KR20210005509A - Method for producing Chunggukjang of Mukeunji and Chunggukjang of Mukeunji produced by the same method - Google Patents

Abstract

The present invention relates to a method for preparing fast-fermented bean paste with aged kimchi, and fast-fermented bean paste with aged kimchi prepared thereby. The method for preparing fast-fermented bean paste with aged kimchi comprises the steps of: (1) inoculating soybeans, which are obtained by soaking the soybeans, draining water, and steaming and cooling the soybeans, with a Bacillus subtilis strain, and fermenting the soybeans to prepare fast-fermented bean paste; (2) salting Chinese cabbage in salt water and washing the salted Chinese cabbage to remove salt therefrom; (3) mixing the salted Chinese cabbage desalted in the step (2) with glutinous rice paste, red pepper powder, high fructose, anchovy sauce, onion, kelp concentrate, garlic, salted shrimps, ginger and green onion to make a kimchi mixture, and aging the kimchi mixture prepare aged kimchi; and (4) mixing the fast-fermented bean paste prepared in the step (1) with the aged kimchi prepared in the step (3). According to the present invention, it is possible to inhibit the growth of harmful microorganisms that occur during a fermentation process.

Description

Method for producing chunggukjang and chunggukjang made by the above method {Method for producing Chunggukjang of Mukeunji and Chunggukjang of Mukeunji produced by the same method}

The present invention comprises the steps of: (1) subtracting water from the soaked soybeans, increasing the increase, and inoculating the cooled soybeans with a Bacillus subtilis strain, followed by fermentation to prepare cheonggukjang; (2) pickling the cabbage in brine, washing and desalting; (3) aging a kimchi mixture of glutinous rice paste, red pepper powder, high fructose, salted anchovy, onion, kelp concentrate, garlic, salted shrimp, ginger, and green onion to the desalted pickled cabbage of step (2) to prepare old paper; And (4) mixing the chunggukjang prepared in step (1) with the chunggukjang prepared in step (3), and the method for preparing the chunggukjang prepared by the method. About.

Cheonggukjang is a traditional soybean fermented food unique to Korea that has been naturally fermented and common sense for about 1,300 years. Boiled soybeans are fermented with Bacillus subtilis in a warm room with a unique taste and aroma. Bacillus , a fermenting bacteria of Cheonggukjang, inhibits the activity of putrefactive bacteria and pathogens in the intestines, thereby reducing carcinogens produced by putrefactive bacteria, carcinogenic substances such as ammonia, indole, and amines, and absorbing and excreting harmful substances. Until now, various effects of Cheonggukjang are known, such as lowering blood cholesterol, preventing high blood pressure, anti-cancer, antioxidant, thrombolysis, preventing osteoporosis, and improving liver function, but consumption is decreasing due to its unique odor.

The excellence of kimchi, a traditional Korean food, is widely known to people around the world and is receiving good responses. Research and application of functional kimchi, along with various types of kimchi, are being marketed, as well as studies of adding various food ingredients to kimchi. Kimchi is a traditional fermented food unique to Korea in which salted fish, seasonings, and spices are added to vegetables. It is an important corrosion indispensable to Koreans' diet. It is a storage food for consumption in winter when vegetables are not produced. The intestinal function that prevents constipation and suppresses diseases such as enteritis and colitis, aids digestion by promoting the decomposition of pectin, a protein-degrading enzyme in the stomach, and lactic acid bacteria released as it matures, has an antibacterial effect that suppresses harmful bacteria. It is known to have an effect of increasing appetite by producing organic acids and alcohols, and has anticancer effects.

Korean Patent Registration No. 0431277 discloses a method of manufacturing functional cheonggukjang, and Korea Registration Patent No. 1628191 discloses a method of manufacturing cheonggukjang containing functional ingredients, but it is different from the method of manufacturing cheonggukjang in the old paper of the present invention. .

The present invention was conceived by the above requirements, and the object of the present invention is to select a strain and prepare and mix old paper under optimal conditions so that it can be well harmonized with cheonggukjang in order to manufacture chunggukjang with excellent quality and preference. Thus, it is to provide a method of manufacturing old paper cheonggukjang in which unpleasant odor peculiar to cheonggukjang is removed and quality and preference are improved.

In order to solve the above problems, the present invention comprises the steps of (1) inoculating a Bacillus subtilis strain on the soaked soybeans after removing the water from the soaked soybeans. (2) pickling the cabbage in brine, washing and desalting; (3) aging a kimchi mixture of glutinous rice paste, red pepper powder, high fructose, salted anchovy, onion, kelp concentrate, garlic, salted shrimp, ginger, and green onion to the desalted pickled cabbage of step (2) to prepare old paper; And (4) it provides a method of manufacturing the chunggukjang, characterized in that it comprises the step of mixing the cheonggukjang prepared in step (1) and the step of mixing the chunggukjang prepared in step (3).

In addition, the present invention provides a chungkukjang prepared by the above method.

Cheonggukjang added to the old paper cheonggukjang of the present invention is fermented as a useful strain to inhibit the proliferation of harmful microorganisms that occur during the fermentation process of cheonggukjang, and does not produce biogenic amines, thus making it possible to prepare safe chunggukjang without toxicity. It is possible to provide a cheonggukjang that can be consumed simply by pouring water and boiling without preparing a separate seasoning.

1 shows the nucleotide sequence of 16S rRNA of the Bacillus subtilis SRCM103727 strain isolated in the present invention.
Figure 2 shows a schematic diagram of the Bacillus certilis SRCM103727 strain isolated in the present invention.
Figure 3 shows the antioxidant activity of Cheonggukjang prepared by inoculating Bacillus certilis SRCM103727 strain isolated in the present invention.
Figure 4 shows the antidiabetic activity of Cheonggukjang prepared by inoculating Bacillus certilis SRCM103727 strain isolated in the present invention.
Figure 5 shows a picture of the old paper cheonggukjang of the present invention.

In order to achieve the object of the present invention, the present invention

(1) step of inoculating a Bacillus subtilis strain on the soaked soybeans after draining the water and increasing the soaked soybeans, and fermenting them to prepare cheonggukjang;

(2) pickling the cabbage in brine, washing and desalting;

(3) aging a kimchi mixture of glutinous rice paste, red pepper powder, high fructose, salted anchovy, onion, kelp concentrate, garlic, salted shrimp, ginger, and green onion to the desalted pickled cabbage of step (2) to prepare old paper; And

(4) It provides a method for producing chungkukjang, characterized in that it comprises the step of mixing the chunggukjang prepared in step (1) and the step of mixing the chunggukjang prepared in step (3).

In the method of manufacturing old paper cheonggukjang of the present invention, the cheonggukjang in step (1) is preferably subtracted from the soybeans soaked for 10 to 14 hours at 3 to 5°C, and then increased for 25 to 35 minutes at 110 to 130°C. It can be prepared by inoculating a Bacillus subtilis strain on soybeans cooled to 40 to 45°C and fermenting at 36 to 38°C for 32 to 40 hours, more preferably at 4°C for 12 hours. After removing the water from the soaked soybeans, it can be prepared by inoculating Bacillus subtilis strain on soybeans that have been steamed at 121°C for 30 minutes and cooled to 40-45°C, and fermented at 37°C for 36 hours. . The production of cheonggukjang under the above-described conditions minimizes unpleasant odors and improves preference and quality due to its palatable taste and aroma.

In addition, in the method for producing old paper cheonggukjang of the present invention, the Bacillus subtilis strain is Bacillus subtilis SRCM103727 strain (accession number: KCCM12483P), protease, cellulase ), amylase, esterase, esterase lipase and naphthol-AS-BI-phosphohydrolase extracellular enzyme secretion ability, thrombus It has antibacterial activity, antioxidant activity and antidiabetic activity against harmful microorganisms of Bacillus cereus , Enterococcus faecalis and Listeria monocytogenes , and histamine and It does not produce the biogenic amines of tyramine, but it does not produce threonine, valine, methionine, isoleucine, leucine, phenylalanine and lysine. It is a strain derived from traditional paste that produces amino acids, and was used as a strain for fermentation of soybeans obtained from the (Re) Fermentation Microorganism Industry Promotion Agency.

In addition, in the manufacturing method of the old paper cheonggukjang of the present invention, the step (2) is preferably pickled cabbage in 8 to 12% (w/v) salt water at 20 to 25°C for 8 to 10 hours and washed to It can be desalted so that the salt concentration is 1.6 to 2.0% (w/w), and more preferably, the cabbage is pickled in 10% (w/v) salt water for 8 to 10 hours at 20 to 25°C and washed to It can be desalted so that the salt concentration becomes 1.8% (w/w). To prepare the pickled cabbage under the same conditions as described above, it was possible to pickle it under conditions having excellent texture without being salty.

In addition, in the manufacturing method of the old paper cheonggukjang of the present invention, step (3) is preferably based on the total weight of the kimchi mixture, in 80 to 83% by weight of desalted pickled cabbage, 2.3 to 2.7% by weight of glutinous rice paste, 3.2 to 3.8 of red pepper powder Wt%, high fructose 0.5~0.7 wt%, salted anchovy 1.8~2.2 wt%, onion 0.8~1.2 wt%, kelp concentrate 2.7~3.3 wt%, garlic 1.3~1.7 wt%, salted shrimp 1.8~2.2 wt%, ginger 0.2 A kimchi mixture mixed with ~0.4% by weight and 2.2-2.6% by weight of Korean leek can be aged to prepare old paper, more preferably, based on the total weight of the kimchi mixture, 2.5% by weight of glutinous rice paste in 81.2% by weight of desalted pickled cabbage, Kimchi mixed with 3.5% by weight of red pepper powder, 0.6% by weight of high fructose, 2% by weight of anchovy sauce, 1% by weight of onion, 3% by weight of kelp concentrate, 1.5% by weight of garlic, 2% by weight of salted shrimp, 0.3% by weight of ginger and 2.4% by weight of green onion The mixture can be aged to prepare old paper. The aged paper prepared with the above ingredients and mixing ratio was well harmonized with spicy, cool, salty, and umami taste, improving the flavor of the aged paper, thereby improving sensory properties.

In addition, in the method for preparing chunggukjang of old paper of the present invention, step (4) is preferably mixed in a weight ratio of 5.5 to 6.5: 3.5 to 4.5 of chunggukjang, more preferably 6: It can be mixed in 4 weight ratio. The taste, aroma, and texture were well harmonized by mixing the cheonggukjang and the aged paper in the same ratio as described above, and thus the preference of cheonggukjang could be further improved.

The manufacturing method of the old paper chungkukjang of the present invention, more specifically

(1) After draining the water from soybeans soaked at 3~5℃ for 10~14 hours, steamed at 110~130℃ for 25~35 minutes, and then cooled to 40~45℃, Bacillus subtilis Inoculating the SRCM103727 strain (accession number: KCCM12483P) and fermenting it at 36 to 38° C. for 32 to 40 hours to prepare cheonggukjang;

(2) Desalting the cabbage so that the salt concentration in the cabbage is 1.6 to 2.0% (w/w) by pickling and washing the cabbage in 8 to 12% (w/v) salt water at 20 to 25° C. for 8 to 10 hours;

(3) Based on the total weight of the kimchi mixture, in 80-83% by weight of the desalted pickled cabbage in step (2) above, 2.3-2.7% by weight of glutinous rice paste, 3.2-3.8% by weight of red pepper powder, 0.5-0.7% by weight of high fructose, anchovy Fish sauce 1.8 to 2.2 wt%, onion 0.8 to 1.2 wt%, kelp concentrate 2.7 to 3.3 wt%, garlic 1.3 to 1.7 wt%, salted shrimp 1.8 to 2.2 wt%, ginger 0.2 to 0.4 wt%, and leek 2.2 to 2.6 wt% Aging the mixture of kimchi tossed to prepare old paper; And

(4) mixing the chungkukjang prepared in the step (1) in a weight ratio of 5.5 to 6.5: 3.5 to 4.5 by weight of the prepared mud from the step (3),

More specifically

(1) Bacillus subtilis SRCM103727 strain (Accession No.: KCCM12483P) on soybeans that were soaked at 4°C for 12 hours and then steamed for 30 minutes at 121°C and cooled to 40-45°C. Fermenting at 37° C. for 36 hours after inoculating the inoculum to prepare cheonggukjang;

(2) desalting the cabbage so that the salt concentration in the cabbage is 1.8% (w/w) by pickling and washing the cabbage in 10% (w/v) salt water at 20 to 25° C. for 8 to 10 hours;

(3) Based on the total weight of the kimchi mixture, 2.5% by weight of glutinous rice paste, 3.5% by weight of red pepper powder, 0.6% by weight of high fructose, 2% by weight of salted anchovy, 1% by weight of onion, to 81.2% by weight of the desalted pickled cabbage from step (2) %, 3% by weight of kelp concentrate, 1.5% by weight of garlic, 2% by weight of salted shrimp, 0.3% by weight of ginger, and 2.4% by weight of green onion to prepare a kimchi mixture by aging it to prepare old paper; And

(4) It may include the step of mixing the cheonggukjang prepared in step (1) with the mudji prepared in step (3) at a weight ratio of 6:4.

Hereinafter, preparation examples and examples of the present invention will be described in detail. However, the following Preparation Examples and Examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following Preparation Examples and Examples.

Manufacturing example 1. Cheonggukjang

After washing with water, the soybeans were soaked at 4°C for 12 hours. Soaked soybeans were drained for 1 hour and then cooked at 121°C for 30 minutes using an autoclave, and then cooled to 40-45°C. Bacillus subtilis SRCM103727 strain (accession number: KCCM12483P) relative to the total weight of the cooled soybean was inoculated to 1% (v/w) (about 1.0×10 ⁶ CFU/ml). After that, it was fermented for 36 hours at 37°C and 80% humidity in a thermo-hygrostat to prepare Cheonggukjang.

Manufacture Example 2. Aged Paper Cheonggukjang

(1) The cabbage was pickled in 10% (w/v) salt water at 20 to 25°C for 8 to 10 hours, washed, and desalted so that the salt concentration in the cabbage was 1.8% (w/w).

(2) Based on the total weight of the kimchi mixture, 2.5% by weight of glutinous rice paste, 3.5% by weight of red pepper powder, 0.6% by weight of high fructose, 2% by weight of salted anchovy, chopped onion 1 A kimchi mixture mixed with weight%, kelp concentrate 3% by weight, minced garlic 1.5% by weight, salted shrimp 2% by weight, minced ginger 0.3% by weight, and chopped green onion 2.4% by weight was aged to prepare old paper.

(3) The prepared cheonggukjang prepared in Preparation Example 1 was mixed with the pasteurized paste prepared in step (2) at a weight ratio of 6:4.

Mixture ratio of kimchi mixture (% by weight) Material type Manufacturing Example 1 Comparative Example 1 Comparative Example 2 Pickled cabbage 81.2 84.2 78 Glutinous rice paste 2.5 2 3 chili powder 3.5 3 4 High fructose 0.6 0.4 0.8 Salted anchovy 2 1.5 2.5 Chopped onion One 0.5 1.5 Kelp concentrate 3 3.8 2.2 chopped garlic 1.5 One 2 Salted shrimp 2 1.5 2.5 Chopped ginger 0.3 0.1 0.5 Fine leeks 2.4 2 3

Comparative example 1 and 2: Old paper Cheonggukjang

The old paper cheonggukjang was prepared by the method of Preparation Example 2, but when the kimchi mixture of step (3) was prepared, it was blended at the material mixing ratio of Table 1 to prepare the old paper cheonggukjang of Comparative Examples 1 and 2.

Materials and methods

(1) Strain isolation and culture conditions

In order to isolate the Bacillus strain, 1 g of 29 samples of Cheonggukjang sold on the market were taken and diluted stepwise in 9 mL of a sterile 0.85% sodium chloride solution. 100 µl of the diluted solution was spread on 1.5% LB agar medium (Luria-Bertani agar, BD Difco, Sparks, MD, USA) and cultured at 30°C for 24 hours, and then strains were primarily selected using the difference in colony morphology. Then, the strain was separated by pure culture again. The purely isolated microorganism was suspended in a 10% skim milk (BD Difco, Sparks, MD, USA) solution and stored at -80°C for use.

(2) Measurement of extracellular enzyme secretion ability

In order to select a strain suitable for fermentation of Cheonggukjang, the activities of starch degrading enzyme (amylase), protease, and fibrinase (cellulase) were verified among the extracellular enzymes released by the isolated microorganism to the outside of the cells. Agar diffusion method using a solid medium containing a component of a substrate that specifically reacts with each enzyme was used. Starch decomposition activity was prepared in a starch agar medium containing 1% soluble starch and 2% agar, and to measure protease activity, a skim milk agar medium was prepared by adding 1.5% agar to 2% skim milk. In addition, for the activity of cellulase, CMC agar medium was prepared by adding 1.5% agar to 1% carboxylmethyl cellulose (CMC) according to Teather and Wood's method, and enzyme using a well diffusion method. Each medium was drilled with an 8 mm hole to measure the activity. The selected strain was cultured with shaking at 30°C for 24 hours in LB liquid medium, and the culture of the selected strain was centrifuged at 13,000 rpm for 10 minutes to take the supernatant, sterilized with a 0.45 μm membrane filter, and 100 μl of each extracellular enzyme activity After dispensing into the hole of the measurement medium and reacting at 25° C. for 24 hours, the size of the ring formed around the well was measured. In the case of starch degrading enzyme activity, the size of the ring formed after dyeing with Lugol's solution after the reaction was measured, and the size of the ring formed after staining with 0.1% Conco red reagent was measured for fibrinolytic enzyme activity. .

(3) Identification of selected strain

The base sequence of the 16S rRNA gene was analyzed to identify the selected strain. For sequence amplification, universal primers 27F (5'-AGA GTT TGA TCC TGG CTC AG-3', SEQ ID NO: 2) and 1492R (5'-GGT TAC CTT GTT ACG ACT T-3', SEQ ID NO: 3) were used. And, 16S rRNA gene sequences of standard strains with high sequence consistency were obtained from NCBI (National Center for Biotechnology Information). Clustal W 2.0 program was used for mutual comparison between sequences, and a schematic diagram was created using Mega 7.0.26 program. The neighbor joining method was used for the analysis of the tree, and the robustness of the generated tree was confirmed by bootstrapping through 1,000 repetitions.

(4) Measurement of antimicrobial activity against food harmful microorganisms

Food accept pre-sale from harmful bacteria Bacillus cereus (Bacillus cereus) KCCM40935, Enterococcus faecalis (Enterococcus faecalis) KCCM11814, Listeria monocytogenes jeneseu (Listeria monocytogenes) Korea Seed Association (KCCM, Korean Culture C enter of Microorganisms) the KCCM43155 instructions It was used as a strain, and the antibacterial activity of the strains selected according to the agar diffusion method was investigated. The selected strains were each inoculated into LB liquid medium and cultured at 30° C. for 24 hours, and after culture, centrifuged at 13,000 rpm for 10 minutes to take a supernatant and sterilize it with a 0.45 μm syringe filter. After making an 8 mm hole in 0.8% agar medium containing each indicator strain, 100 µl of each selected microorganism culture supernatant was dispensed, incubated at 30°C for 24 hours, and the diameter of the inhibitor ring formed around the hole was measured. The antagonistic ability of the strain against harmful microorganisms was confirmed.

(5) Antioxidant activity assay

Antioxidant activity was analyzed using the method of Kedare & Singh. Specifically, 20 µl of the strain culture supernatant (Tryptic soy broth) was mixed with 180 µl of 0.1mM DPPH (2,2-diphenyl-1-picrylhydrazyl in methanol), and the absorbance was measured at 517 nm after a dark reaction at room temperature for 30 minutes. Antioxidant activity was calculated according to the following formula. TSB liquid medium was used as the negative control, and the measured value was measured after treatment in the same manner compared to the control.

Antioxidant activity (%) = [1-(A/B)]×100

A: Absorbance of DPPH solution with sample at 517 ㎚

B: Absorbance of DPPH solution without sample at 517nm

(6) antidiabetic activity

In order to measure the α-glucosidase inhibitory activity of the selected strain, it was evaluated under the conditions disclosed in Table 2 below. After adding 50 µl of 100mM PPB (potassium phosphate buffer, pH 7.0 at 37°C) to 12.5 µl of the culture supernatant of the selected strain, 12.5 µl of 0.5 U/ml α-glucosidase solution was added, and 20 mM PNPG (p-nitrophenyl -β-D-glucanopyranoside) solution was added and reacted at 37°C for 20 minutes. Thereafter, 100 µl of 200 mM sodium carbonate aqueous solution was added, and absorbance was measured at 400 nm using a microplate reader (SPARK, TECAN, Austria), and α-glucosidase inhibitory activity was expressed as a percentage (%).

α-glucosidase inhibitory activity = 1-(sample volume activity/enzyme volume activity) × 100%

Enzyme volume activity(U/ml) = (A _t1 -A _b ) × 0.0442

Sample volume activity(U/ml) = (A _t2 -A _b ) × 0.0442

α-glucosidase inhibitory activity measurement conditions solution Test 1(t1) Test 2(t2) Blank Mix by inversion and equilibrate to 37℃ DW 12.5µl - 25µl Sample solution - 12.5µl - 100mM PPB 50µl 50µl 50µl 0.5 U/ml α-glucosidase solution 12.5µl 12.5µl - 20mM PNPG 25µl 25µl 25µl 200mM sodium carbonate solution 100 μl 100 μl 100 μl

(7) Generation of Biogenic amines

Qualitative tests to determine whether or not biogenic amines were produced were confirmed according to the method of Chang et al. Specifically, the solid medium for confirming the production of biogenic amines of the selected strain was used to prepare an LB agar medium containing 0.25% glycerol, 0.006% bromocresol purple, and 0.1% histidine or tyrosine. The selected strain was cultivated at 37° C. for 24 hours after streak smearing on each medium, and the level of color development was compared with a control medium, a general LB medium, to determine whether or not biogenic amine was produced.

(8) Enzyme activity analysis using API ZYM kit

In order to investigate whether the selected strain produced enzymes, an API ZYM kit (bioMeriux Co., France), which was prepared based on the availability of substrates of a total of 20 types of enzymes, was used. After culturing the selected strain in LB solid medium, the cells were recovered, suspended in 0.85% NaCl solution, and adjusted to a turbidity of 5-6 with Macfarland. After dispensing the suspension into each tube of the API ZYM kit, incubating at 37°C for 4 hours, dropping the ZYM-A and ZYM-B reagents into each tube and reacting at room temperature for 5 minutes to change the color to each substrate enzyme. It was read whether it was active or not. Values ranged from 0 to 5 depending on the degree of color change, 0 being negative, 5 (=40 nanomoles) being the maximum intensity response, and 4 to 1 being the intermediate values of 30, 20, 10 and 5 nanomoles, respectively. If it was 3 or more, it was determined as positive.

(9) API 50 CH Analysis of sugar availability using kit

In order to confirm the sugar availability of the selected strain, an API 50 CH kit (BioMerieux, France) prepared based on the availability of a total of 49 types of carbohydrates was used. The selected strain was subcultured in 5 ml of LB liquid medium, cultured at 30°C for 24 hours, and then 1 ml of the culture solution was centrifuged at 13,000 rpm for 20 minutes to remove the supernatant, and only the cell pellets were recovered and PBS (phosphate buffered saline, 0.1 M, pH 7.0) was used in the experiment after washing twice. A suspension was prepared by adjusting the turbidity to 2 with McFarland (BioMerieux), and 1 ml of the strain culture solution suspended in the API 50 CHB/E medium was added and mixed, and 120 μl was dispensed into API 50 CH strips, and in an incubator at 37° C. for 24 hours and After incubation for 48 hours, each sugar fermentation pattern was compared. The sugar fermentation pattern was read as positive and negative by checking the color change of the tube.

(10) Measurement of thrombolytic activity

The thrombolytic activity was measured according to the fibrin plate method. Specifically, 100 µl of a 50 U/ml thrombin solution was dispensed into a 90 mm Petri dish, followed by 10 ml of a 0.5% fibrinogen solution (in 67 mM sodium phosphate buffer) and 10 ml of a 1.0% agarose solution in order. It was added and mixed so that the thrombus was formed evenly. After allowing the medium to coagulate while leaving at room temperature for 10 to 30 minutes, 100 µl of the strain culture solution was injected into the hole formed by making an 8 mm hole for sample injection, and reacted at 30°C for 24 hours. The diameter of the ring was measured to measure the ability to dissolve thrombi.

(11) Manufacture of Cheonggukjang inoculated with selected strains

After purchasing domestic soybeans (baektae), the beans were sorted and immersed in water for 12 hours. After dehydration for 1 hour, 500 g was added to a glass bottle to increase soybeans at 121°C for 30 minutes.After cooling at room temperature to 40 to 45°C, the selected strain was added to the bottle at 1% (v/w) of the amount of soybeans (approx. 1.0×10 ⁶ CFU/ml). After that, it was fermented for 36 hours at 37°C and 80% humidity in a thermo-hygrostat.

(12) Analysis of general ingredients of Cheonggukjang

The moisture content of Cheonggukjang was measured using a moisture meter (FD-720, KEtt Co,. Japan), and the pH was measured using a pH meter after diluting 5 g of a sample in 45 ml of distilled water and shaking.

(13) Analysis of Amino Nitrogen in Cheonggukjang

Amino nitrogen was measured by the Formol method. Take a sample (2 g) into an Erlenmeyer flask (250 ml), add distilled water (100 ml), stir (1 hour), and titrate with 0.1N NaOH solution to pH 8.4. Neutral formalin (20 ml) was added thereto, followed by neutralization titration with 0.1N NaOH solution to pH 8.4. Separately, a background test was performed on distilled water, and the amino nitrogen content was calculated according to the following equation.

Amino-type nitrogen =

A: Sample titration amount of 0.1N NaOH solution (ml)

B: 0.1N NaOH solution blank test (ml)

F: titer of 0.1N NaOH solution

(14) Cheonggukjang's Bacillus cereus ( Bacillus cereus ) Content analysis

In order to analyze the content of Bacillus cereus in Cheonggukjang prepared by inoculating with the selected strain, an experiment was conducted according to the Food Code. Each 50 g of Cheonggukjang was diluted in decimal with 450 ml of sterile diluent, and 100 µl of each dilution was spread on MYP (Mannitol Egg Yolk Polymyxin) agar medium and cultured in an incubator at 30°C for 24 hours. Among the colonies formed on the surface of the medium, the number of pink colonies having turbid rings generating lecithinase around them was counted.

(15) Analysis of free amino acids in Cheonggukjang

In order to analyze the essential free amino acid content of cheonggukjang prepared by inoculation with the selected strain, 2 g of cheonggukjang was taken, 30 ㎖ of tertiary distilled water was added, stirred, and then adjusted to 50 ㎖. After extraction for 20 minutes using ultrasonic waves, 13,000 rpm Centrifuged at for 10 minutes. After adding 2 ml of 5% trichloroacetic acid (TCA) to 2 ml of the centrifuged supernatant, centrifugation at 13,000 rpm for 10 minutes, the supernatant was taken, diluted with 0.02N HCl, and filtered using a 0.2 μm syringe filter. Essential free amino acids areoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine under the conditions disclosed in Table 3 below. valine) 8 kinds of amino acids were analyzed.

Essential free amino acid content analysis conditions Instrument Amino acid analysis (Hitach L-8900) Detector UV/Vis (440nm-570nm) Column Hitachi HPLC packed column, #2622PF Column (4.6x60mm) Ion Exchange column Buffer flow rate 0.40ml/min Ninhydrin flow rate 0.35ml/min Temperature 50℃ Injection volume 20µl Mobile phase Buffer set (PH-SET KANTO)

(16) Analysis of antioxidant, thrombolytic and antidiabetic activity of Cheonggukjang

The antioxidant, thrombolytic, and antidiabetic activities of Cheonggukjang prepared by inoculation with the selected strain were performed in the same manner as the above-described method for analyzing antioxidant, thrombolytic and antidiabetic activity of the selected strain.

Example 1. Selection of strains for cheonggukjang production

Were isolated the Bacillus (Bacillus) in the strain of the total 164-29 kinds of sauces, it can be used as is in the food material, and without creating a biogenic amine, the extracellular secretion of the enzyme activity, antimicrobial activity, the antioxidant activity of the microorganisms , The SRCM103727 strain having excellent antidiabetic activity and thrombolytic activity was selected, and the 16S rRNA gene nucleotide sequence (SEQ ID NO: 1, FIG. 1) was analyzed for identification of the selected SRCM103727 strain, and the NCBI BLAST search result, SRCM103727 strain showed 99% homology with Bacillus subtilis , and as a result of creating a phylogenetic tree based on the 16S rRNA sequence of the standard strain, the closest relationship with Bacillus certilis NBRC13719 was found. Confirmed (Fig. 2). Finally, the selected strain was named Bacillus subtilis SRCM103727, and was deposited with the Korea Microbiological Conservation Center (KCCM) on April 04, 2019, and was given the deposit number KCCM12483P, which was used as a strain for the production of Cheonggukjang. It was finally selected.

Example 2. Characterization of SRCM103727 strain

The extracellular enzyme activity of the finally selected SRCM103727 strain was confirmed. As a result, amylase, protease, and cellulase activity were all excellent (Table 4), and when various extracellular enzyme activities were confirmed using the API ZYM kit, esterase, esterase lipase, and It was confirmed that there was Naphthol-AS-BI-phosphohydrolase activity (Table 5).

In addition, the finally selected SRCM103727 strain showed excellent antimicrobial activity against food harmful microorganisms Bacillus cereus KCCM40935, Enterococcus faecalis KCCM11814, Listeria monocytogenes KCCM43155, and anti-acid It was confirmed that it has active and thrombolytic activity, and does not produce biogenic amines, histamine and tyramine (Table 4). In addition, it was confirmed that the SRCM103727 strain had anti-diabetic activity through α-glucosidase inhibitory activity (Table 4). As a result of checking the sugar availability of SRCM103727 strain using the API 50 CH kit, glycerol (Glycerol, GLY), L-arabinose (LARA), glucose (GLU), D-mannose (D-Mannose, MNE) ), inositol (INO), mannitol (MAN), sorbitol (SOR), cellobiose (CEL) and maltose (Maltose, MAL) can be used as carbon sources (Table 6) .

Confirmation of extracellular enzyme activity, antibacterial activity, antioxidant activity, thrombolytic activity, biogenic amine production and antidiabetic activity of SRCM103727 strain Extracellular enzyme activity (mm) Amylase 22 Protease 19 Cellulase 26 Antibacterial activity (mm) Bacillus cereus KCCM40935 17 Enterococcus Pacalis KCCM11814 20 Listeria monocytogenes KCCM43155 12 Antioxidant activity (%) 48.52 Thrombolytic activity (mm) 17 Produces biogenic amines (histamine or tyramine) ND Antidiabetic activity (%) 30.97

ND: not detected

Enzyme activity confirmation using API ZYM kit of SRCM103727 strain One 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 - + + - - - - - - - + - - - - - - - - -

1: alkaline phosphatase; 2: esterase (C4); 3: esterase lipase (C8); 4: lipase (C14); 5: leucine arylamidase; 6: valine arylamidase; 7: cystine arylamidase; 8: trypsin; 9: α-chymotrypsin; 10: acid phosphatase; 11: naphthol-AS-BI-phosphohydrolase; 12: α-galactosidase; 13: β-galactosidase; 14: β-glucuronidase; 15: α-glucosidase; 16: β-glucosidase; 17: N-acetyl-β-glucosaminidase; 18: α-nabbisudase; 19: α-fucosidase; 20: control-: negative reaction, +: positive reaction.

Confirmation of sugar availability using API 50 CH kit of SRCM103727 strain Party reaction Party reaction control - ESC - GLY + SAL - ERY - CEL + DARA - MAL + LARA + LAC - RIB - MEL - DXYL - SAC - LXYL - TRE - ADO - INU - MDX - MLZ - GAL - RAF - GLU + AMD - FRU - GLYG - MNE + XLT - SBE - GEN - RHA - TUR - DUL - LYX - INO + TAG - MAN + DFUC - SOR + LFUC - MDM - DARL - MDG - LARL - NAG - GNT - AMY - 2KG - ARB - 5KG -

GLY: Glycerol; Ery: Erythritol; DARA: D-Arabinose; LARA: L-Arabinose; RIB: Ribose; DXYL: D-Xylose; LXYL: L-Xylose; ADO: Adonithol; MDX: Methyl xyloside; GAL: Galactose; GLU: Glucose; FRU: D-Fructose; MNE: D-Mannose; SBE: Sorbose; RHA: Rhamnose; DUL: Dulcitol; INO: Inositol; MAN: Mannitol; SOR: Sorbitol; MDM: Methyl-D-mannoside; MDG: Methyl-D-glucose; NAG: N-acetyl-glucosamine; AMY: Amygdalin; ARB: Arbutin; ESC: Esculine; SAL: Salicin; CEL: Cellobiose; MAL: Malotose; LAC: Lactose; MEL: Melibiose; SAC: Sucrose; TRE: Trehalose; INU: Inulin; MLZ: Melizitose; RAF: D-raffinose; AMD: Starch; GLYG: Glycogen,; XLT: Xylitol; GEN: Gentibiose; TUR: Turanose; LYX: Lyxose; TAG: Tagatose; DFUC: D-Fucose; LFUC: L-Fucose; DARL: D-Arabitol; LARAL: L-arabitol; GNT: Gluconate; 2KG: 2, Keto-gluconate; 5KG: 5, Keto-gluconate-: negative, +: positive.

Example 3. Analysis of Physicochemical Characteristics of Cheonggukjang Prepared by Inoculating SRCM103727 Strain

As a result of analyzing the physicochemical properties of Cheonggukjang prepared using the finally selected SRCM103727 strain, the water content and pH were higher in Cheonggukjang (test area) prepared by inoculating the SRCM103727 strain compared to Cheonggukjang (control) without inoculating the strain. It was high.

Amino-type nitrogen (AN: Amino type Nitrogen) is a factor of the delicious taste component of cheonggukjang, and it was confirmed that the amino-type nitrogen content was also higher in cheonggukjang (test group) prepared by inoculating the strain compared to the control (Table 7). .

In addition, as a result of measuring the amino acid content of Cheonggukjang prepared using the SRCM103727 strain, threonine, valine, methionine, isoleucine, compared to Cheonggukjang (control) without inoculation of the strain. It was confirmed that the contents of leucine, phenylalanine, and lysine were significantly enhanced (Table 8).

Physicochemical Characteristics of Cheonggukjang Prepared by Inoculating SRCM103727 Strain characteristic Strains uninoculated control SRCM103727 strain inoculation test area Moisture content (%) 51.29 54.98 pH 6.71 8.58 Bacillus cereus (CFU/ml) 0 0 Amino nitrogen (mg%) 189.53 440.83

Free amino acid content of Cheonggukjang prepared by inoculating strain SRCM103727 Amino acid (mg/kg) Strains uninoculated control SRCM103727 strain inoculation test area Threonine 3.2 8.85 Valine 18.35 65.5 Methionine 4.45 48.25 Isoleucine 0.6 13.45 Leucine 2.45 58 Phenylalanine 12.35 93.15 Lee Sin 9.2 78 Tryptophan 0 0 gun 50.6 365.2

In addition, in the case of Cheonggukjang prepared using the finally selected SRCM103727 strain, it was confirmed that the anti-oxidant activity was not only excellent, but also the antidiabetic activity was remarkably enhanced compared to Cheonggukjang (control) without inoculation of the strain (Fig. 3). And Fig. 4).

Example 4. Old paper Cheonggukjang's sensory test

A sensory test was performed on 30 sensory test personnel with the cheonggukjang prepared by the method of Preparation Example 2 and the old paper cheonggukjang of the comparative examples, and the results are shown in Table 9 below. For the sensory test of Cheonggukjang, 200 g of Cheonggukjang was added to 500 mL of water and boiled for 5 minutes to make Cheonggukjang Jjigae. Then, the sensory test items with rice were conducted for color, aroma, taste and overall acceptability. Accordingly, the score was recorded by the test subject according to the following evaluation criteria with 5 points as a perfect score, and then the average value was calculated and recorded. 5: very good, 4: good, 3: normal, 2: bad, 1: very bad.

Cheonggukjang's sensory test division color incense flavor Overall preference Manufacturing Example 2 4.04 4.56 4.40 4.44 Comparative Example 1 3.86 4.02 4.12 4.06 Comparative Example 2 3.90 3.94 4.00 3.98

As a result, it was confirmed that it was more preferable to add an appropriate amount of chungkukjang prepared by blending with the material blending ratio of Preparation Example 2, showing a higher score than that of the chungkukjang of jungjii chungkukjang of Preparation Example 2.

Korea Microorganism Conservation Center (overseas) KCCM12483P 20190404

<110> Agricultural Corporation Sunchang Sung Ga Jung Foods Co., Ltd. <120> Method for producing Chunggukjang of Mukeunji and Chunggukjang of Mukeunji produced by the same method <130> PN20001 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 1487 <212> DNA <213> Bacillus subtilis SRCM103727 <400> 1 ctgctcagga cgaacgctgg cggcgtgcct aatacatgca agtcgagcgg acagatggga 60 gcttgctccc tgatgttagc ggcggacggg tgagtaacac gtgggtaacc tgcctgtaag 120 actgggataa ctccgggaaa ccggggctaa taccggatgg ttgtttgaac cgcatggttc 180 aaacataaaa ggtggcttcg gctaccactt acagatggac ccgcggcgca ttagctagtt 240 ggtgaggtaa cggctcacca aggcaacgat gcgtagccga cctgagaggg tgatcggcca 300 cactgggact gagacacggc ccagactcct acgggaggca gcagtaggga atcttccgca 360 atggacgaaa gtctgacgga gcaacgccgc gtgagtgatg aaggttttcg gatcgtaaag 420 ctctgttgtt agggaagaac aagtaccgtt cgaatagggc ggtaccttga cggtacctaa 480 ccagaaagcc acggctaact acgtgccagc agccgcggta atacgtaggt ggcaagcgtt 540 gtccggaatt attgggcgta aagggctcgc aggcggtttc ttaagtctga tgtgaaagcc 600 cccggctcaa ccggggaggg tcattggaaa ctggggaact tgagtgcaga agaggagagt 660 ggaattccac gtgtagcggt gaaatgcgta gagatgtgga ggaacaccag tggcgaaggc 720 gactctctgg tctgtaactg acgctgagga gcgaaagcgt ggggagcgaa caggattaga 780 taccctggta gtccacgccg taaacgatga gtgctaagtg ttagggggtt tccgcccctt 840 agtgctgcag ctaacgcatt aagcactccg cctggggagt acggtcgcaa gactgaaact 900 caaaggaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg 960 cgaagaacct taccaggtct tgacatcctc tgacaatcct agagatagga cgtccccttc 1020 gggggcagag tgacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt 1080 aagtcccgca acgagcgcaa cccttgatct tagttgccag cattcagttg ggcactctaa 1140 ggtgactgcc ggtgacaaac cggaggaagg tggggatgac gtcaaatcat catgcccctt 1200 atgacctggg ctacacacgt gctacaatgg acagaacaaa gggcagcgaa accgcgaggt 1260 taagccaatc ccacaaatct gttctcagtt cggatcgcag tctgcaactc gactgcgtga 1320 agctggaatc gctagtaatc gcggatcagc atgccgcggt gaatacgttc ccgggccttg 1380 tacacaccgc ccgtcacacc acgagagttt gtaacacccg aagtcggtga ggtaaccttt 1440 taggagccag ccgccgaagg tgggacagat gattggggtg aagtcgt 1487 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 agagtttgat cctggctcag 20 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ggttaccttg ttacgactt 19

Claims (5)

(1) step of inoculating a Bacillus subtilis strain on the soaked soybeans after draining the water and increasing the soaked soybeans, and fermenting them to prepare cheonggukjang;
(2) pickling the cabbage in brine, washing and desalting;
(3) aging a kimchi mixture of glutinous rice paste, red pepper powder, high fructose, salted anchovy, onion, kelp concentrate, garlic, salted shrimp, ginger, and green onion to the desalted pickled cabbage of step (2) to prepare old paper; And
(4) A method of manufacturing old paper cheonggukjang, characterized in that it comprises the step of mixing the chunggukjang prepared in step (1) with the chunggukjang prepared in step (3). The method of claim 1, wherein the Bacillus subtilis strain is Bacillus subtilis SRCM103727 strain (accession number: KCCM12483P). The method of claim 2,
(1) After draining the water from soybeans soaked at 3~5℃ for 10~14 hours, steamed at 110~130℃ for 25~35 minutes, and then cooled to 40~45℃, Bacillus subtilis Inoculating the SRCM103727 strain (accession number: KCCM12483P) and fermenting it at 36 to 38° C. for 32 to 40 hours to prepare cheonggukjang;
(2) Desalting the cabbage so that the salt concentration in the cabbage is 1.6 to 2.0% (w/w) by pickling and washing the cabbage in 8 to 12% (w/v) salt water at 20 to 25° C. for 8 to 10 hours;
(3) Based on the total weight of the kimchi mixture, in 80-83% by weight of the desalted pickled cabbage in step (2) above, 2.3-2.7% by weight of glutinous rice paste, 3.2-3.8% by weight of red pepper powder, 0.5-0.7% by weight of high fructose, anchovy Fish sauce 1.8 to 2.2 wt%, onion 0.8 to 1.2 wt%, kelp concentrate 2.7 to 3.3 wt%, garlic 1.3 to 1.7 wt%, salted shrimp 1.8 to 2.2 wt%, ginger 0.2 to 0.4 wt%, and leek 2.2 to 2.6 wt% Aging the mixture of kimchi tossed to prepare old paper; And
(4) A method for producing chunggukjang, characterized in that it comprises mixing the chunggukjang prepared in step (1) with the chunggukjang prepared in step (3) at a weight ratio of 5.5 to 6.5: 3.5 to 4.5 . The method of claim 3, wherein the Bacillus subtilis SRCM103727 strain is a protease, cellulase, amylase, esterase, esterase lipase, and Extracellular enzyme secretion ability, thrombolytic activity of naphthol-AS-BI-phosphohydrolase, Bacillus cereus , Enterococcus faecalis and Listeria monocyto Genes ( Listeria monocytogenes ) has antibacterial activity, antioxidant activity and antidiabetic activity against harmful microorganisms, and does not produce biogenic amines such as histamine and tyramine, threonine, valine , Methionine (methionine), isoleucine (isoleucine), leucine (leucine), phenylalanine (phenylalanine), and Bacillus subtilis ( Bacillus subtilis ) producing amino acids of lysine ( Bacillus subtilis ) Preparation of the old paper Cheonggukjang characterized in that it is a strain SRCM103727 Way. Cheonggukjang prepared by the method of any one of claims 1 to 4.

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