillus amyloliquefaciens CJ 14-6 strain and conventional Aspergillus oryzae strain Strains Protease activity (U/g) Novel Bacillus amyloliquefaciens CJ 14-6 225 Conventional Aspergillus oryzae 102

(53) As can be seen from the results of Table 4, the soybean koji using the novel Bacillus amyloliquefaciens CJ 14-6 strain was 120.6% higher in the protease activity than the soybean koji using the convention Aspergillus oryzae strain. This shows that the use of the soybean koji using the novel Bacillus amyloliquefaciens CJ 14-6 strain can promote the protein degradation activity when used in gochujang or doenjang and contribute to the enhancement of the taste qualities.

Experimental Example 1: Measurement of Anti-Obesity Effect of Soybean Koji Using Novel Strain (CJ 14-6)

(54) An animal experiment was conducted in order to evaluate the anti-obesity effect of the soybean koji using the novel Bacillus amyloliquefaciens CJ 14-6 strain. The animals used in the experiment were male white rats (S.D. rat, 5 weeks old), which were purchased from Damool Science (South Korea) and kept in vivariums maintained at 18±2° C. under illumination regulated on a cycle of 12 hours (from 08:00 to 20:00).

(55) Rat objects were divided into two groups, each group consisting of seven objects. In the control group, 20% of lard was added to the powdery feed for white rat, and the rat objects were fed on a high-fat diet. In the Example-2 group, the rat objects were fed with 0.91% of the soybean koji using the novel strain of the present disclosure on the same high-fat diet. The weight and the adipose-tissue weight of the rat objects are presented in Tables 5 and 6, respectively.

(56) The weight and the diet intake were measured every week, and the adipose-tissue weight and the lipid content were determined after a 12-hour fasting prior to the termination of the testing. The collected blood was centrifugally separated at 1,900×G for 20 minutes to isolate the blood serum, which was used as a sample for determination of the lipid content in blood serum. In order to analyze the total lipid content, the neutral fat content, and the total cholesterol content of liver and adipose tissues, chloroform-methanol (2:1, v/v) was added to 0.1 g of the collected liver and adipose tissues, which were then kept refrigerated for 3 days and soaked in distilled water. The liver and adipose tissues were centrifugally separated at 1,150×G for 20 minutes and measured in regards to the total lipid content in the lipid layer, that is, the bottom layer. The lipid tissue was diluted and used to determine the total cholesterol content and the neutral fat content. The lipid content in the blood serum and tissues through the content analyses are presented in Tables 7 to 10.

(57) In order to measure the enzyme activity related to the biosynthesis of fatty acid in the adipose tissue, a 0.1M potassium phosphate buffer (pH 7.4, 37° C.) in a volume three times as much as the weight of the adipose tissue was added, and after homogenization, the adipose tissue was centrifugally separated at 3,000 rpm for 15 minutes. The supernatant thus obtained was centrifugally separated at 15,000 rpm for 30 minutes to collect a second supernatant. The enzyme activity measurements are presented in Tables 11 and 12.

(58) To determine the size of the adipocytes, the adipocytes collected were immobilized with a Bouin solution to make a paraffin block, which was cut into slices and stained with an anillin blue dye. Subsequently, the images of the adipocytes were taken with an electronic microscope to compare the adipocytes in each treated region.

(59) TABLE-US-00005 TABLE 5 Body weight Daily diet Initial body Final body increment intake Groups weight (g) weight (g) (g/day) (g/day) Control 171.79 544.00 6.84 ± 0.40 17.49 ± 0.80 group Example-2 172.57 513.29 6.26 ± 0.40 17.33 ± 1.78 group

(60) TABLE-US-00006 TABLE 6 Adipose tissue weight (g/100 g body wt.) Fat in Fat around Groups epididymis kidney Total white fat Control group 2.43 0.78 6.80 Example-2 group 2.24 0.65 6.29

(61) TABLE-US-00007 TABLE 7 Lipid content in blood serum (mg/dl) Total LDL- Groups Neutral fat cholesterol cholesterol Control 225.57 ± 25.64 128.43 ± 12.92 125.40 ± 15.43 group Example-2   152.57 ± 10.21***   95.86 ± 8.28***   63.66 ± 8.75*** group

(62) TABLE-US-00008 TABLE 8 Lipid content in liver tissue (mg/g) Total Groups Total fat Neutral fat cholesterol Control group 247.02 ± 21.87 94.91 ± 18.43 360.45 ± 57.55 Example-2 group  220.99 ± 16.77*  64.57 ± 3.00** 292.48 ± 76.68

(63) TABLE-US-00009 TABLE 9 Lipid content in epididymis adipose tissue (mg/g) Total Groups Total fat Neutral fat cholesterol Control group 501.55 ± 57.14 166.15 ± 16.18 294.90 ± 15.00 Example-2 group 459.50 ± 35.17   118.44 ± 18.44***  250.48 ± 26.74**

(64) TABLE-US-00010 TABLE 10 Lipid content in mesenteric adipose tissue (mg/g) Total Groups Total fat Neutral fat cholesterol Control group 412.22 ± 37.59 122.01 ± 6.02 178.23 ± 24.41 Example-2 group  368.37 ± 23.66*  108.81 ± 9.02**  147.77 ± 12.72*

(65) TABLE-US-00011 TABLE 11 Enzyme related to fatty acid synthesis in liver tissue (nmol/min/mg protein) Groups FAS ME G6PDH Control group 12.22 ± 1.07 31.47 ± 1.39 24.68 ± 1.45 Example-2 group  9.77 ± 0.54 26.96 ± 1.74  17.31 ± 1.19**

(66) TABLE-US-00012 TABLE 12 Enzyme related to fatty acid synthesis in adipose tissue (nmol/min/mg protein) Groups FAS ME G6PDH Control group 7.29 ± 0.59 8.00 ± 0.31 24.62 ± 1.26 Example-2 group  5.35 ± 0.35*  5.51 ± 0.62**  20.21 ± 1.30*

(67) TABLE-US-00013 TABLE 13 Groups Control group Example-2 group Image of adipocyte Refer to FIG. 1 Refer to FIG. 2 Size of adipocyte 21.41 ± 2.45 18.18 ± 1.11 (μm.sup.2)

(68) TABLE-US-00014 TABLE 14 Body fat accumulation index factors Leptin HR-LPL TE-LPL Groups (μg/ml) (unit/g) (unit/g) Control group 5.75 ± 0.70 7.03 ± 1.08 36.12 ± 5.50 Example-2 group   3.97 ± 0.17***  4.30 ± 1.06**   22.74 ± 2.08***

(69) Statistic calibration: student-T test

(70) *: P<0.05

(71) **: P<0.01

(72) ***: P<0.001

(73) As can be seen from the results of Table 5, the Example-2 group had a body weight increment accounting for no more than 91.5% of the body weight increment of the control group. According to the results of Table 6, the Example-2 group was lower in the weight of the adipose tissue around the kidney than the control group; so the weight of the adipose tissue around the kidney in the Example-2 group accounted for no more than 83.3% of that in the control group.

(74) Accordingly, the results of the example show that the soybean koji using a novel strain (CJ 14-6) makes an effect to reduce the body weight.

(75) [Accession Number]

(76) Depository Institution: Korean Culture Center of Microorganisms (KCCM), Yoorim Bldg. 45 Hongjenae 2-ga, Seodaemoon-gu, Seoul, 120-861, Rep. of Korea

(77) (Abroad)

(78) Accession No.: KCCM11718P

(79) Date of Deposit: Jul. 1, 2015